Heil, H.S., Schreiber, B., Götz, R., Emmerling, M., Dabauvalle, M.-C., Krohne, G., Höfling, S., Kamp, M., Sauer, M., Heinze, K.G.: Sharpening emitter localization in front of a tuned mirror.Light: Science & Applications.7,99-- (2018).
Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
Adam, A., Deimel, S., Pardo-Medina, J., García-Martínez, J., Konte, T., Limón, M., Avalos, J., Terpitz, U.: Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction.International Journal of Molecular Sciences.19,215 (2018).
Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.
Brunk, M., Sputh, S., Doose, S., van de Linde, S., Terpitz, U.: HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi.Scientific Reports.8,605-- (2018).
The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C.S., Sauer, M., Sukhorukov, V.L.: Nanostructure of DNA repair foci revealed by superresolution microscopy.The FASEB Journal.fj.201701435-- (2018).
Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
Rat, C., Heiby, J.C., Bunz, J.P., Neuweiler, H.: Two-step self-assembly of a spider silk molecular clamp.Nature Communications.9,4779-- (2018).
Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.
Neubert, F., Beliu, G., Terpitz, U., Werner, C., Geis, C., Sauer, M., Doose, S.: Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging.Angewandte Chemie International Edition.57,16364--16369 (2018).
Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.
Börtlein, C., Draeger, A., Schoenauer, R., Kuhlemann, A., Sauer, M., Schneider-Schaulies, S., Avota, E.: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling.Frontiers in Immunology.9,815-- (2018).
By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.
Haselmann, H., Mannara, F., Werner, C., Planagumà, J., Miguez-Cabello, F., Schmidl, L., Grünewald, B., Petit-Pedrol, M., Kirmse, K., Classen, J., Demir, F., Klöcker, N., Soto, D., Doose, S., Dalmau, J., Hallermann, S., Geis, C.: Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction.Neuron.100,91--105.e9 (2018).
AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.
Tauscher, S., Nakagawa, H., Völker, K., Werner, F., Krebes, L., Potapenko, T., Doose, S., Birkenfeld, A.L., Baba, H.A., Kuhn, M.: β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice.Cardiovascular Diabetology.17,103 (2018).
The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
Grabenbauer, F., Katzer, A., Sisario, D., Memmel, S., Flentje, M., Sukhorukov, V.L., Djuzenova, C.S.: MEK-inhibitor PD184352 enhances the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922: the role of cell type and drug-irradiation schedule.Oncotarget.9,37379-37392 (2018).
Targeting MEK protein in cancer cells usually leads to acquired resistance to MEK inhibitors and activation of the prosurvival protein Akt. Since both MEK and Akt are clients of the Hsp90 chaperone system, the present study explores the responses of irradiated lung carcinoma A549 and glioblastoma SNB19 cell lines to combined MEK and Hsp90 inhibition. Unexpectedly, the MEK inhibitor PD184352 administered 24 h prior to irradiation, enhanced cell survival through upregulation of not only MEK and Erk1/2 but also of Akt. In contrast, PD184352 added 1 h before irradiation strongly reduced the expression of Erk and did not upregulate Akt in both cell lines. As a result, the MEK inhibitor increased the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in glioblastoma SNB19 cells. Possible reasons for the enhanced cell killing under this short-term pretreatment schedule may be a down-regulation of Erk during or directly after irradiation, increased DNA damage and/or a strong G2/M arrest 24 h after irradiation. In addition, an 1-h pretreatment with PD184352 and/or NVP-AUY922 under schedule II induced neither G1 arrest nor up-regulation of p-Akt in both cell lines as it did under schedule I. Yet, a long-term treatment with the MEK inhibitor alone caused a strong cytostatical effect. We conclude that the duration of drug pretreatment before irradiation plays a key role in the targeting of MEK in tumor cells. However, due to an aberrant activation of prosurvival proteins, the therapeutic window needs to be carefully defined, or a combination of inhibitors should be considered.
Forero, A., Rivero, O., Wäldchen, S., Ku, H.-P., Kiser, D.P., Gärtner, Y., Pennington, L.S., Waider, J., Gaspar, P., Jansch, C., Edenhofer, F., Resink, T.J., Blum, R., Sauer, M., Lesch, K.-P.: Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain.Frontiers in Cellular Neuroscience.11,307-- (2017).
Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
Scholz, N., Guan, C., Nieberler, M., Grotemeyer, A., Maiellaro, I., Gao, S., Beck, S., Pawlak, M., Sauer, M., Asan, E., Rothemund, S., Winkler, J., Prömel, S., Nagel, G., Langenhan, T., Kittel, R.J.: Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.eLife.6,e28360-- (2017).
Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
Brühlmann, D., Sokolov, M., Butté, A., Sauer, M., Hemberger, J., Souquet, J., Broly, H., Jordan, M.: Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality.Biotechnology and Bioengineering.114,1448--1458 (2017).
Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.
Walter, T., Schlegel, J., Burgert, A., Kurz, A., Seibel, J., Sauer, M.: Incorporation studies of clickable ceramides in Jurkat cell plasma membranes.Chem. Commun.53,6836--6839 (2017).
The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.
Markert, S.M., Bauer, V., Muenz, T.S., Jones, N.G., Helmprobst, F., Britz, S., Sauer, M., Rössler, W., Engstler, M., Stigloher, C.: Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species. In: Müller-Reichert, T. und Verkade, P. (hrsg.) Correlative Light and Electron Microscopy III. S. 21--47. Academic Press (2017).
Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.
Brühlmann, D., Muhr, A., Parker, R., Vuillemin, T., Bucsella, B., Kalman, F., Torre, S., La Neve, F., Lembo, A., Haas, T., Sauer, M., Souquet, J., Broly, H., Hemberger, J., Jordan, M.: Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation.Journal of Biotechnology.252,32--42 (2017).
Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.
Sauer, M., Heilemann, M.: Single-Molecule Localization Microscopy in Eukaryotes.Chem. Rev. (2017).
Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.
Burgert, A., Schlegel, J., Bécam, J., Doose, S., Bieberich, E., Schubert-Unkmeir, A., Sauer, M.: Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy.Angewandte Chemie. (2017).
The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.
Memmel, S., Sisario, D., Zöller, C., Fiedler, V., Katzer, A., Heiden, R., Becker, N., Eing, L., Ferreira, F.L.R., Zimmermann, H., Sauer, M., Flentje, M., Sukhorukov, V.L., Djuzenova, C.S.: Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities.Oncotarget.8,45298–45310 (2017).
High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
Michie, M.S., Götz, R., Franke, C., Bowler, M., Kumari, N., Magidson, V., Levitus, M., Loncarek, J., Sauer, M., Schnermann, M.J.: Cyanine Conformational Restraint in the Far-Red Range.J. Am. Chem. Soc.139,12406--12409 (2017).
Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
Lukeš, T., Glatzová, D., Kvíčalová, Z., Levet, F., Benda, A., Letschert, S., Sauer, M., Brdička, T., Lasser, T., Cebecauer, M.: Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging.Nature Communications.8,1731-- (2017).
Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
Ziegler, S., Weiss, E., Schmitt, A.-L., Schlegel, J., Burgert, A., Terpitz, U., Sauer, M., Moretta, L., Sivori, S., Leonhardt, I., Kurzai, O., Einsele, H., Loeffler, J.: CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells.Scientific Reports.7,6138-- (2017).
Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
Heiby, J.C., Rajab, S., Rat, C., Johnson, C.M., Neuweiler, H.: Conservation of folding and association within a family of spidroin N-terminal domains.Scientific Reports.7,16789 (2017).
Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.
Maric, H.M., Hausrat, T.J., Neubert, F., Dalby, N.O., Doose, S., Sauer, M., Kneussel, M., Strømgaard, K.: Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.Nature Chemical Biology.13,153-- (2017).
γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.
Collenburg, L., Walter, T., Burgert, A., Müller, N., Seibel, J., Japtok, L., Kleuser, B., Sauer, M., Schneider-Schaulies, S.: A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells.The Journal of Immunology.196,3951--3962 (2016).
Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
Mateos-Gil, P., Letschert, S., Doose, S., Sauer, M.: Super-resolution imaging of plasma membrane proteins with click chemistry.Frontiers in Cell and Developmental Biology.4,98-- (2016).
Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.
Markert, S.M., Britz, S., Proppert, S., Lang, M., Witvliet, D., Mulcahy, B., Sauer, M., Zhen, M., Bessereau, J.-L., Stigloher, C.: Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.Neurophotonics.3,041802--041802 (2016).
Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.
Franke, C., Sauer, M., van de Linde, S.: Photometry unlocks 3D information from 2D localization microscopy data.Nat Meth.advance online publication, (2016).
We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.
Dugar, G., Svensson, S.L., Bischler, T., Wäldchen, S., Reinhardt, R., Sauer, M., Sharma, C.M.: The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni.Nature Communications.7,11667-- (2016).
The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.
Yadav, P., Selvaraj, B.T., Bender, F.L.P., Behringer, M., Moradi, M., Sivadasan, R., Dombert, B., Blum, R., Asan, E., Sauer, M., Julien, J.-P., Sendtner, M.: Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.Acta Neuropathologica.132,93--110 (2016).
In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
Konte, T., Terpitz, U., Plemenitas, A.: Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae.Frontiers in Microbiology.7, (2016).
The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.
Djuzenova, C., Fiedler, V., Katzer, A., Michel, K., Deckert, S., Zimmermann, H., Sukhorukov, V., Flentje, M.: Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule.Oncotarget.5, (2016).
Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells
Niehorster, T., Loschberger, A., Gregor, I., Kramer, B., Rahn, H.-J., Patting, M., Koberling, F., Enderlein, J., Sauer, M.: Multi-target spectrally resolved fluorescence lifetime imaging microscopy.Nat Meth.advance online publication, (2016).
We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).
Schulze, A., Beliu, G., Helmerich, D.A., Schubert, J., Pearl, L.H., Prodromou, C., Neuweiler, H.: Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism.Nat Chem Biol.advance online publication, (2016).
The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.
Feldbauer, K., Schlegel, J., Weissbecker, J., Sauer, F., Wood, P.G., Bamberg, E., Terpitz, U.: Optochemokine Tandem for Light-Control of Intracellular Ca2+.PLoS ONE.11,e0165344-- (2016).
<p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca<sup>2+</sup>-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca<sup>2+</sup> by tandem endosomes into the cytosol via CatCh was visualized using the Ca<sup>2+</sup>-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca<sup>2+</sup> in response to light.</p>
Jung, L.A., Gebhardt, A., Koelmel, W., Ade, C.P., Walz, S., Kuper, J., von Eyss, B., Letschert, S., Redel, C., d'Artista, L., Biankin, A., Zender, L., Sauer, M., Wolf, E., Evan, G., Kisker, C., Eilers, M.: OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.Oncogene.36,1911-- (2016).
MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.
Alexander, M., Sebastian, L., Elisabeth, M., Markus, S., Jürgen, S.: Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates, https://www.degruyter.com/view/j/znc.2016.71.issue-9-10/znc-2016-0123/znc-2016-0123.xml, (2016).
The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.
Werner, C., Pauli, M., Doose, S., Weishaupt, A., Haselmann, H., Grünewald, B., Sauer, M., Heckmann, M., Toyka, K.V., Asan, E., Sommer, C., Geis, C.: Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition.Brain.139,365--379 (2016).
See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.
Paul, M.M., Pauli, M., Ehmann, N., Hallermann, S., Sauer, M., Kittel, R.J., Heckmann, M.: Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation.Frontiers in Cellular Neuroscience.9, (2015).
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
Hennig, S., van de Linde, S., Bergmann, S., Huser, T., Sauer, M.: Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.ACS Nano.9,8122--8130 (2015).
We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.
Brühlmann, D., Jordan, M., Hemberger, J., Sauer, M., Stettler, M., Broly, H.: Tailoring recombinant protein quality by rational media design.Biotechnology Progress.31,615--629 (2015).
Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015
Humpert, F., Yahiatène, I., Lummer, M., Sauer, M., Huser, T.: Quantifying molecular colocalization in live cell fluorescence microscopy.Journal of Biophotonics.8,124--132 (2015).
One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)
Hennig, S., van de Linde, S., Lummer, M., Simonis, M., Huser, T., Sauer, M.: Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes.Nano Lett.15,1374--1381 (2015).
Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.
Ehmann, N., Sauer, M., Kittel, R.J.: Super-resolution microscopy of the synaptic active zone.Frontiers in Cellular Neuroscience.9, (2015).
Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.
Shirakashi, R., Yasui, T., Memmel, S., Sukhorukov, V.L.: Electro-microinjection of fish eggs with an immobile capillary electrode.Biomicrofluidics.9,064109 (2015).
Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.
Schneider, J., Klein, T., Mielich-Süss, B., Koch, G., Franke, C., Kuipers, O.P., Kovács, Á.T., Sauer, M., Lopez, D.: Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium.PLoS Genet.11,e1005140-- (2015).
<title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>
Burgert, A., Letschert, S., Doose, S., Sauer, M.: Artifacts in single-molecule localization microscopy.Histochemistry and Cell Biology.144,123-131 (2015).
Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.
Wäldchen, S., Lehmann, J., Klein, T., van de Linde, S., Sauer, M.: Light-induced cell damage in live-cell super-resolution microscopy.Scientific Reports.5,15348-- (2015).
Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
García-Martínez, J., Brunk, M., Avalos, J., Terpitz, U.: The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination.Sci. Rep.5, (2015).
Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.
Grimm, J.B., Klein, T., Kopek, B.G., Shtengel, G., Hess, H.F., Sauer, M., Lavis, L.D.: Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.Angewandte Chemie.n/a--n/a (2015).
The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.
von Eyss, B., Jaenicke, L. A., Kortlever, R. M., Royla, N., Wiese, K. E., Letschert, S., McDuffus, L.-A., Sauer, M., Rosenwald, A., Evan, G. I., Kempa, S., Eilers, M.: A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer.Cancer Cell.28,743--757 (2015).
Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.
Terpitz, U.: Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung.Naturwissenschaftliche Rundschau.68,465-466 (2015).
Thukral, L., Schwarze, S., Daidone, I., Neuweiler, H.: β-Structure within the Denatured State of the Helical Protein Domain BBL.Journal of Molecular Biology. (2015).
Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.
Djuzenova, C.S., Fiedler, V., Memmel, S., Katzer, A., Hartmann, S., Krohne, G., Zimmermann, H., Scholz, C.-J., Polat, B., Flentje, M., Sukhorukov, V.L.: Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status.Experimental Cell Research.330,346 - 357 (2015).
Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.
Andronic, J., Shirakashi, R., Pickel, S.U., Westerling, K.M., Klein, T., Holm, T., Sauer, M., Sukhorukov, V.L.: Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy.PLoS ONE.10,e0119990 (2015).
Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
Laine, R.F., Albecka, A., van de Linde, S., Rees, E.J., Crump, C.M., Kaminski, C.F.: Structural analysis of herpes simplex virus by optical super-resolution imaging.Nat Commun.6, (2015).
Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.
Schücker, K., Holm, T., Franke, C., Sauer, M., Benavente, R.: Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution.Proceedings of the National Academy of Sciences.112,2029-2033 (2015).
Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.
Letschert, S., Göhler, A., Franke, C., Bertleff-Zieschang, N., Memmel, E., Doose, S., Seibel, J., Sauer, M.: Super-Resolution Imaging of Plasma Membrane Glycans.Angewandte Chemie International Edition.n/a--n/a (2014).
Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.
van de Linde, S., Sauer, M.: How to switch a fluorophore: from undesired blinking to controlled photoswitching.Chem. Soc. Rev. 2014.43(4),1076-1087 (2014).
Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching, bio-labeling compatibility, and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore, we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.
Sauer, M., Neuweiler, H.: PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching. In: Engelborghs, Y. und Visser, A.J.W.G. (hrsg.) Methods Mol Biol - Fluorescence Spectroscopy and Microscopy. S. 597-615. Humana Press (2014).
Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.
Holm, T., Klein, T., Löschberger, A., Klamp, T., Wiebusch, G., van de Linde, S., Sauer, M.: A Blueprint for Cost-Efficient Localization Microscopy.ChemPhysChem.15,651--654 (2014).
Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.
Memmel, S., Sukhorukov, V.L., Höring, M., Westerling, K., Fiedler, V., Katzer, A., Krohne, G., Flentje, M., Djuzenova, C.S.: Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status.PLoS ONE.9,e87052 (2014).
Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
Whelan, D.R., Holm, T., Sauer, M., Bell, T.D.M.: Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM).Australian Journal of Chemistry.67,179-183 (2014).
The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.
Löschberger, A., Niehörster, T., Sauer, M.: Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.Biotechnology Journal.n/a--n/a (2014).
Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.
Andreska, T., Aufmkolk, S., Sauer, M., Blum, R.: High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons.Frontiers in Cellular Neuroscience.8, (2014).
In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.
Mehlitz, A., Karunakaran, K., Herweg, J.-A., Krohne, G., van de Linde, S., Rieck, E., Sauer, M., Rudel, T.: The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress.Cellular Microbiology.n/a--n/a (2014).
Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.
Proppert, S., Wolter, S., Holm, T., Klein, T., van de Linde, S., Sauer, M.: Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging.Opt. Express.22,10304--10316 (2014).
In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
Ehmann, N., van de Linde, S., Alon, A., Ljaschenko, D., Keung, X.Z., Holm, T., Rings, A., DiAntonio, A., Hallermann, S., Ashery, U., Heckmann, M., Sauer, M., Kittel, R.J.: Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states.Nat Commun.5, (2014).
The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
Ries, J., Schwarze, S., Johnson, C.M., Neuweiler, H.: Microsecond folding and domain motions of a spider silk protein structural switch.Journal of the American Chemical Society.null (2014).
Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ~100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.
Löschberger, A., Franke, C., Krohne, G., van de Linde, S., Sauer, M.: Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.Journal of Cell Science.127,4351-4355 (2014).
Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
Sauer, M.: Timing Protein Assembly in Neurons.Chemistry & Biology.21,703--704 (2014).
Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.
Bielopolski, N., Lam, A.D., Bar-On, D., Sauer, M., Stuenkel, E.L., Ashery, U.: Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity.Journal of Biological Chemistry.289,17087-17099 (2014).
Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.
Klein, T., Proppert, S., Sauer, M.: Eight years of single-molecule localization microscopy.Histochemistry and Cell Biology.141,561--575 (2014).
Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.
Hartmann, S., Günther, N., Biehl, M., Katzer, A., Kuger, S., Worschech, E., Sukhorukov, V.L., Krohne, G., Zimmermann, H., Flentje, M., Djuzenova, C.S.: Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines.Cancer Letters.331,200 - 210 (2013).
This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.
Andronic, J., Bobak, N., Bittner, S., Ehling, P., Kleinschnitz, C., Herrmann, A.M., Zimmermann, H., Sauer, M., Wiendl, H., Budde, T., Meuth, S.G., Sukhorukov, V.L.: Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes.Biochimica et Biophysica Acta (BBA) - Biomembranes.1828,699 - 707 (2013).
Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.
Seibel, J., König, S., Göhler, A., Doose, S., Memmel, E., Bertleff, N., Sauer, M.: Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics.Expert Rev Proteomics.10,25--31 (2013).
Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.
Schäfer, P., van de Linde, S., Lehmann, J., Sauer, M., Doose, S.: Methylene blue- and thiol-based oxygen depletion for super-resolution imaging.Analytical Chemistry.85,3393 - 3400 (2013).
Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.
Steger, K., Bollmann, S., Noe, F., Doose, S.: Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale.Phys. Chem. Chem. Phys.- (2013).
Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions, e.g. in a solvent of high viscosity, a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion, fluorescence quenching interactions, and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.
Klamp, T., Camps, M., Nieto, B., Guasch, F., Ranasinghe, R.T., Wiedemann, J., Petrášek, Z., Schwille, P., Klenerman, D., Sauer, M.: Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay.Sci. Rep.3, (2013).
There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.
van de Linde, S., Aufmkolk, S., Franke, C., Holm, T., Klein, T., Löschberger, A., Proppert, S., Wolter, S., Sauer, M.: Investigating Cellular Structures at the Nanoscale with Organic Fluorophores.Chemistry & Biology.20,8 - 18 (2013).
Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.
Kolesinska, B., Podwysocka, D.J., Rueping, M.A., Seebach, D., Kamena, F., Walde, P., Sauer, M., Windschiegl, B., Meyer-Ács, M., Vor der Brüggen, M., Giehring, S.: Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives.Chemistry & Biodiversity.10,1--38 (2013).
After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.
Terpitz, U., Sukhorukov, V.L., Zimmermann, D.: Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes.ASSAY and Drug Development Technologies.11,9-16 (2013).
Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.
Kuger, S., Graus, D., Brendtke, R., Gunther, N., Katzer, A., Lutyj, P., Polat, B., Chatterjee, M., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule.Transl Oncol.6,169-179 (2013).
Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.
Zimmermann, U., Bitter, R., Marchiori, P.E.R., Rüger, S., Ehrenberger, W., Sukhorukov, V.L., Schüttler, A., Ribeiro, R.V.: A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology.Theoretical and Experimental Plant Physiology.25,2 - 11 (2013).
The non-invasive, magnetic leaf patch clamp pressure probe (also termed ZIM-probe) allows for the first time to measure continuously turgor pressure changes of plant leaves over long periods of time with high precision and in real time. The probe has become an important tool in plant physiology, molecular biology and ecology, but also in agriculture because the probe is very robust and user-friendly. Growers receive the information about the water status of their plants by wireless telemetry, mobile network and internet on an as-needed basis and can thus adjust very precisely both the timing of irrigation and the quantity of water to apply. Effects of air and leaf temperature, relative humidity, illumination and wind on turgor pressure can be monitored very sensitively both under indoor and outdoor conditions. Even the effects of blue and red light as well as of oscillations of stomata aperture on turgor pressure can be monitored by the probe with high sensitivity. Similarly, water deficit due to increase of the osmotic pressure in the nutrition solutions resulted in significant changes of the probe signals. Multiple probe readings open up new possibilities to resolve (together with other techniques) the mechanisms of short- and long-distance water transport, particularly how plants can cope with water shortage. The applications of the magnetic probe are numerous and one can expect highly interesting developments in plant water relations in the nearest future.
Sauer, M.: Localization microscopy coming of age: from concepts to biological impact.Journal of Cell Science.126,3505-3513 (2013).
Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.
Schwarze, S., Zwettler, F.U., Johnson, C.M., Neuweiler, H.: The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening.Nat Commun.4, (2013).
Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.
Wolf, A., Akrap, N., Marg, B., Galliardt, H., Heiligentag, M., Humpert, F., Sauer, M., Kaltschmidt, B., Kaltschmidt, C., Seidel, T.: Elements of Transcriptional Machinery Are Compatible among Plants and Mammals.PLOS ONE.8,e53737-- (2013).
In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
Lummer, M., Humpert, F., Wiedenlübbert, M., Sauer, M., Schüttpelz, M., Staiger, D.: A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants.Molecular Plant.6,1518--1530 (2013).
Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.
SHIRAKASHI, R., SUKHORUKOV, V.L., REUSS, R., SCHULZ, A., ZIMMERMANN, U.: Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV)-Jurkat Cell.Journal of Thermal Science and Technology.7,589-602 (2012).
Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.
van de Linde, S., Heilemann, M., Sauer, M.: Live-Cell Super-Resolution Imaging with Synthetic Fluorophores.Annual Review of Physical Chemistry.63,null (2012).
Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Rodriguez-Dominguez, C.M., Ehrenberger, W., Sann, C., Rüger, S., Sukhorukov, V., Martín-Palomo, M.J., Diaz-Espejo, A., Cuevas, M.V., Torres-Ruiz, J.M., Perez-Martin, A., Zimmermann, U., Fernández, J.E.: Concomitant measurements of stem sap flow and leaf turgor pressure in olive trees using the leaf patch clamp pressure probe.Agricultural Water Management.114,50 - 58 (2012).
Stem sap flow (Q) and leaf turgor pressure (Pc) were measured simultaneously on 4-year-old, 2.4 m tall ‘Arbequina’ olive trees in a hedgerow orchard. Measurements were performed on well-watered control trees as well as on 60RDI and 30RDI trees (RDI = regulated deficit irrigation). The 60RDI trees received 59.2% of the crop water needs (ETc), and the 30RDI trees received 29.4% of ETc. Pc was determined non-invasively using the magnetic leaf patch clamp pressure probe (ZIM probe). The patch pressure Pp measured by the probe is inversely correlated with turgor pressure at Pc > ca. 50 kPa. Pc is coupled with xylem pressure; thus Pp yields information about the development of tension in xylem. In the case of the control trees a positive correlation between Q and Pp was generally found, i.e. Q increased usually with increasing Pp and decreased with decreasing Pp, as expected. However, Q peaking did not always coincided with Pp peaking at noon. Occasionally, Q peaking preceded or followed Pp peaking with a time difference of up to 3 h in both cases. Under some circumstances, the onset of Q after sunrise was greatly delayed, even though a pronounced increase of Pp was observed. A delayed onset of Q after sunrise resulted in hysteresis phenomena, i.e. the linear increase of Q and Pp in the morning hours did not coincide with the corresponding decrease of Q and Pp in the afternoon. The development of severe water stress (Pc < ca. 50 kPa) associated with the increase in the intercellular spaces of the spongy tissue in the leaves resulted in inverted diurnal Pp curves, i.e. minimum Pp values were recorded at noon and maximum Pp values during the night on the 30RDI trees. The effects were reversible as shown by re-watering. By contrast, the magnitude of Q decreased continuously from the turgescent state to the state of severe water stress; maximum Q values were still recorded around noon. The data suggests that short-range tension forces are responsible for water lifting in olive trees and that water uptake from water storage reservoirs must play an important role in the supply of the leaves with water. Furthermore, for setting of irrigation thresholds the finding of shape changes of the Pp curves upon severe water stress seems to be a useful indicator. Such shape changes are detected and monitored more sensitively than changes in the magnitude of sap flow rates or of turgor pressure.
Wolter, S., Loschberger, A., Holm, T., Aufmkolk, S., Dabauvalle, M.-C., van de Linde, S., Sauer, M.: rapidSTORM: accurate, fast open-source software for localization microscopy.Nat Meth.9,1040--1041 (2012).
To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.
Lando, D., Endesfelder, U., Berger, H., Subramanian, L., Dunne, P.D., McColl, J., Klenerman, D., Carr, A.M., Sauer, M., Allshire, R.C., Heilemann, M., Laue, E.D.: Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast.Open Biology.2, (2012).
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
Göhler, A., Büchner, C., André, S., Doose, S., Kaltner, H., Gabius, H.-J.: Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding.Biochimie.94,2649 - 2655 (2012).
Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.
Yahiatène, I., Doose, S., Huser, T., Sauer, M.: Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments.Analytical Chemistry.84,2729-2736 (2012).
We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.
Klein, T., van de Linde, S., Sauer, M.: Live-Cell Super-Resolution Imaging Goes Multicolor.ChemBioChem.13,1861--1863 (2012).
New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.
Niewidok, N., Wack, L.-J., Schiessl, S., Stingl, L., Katzer, A., Polat, B., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Hsp90 inhibitors, NVP-AUY922 and NVP-BEP800 may exert a significant radiosensitization on tumor cells along with a cell type-specific cytotoxicity.Transl. Oncology.5,356-369 (2012).
Targeting Hsp90 provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration is analyzed. Cellular response was evaluated at different time intervals (0.5, 24 and 48 h) after IR-alone, drug-alone, or combined drug-IR treatments by colony counts, expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR/drug-induced cell cycle arrest, DNA damage and repair. A short 30-min exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone, and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the anti-apoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells. The data show that, simultaneous Hsp90 inhibition and irradiation may induce cell-type specific radiosensitization as well as cytotoxicity against tumor cells.
Terpitz, U., Letschert, S., Bonda, U., Spahn, C., Guan, C., Sauer, M., Zimmermann, U., Bamberg, E., Zimmermann, D., Sukhorukov, V.: Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies.Journal of Membrane Biology.1-12 (2012).
The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
Dertinger, T., Colyer, R., Vogel, R., Heilemann, M., Sauer, M., Enderlein, J., Weiss, S.: Superresolution Optical Fluctuation Imaging (SOFI).Advances in Experimental Medicine and Biology.733,17-21 (2012).
Superresolution microscopy has shifted the limits for fluorescence microscopy in cell biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.
CS, D., C, B., K, R., S, K., A, K., N, N., N, G., B, P., VL, S., M, F.: Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia.Cancer Biol Ther.13,425-434 (2012).
NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone gammaH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone gammaH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.
Ehrenberger, W., Rüger, S., Rodríguez-Domínguez, C.M., Díaz-Espejo, A., Fernández, J.E., Moreno, J., Zimmermann, D., Sukhorukov, V.L., Zimmermann, U.: Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state.Plant Biology.14,666--674 (2012).
The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, Pp, in response to an externally applied magnetic force. Pp is inversely coupled with leaf turgor pressure, Pc, i.e. at high Pc values the Pp values are small and at low Pc values the Pp values are high. This relationship between Pc and Pp could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (Pc dropped below ca. 50 kPa), Pp curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum Pp value, and during darkness a peak Pp value is recorded. This reversal of the Pp curves was completely reversible. Upon watering, the original diurnal Pp changes were re-established within 2–3 days. Olive trees in the field showed a similar turnover of the shape of the Pp curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the Pp curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal Pp curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental Pp curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero Pc.
WOLTER, S., SAUER, M.: Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images.Journal of Microscopy.245,109--109 (2012).
Bar-On, D., Wolter, S., van de Linde, S., Heilemann, M., Nudelman, G., Nachliel, E., Gutman, M., Sauer, M., Ashery, U.: Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters.Journal of Biological Chemistry. (2012).
Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.
Owen, D.M., Sauer, M., Gaus, K.: Fluorescence localization microscopy: The transition from concept to biological research tool.cib.5,0---1 (2012).
Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.
Löschberger, A., van de Linde, S., Dabauvalle, M.-C., Rieger, B., Heilemann, M., Krohne, G., Sauer, M.: Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.Journal of Cell Science.125,570-575 (2012).
One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.
Lum, J.K., Neuweiler, H., Fersht, A.R.: Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53.Journal of the American Chemical Society.134,1617-1622 (2012).
The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.
Shirakashi, R., Mischke, M., Fischer, P., Memmel, S., Krohne, G., Fuhr, G.R., Zimmermann, H., Sukhorukov, V.L.: Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation.Biochemical and biophysical research communications. (2012).
The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during prehatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.
Teufel, D.P., Johnson, C.M., Lum, J.K., Neuweiler, H.: Backbone-Driven Collapse in Unfolded Protein Chains.Journal of Molecular Biology.409,250 - 262 (2011).
Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.
Kaminski Schierle, G.S., van de Linde, S., Erdelyi, M., Esbjörner, E.K., Klein, T., Rees, E., Bertoncini, C.W., Dobson, C.M., Sauer, M., Kaminski, C.F.: In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging.Journal of the American Chemical Society.133,12902-12905 (2011).
Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.
Gohler, A., Buchner, C., Andre, S., Doose, S., Kaltner, H., Gabius, H.-J.: Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy.Analyst.136,5270-5276 (2011).
Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan's presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins.
Lummer, M., Humpert, F., Steuwe, C., Caesar, K., Schüttpelz, M., Sauer, M., Staiger, D.: Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants.Traffic.12,693--702 (2011).
Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.
Niewidok, N., Wack, L.-J., Stingl, L., Katzer, A., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Simultaneous radiation and Hsp90 inhibition by NVP-AUY922 or NVP-BEP800 is more efficient in tumor cell killing than drug-first modality followed by irradiation.STRAHLENTHERAPIE UND ONKOLOGIE.187,125-126 (2011).
Bobak, N., Bittner, S., Andronic, J., Hartmann, S., Mühlpfordt, F., Schneider-Hohendorf, T., Wolf, K., Schmelter, C., Göbel, K., Meuth, P., Zimmermann, H., Döring, F., Wischmeyer, E., Budde, T., Wiendl, H., Meuth, S.G., Sukhorukov, V.L.: Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels.Biochimica et Biophysica Acta (BBA) - Biomembranes.1808,2036 - 2044 (2011).
A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel KV1.3 and the calcium-activated potassium channel KCa3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of KV1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K2P channels.
SHIRAKASHI, R., REUSS, R., SCHULZ, A., SUKHORUKOV, V.L., ZIMMERMANN, U.: Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV) -Jurkat Cell (Measurement of Fusion Ratio and Electric Field Analysis of Pulsed GUV-Jurkat Cell).TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B.77,1269-1278 (2011).
Electrofusion of Giant Unilamellar Vesicle (GUV) and living cell is a gentle method to deliver membrane impermeable cryo-/lyo-protective molecules into living cell for cryo-/lyopreservation. In this method, cellular fusion is believed to be triggered by the irreversible electical breakdown of the membrane at contact region. Nevertheless, as the time change of the membrane potential distribution during an electric pulse is still not clear, the length and strength of electric pulse are hardly designed. In this study, the GUV-Jurkat electrofusion ratios under various pulse strength and length were measured. In addition, we calculated the time change of membrane potential distribution of a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The measured cellular and GUV's electric properties and a GUV-Jurkat profile were used for the analysis to validate the quantitative calculation results. Both results of experiment and calculation suggested that; 1) GUV-Jurkat should be stretched for the electrofusion, 2) whole contact region of membrane should breakdown at the same time to process electrofusion, 3) after applying an electric field for around 1μsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10μsec, membrane potential is relaxed, 5) the irreversible electric breakdown is around 3V.
Seefeldt, B., Altenhoner, K., Tosic, O., Geisler, T., Sauer, M., Mattay, J.: Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes.Photochem. Photobiol. Sci.10,1488--1495 (2011).
We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.
Noe, F., Doose, S., Daidone, I., Löllmann, M., Sauer, M., Chodera, J.D., Smith, J.C.: Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments.Proceedings of the National Academy of Sciences. (2011).
There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprints� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.
Jensen, M.H., Sukumaran, M., Johnson, C.M., Greger, I.H., Neuweiler, H.: Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy.Journal of Molecular Biology.414,96 - 105 (2011).
Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.
Wolter, S., Endesfelder, U., van de Linde, S., Heilemann, M., Sauer, M.: Measuring localization performance of super-resolution algorithms on very active samples.Optics Express.19,7020--33 (2011).
Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.
Bollmann, S., Lollmann, M., Sauer, M., Doose, S.: Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions.Phys. Chem. Chem. Phys.13,12874-12882 (2011).
Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.
van de Linde, S., Wolter, S., Sauer, M.: Single-molecule Photoswitching and Localization.Aust. J. Chem.64,503--511 (2011).
Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.
Vogel, B., Löschberger, A., Sauer, M., Hock, R.: Cross-linking of DNA through HMGA1 suggests a DNA scaffold.Nucleic Acids Research. (2011).
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
Klein, T., Loeschberger, A., Proppert, S., Wolter, S., van de Linde, S., Sauer, M.: Live-cell dSTORM with SNAP-tag fusion proteins.NATURE METHODS.8,7-9 (2011).
In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.
van de Linde, S., Loschberger, A., Klein, T., Heidbreder, M., Wolter, S., Heilemann, M., Sauer, M.: Direct stochastic optical reconstruction microscopy with standard fluorescent probes.Nat. Protocols.6,991--1009 (2011).
Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.
van de Linde, S., Krstic, I., Prisner, T., Doose, S., Heilemann, M., Sauer, M.: Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging.Photochem. Photobiol. Sci.10,499-506 (2011).
Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.
Kleinlogel, S., Terpitz, U., Legrum, B., Gokbuget, D., Boyden, E.S., Bamann, C., Wood, P.G., Bamberg, E.: A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.Nat Meth.8,1083--1088 (2011).
The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.
Bollmann, S., Burgert, A., Plattner, C., Nagel, L., Sewald, N., Löllmann, M., Sauer, M., Doose, S.: Conformational Flexibility of Glycosylated Peptides.ChemPhysChem.12,2907--2911 (2011).
With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides
Nottbohm, C.T., Sopher, R., Heilemann, M., Sauer, M., Goelzhaeuser, A.: Fluorescently labeled 1 nm thin nanomembranes.JOURNAL OF BIOTECHNOLOGY.149,267-271 (2010).
Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.
Kasper, R., Harke, B., Forthmann, C., Tinnefeld, P., Hell, S.W., Sauer, M.: Single-Molecule STED Microscopy with Photostable Organic Fluorophores.Small.6,1379--1384 (2010).
The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer
Kottke, T., van de Linde, S., Sauer, M., Kakorin, S., Heilemann, M.: Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy.The Journal of Physical Chemistry Letters.1,3156-3159 (2010).
The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.
Goelzhaeuser, A., Nattkemper, T., Niehaus, K., Puehler, A., Sauer, M.: BioImaging: Contributions from biology, physics and informatics.JOURNAL OF BIOTECHNOLOGY.149,227-228 (2010).
Endesfelder, U., van de Linde, S., Wolter, S., Sauer, M., Heilemann, M.: Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility.CHEMPHYSCHEM.11,836-840 (2010).
Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.
Seefeldt, B., Kasper, R., Beining, M., Mattay, J., Arden-Jacob, J., Kemnitzer, N., Drexhage, K.H., Heilemann, M., Sauer, M.: Spiropyrans as molecular optical switches.PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES.9,213-220 (2010).
Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.
Neuweiler, H., Banachewicz, W., Fersht, A.R.: Kinetics of chain motions within a protein-folding intermediate.PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA.107,22106-22110 (2010).
Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.
Seidel, T., Seefeldt, B., Sauer, M., Dietz, K.-J.: In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET.JOURNAL OF BIOTECHNOLOGY.149,272-279 (2010).
Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.
Wombacher, R., Heidbreder, M., van de Linde, S., Sheetz, M.P., Heilemann, M., Cornish, V.W., Sauer, M.: Live-cell super-resolution imaging with trimethoprim conjugates.NATURE METHODS.7,717-U72 (2010).
The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.
van de Linde, S., Wolter, S., Heilemann, M., Sauer, M.: The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging.JOURNAL OF BIOTECHNOLOGY.149,260-266 (2010).
Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.
Goehler, A., Andre, S., Kaltner, H., Sauer, M., Gabius, H.-J., Doose, S.: Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy.BIOPHYSICAL JOURNAL.98,3044-3053 (2010).
Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.
Heidbreder, M., Endesfelder, U., van de Linde, S., Hennig, S., Widera, D., Kaltschmidt, B., Kaltschmidt, C., Heilemann, M.: Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots.BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH.1803,1224-1229 (2010).
We introduce semiconductor quantum dot-based fluorescence imaging with similar to 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells. (C) 2010 Elsevier B.V. All rights reserved.
Daidone, I., Neuweiler, H., Doose, S., Sauer, M., Smith, J.C.: Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains.PLOS COMPUTATIONAL BIOLOGY.6, (2010).
Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.
Dertinger, T., Heilemann, M., Vogel, R., Sauer, M., Weiss, S.: Superresolution Optical Fluctuation Imaging with Organic Dyes.ANGEWANDTE CHEMIE-INTERNATIONAL EDITION.49,9441-9443 (2010).
SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.
Zheng, Z., Nottbohm, C.T., Turchanin, A., Muzik, H., Beyer, A., Heilemann, M., Sauer, M., Goelzhaeuser, A.: Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions.ANGEWANDTE CHEMIE-INTERNATIONAL EDITION.49,8493-8497 (2010).
Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.
Uphoff, S., Holden, S.J., Reste, L.L., Periz, J., van de Linde, S., Heilemann, M., Kapanidis, A.N.: Monitoring multiple distances within a single molecule using switchable FRET.NATURE METHODS.7,831-U90 (2010).
The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called `switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.
