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Reinhard, S., Aufmkolk, S., Sauer, M., Doose, S.: Registration and Visualization of Correlative Super-Resolution Microscopy Data. Biophysical Journal. 116, 2073--2078 (2019).
We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
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Beliu, G., Kurz, A.J., Kuhlemann, A.C., Behringer-Pliess, L., Meub, M., Wolf, N., Seibel, J., Shi, Z.-D., Schnermann, M., Grimm, J.B., Lavis, L.D., Doose, S., Sauer, M.: Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Communications Biology. 2, 261-- (2019).
Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
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Gambarotto, D., Zwettler, F.U., Le Guennec, M., Schmidt-Cernohorska, M., Fortun, D., Borgers, S., Heine, J., Schloetel, J.-G., Reuss, M., Unser, M., Boyden, E.S., Sauer, M., Hamel, V., Guichard, P.: Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nature Methods. 16, 71--74 (2019).
Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
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Nerreter, T., Letschert, S., Götz, R., Doose, S., Danhof, S., Einsele, H., Sauer, M., Hudecek, M.: Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T. Nature Communications. 10, 3137-- (2019).
Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
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Heiby, J.C., Goretzki, B., Johnson, C.M., Hellmich, U.A., Neuweiler, H.: Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk. Nature Communications. 10, 4378-- (2019).
Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.
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Banaszek, A., Bumm, T.G.P., Nowotny, B., Geis, M., Jacob, K., Wölfl, M., Trebing, J., Kucka, K., Kouhestani, D., Gogishvili, T., Krenz, B., Lutz, J., Rasche, L., Hönemann, D., Neuweiler, H., Heiby, J.C., Bargou, R.C., Wajant, H., Einsele, H., Riethmüller, G., Stuhler, G.: On-target restoration of a split T cell-engaging antibody for precision immunotherapy. Nature Communications. 10, 5387-- (2019).
T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
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Derakhshani, S., Kurz, A., Japtok, L., Schumacher, F., Pilgram, L., Steinke, M., Kleuser, B., Sauer, M., Schneider-Schaulies, S., Avota, E.: Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium. Frontiers in Immunology. 10, 1294-- (2019).
Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.
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Goretzki, B., Heiby, J.C., Hacker, C., Neuweiler, H., Hellmich, U.A.: NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis. Biomolecular NMR Assignments. (2019).
Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.
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Zilkowski, I., Ziouti, F., Schulze, A., Hauck, S., Schmidt, S., Mainz, L., Sauer, M., Albrecht, K., Jundt, F., Groll, J.: Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells. Biomacromolecules. 20, 916--926 (2019).
Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.
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Brühlmann, D., Vuillemin, T., Satwekar, A., Galano, E., Palmese, A., D'Angelo, A., Manco, Z., Souquet, J., Broly, H., Sauer, M., Hemberger, J., Jordan, M.: Generation of site-distinct N-glycan variants for in vitro bioactivity testing. Biotechnology and Bioengineering. 116, 1017--1028 (2019).
Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.
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Khayenko, V., Maric, H.M.: Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts. Frontiers in Molecular Neuroscience. 12, 162-- (2019).
γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.
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Panzer, S., Brych, A., Batschauer, A., Terpitz, U.: Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps. Frontiers in Microbiology. 10, (2019).
In fungi green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371) and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of White Collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids, especially by acetic acid and indole-3-acetic acid. In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.
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Djuzenova, C.S., Fiedler, V., Memmel, S., Katzer, A., Sisario, D., Brosch, P.K., Göhrung, A., Frister, S., Zimmermann, H., Flentje, M., Sukhorukov, V.L.: Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells. BMC Cancer. 19, 299-- (2019).
Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.
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Schermelleh, L., Ferrand, A., Huser, T., Eggeling, C., Sauer, M., Biehlmaier, O., Drummen, G.P.C.: Super-resolution microscopy demystified. Nature Cell Biology. 21, 72--84 (2019).
Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
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Mercier, R., Wolmarans, A., Schubert, J., Neuweiler, H., Johnson, J.L., LaPointe, P.: The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation. Nature Communications. 10, 1273-- (2019).
Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.
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Kasaragod, V.B., Hausrat, T.J., Schaefer, N., Kuhn, M., Christensen, N.R., Tessmer, I., Maric, H.M., Madsen, K.L., Sotriffer, C., Villmann, C., Kneussel, M., Schindelin, H.: Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins. Neuron. 101, 673--689.e11 (2019).
Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.
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Baluapuri, A., Hofstetter, J., Dudvarski Stankovic, N., Endres, T., Bhandare, P., Vos, S.M., Adhikari, B., Schwarz, J.D., Narain, A., Vogt, M., Wang, S.-Y., Düster, R., Jung, L.A., Vanselow, J.T., Wiegering, A., Geyer, M., Maric, H.M., Gallant, P., Walz, S., Schlosser, A., Cramer, P., Eilers, M., Wolf, E.: MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation. Molecular Cell. 74, 674--687.e11 (2019).
Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
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Sdelci, S., Rendeiro, A.F., Rathert, P., You, W., Lin, J.-M.G., Ringler, A., Hofstätter, G., Moll, H.P., Gürtl, B., Farlik, M., Schick, S., Klepsch, F., Oldach, M., Buphamalai, P., Schischlik, F., Májek, P., Parapatics, K., Schmidl, C., Schuster, M., Penz, T., Buckley, D.L., Hudecz, O., Imre, R., Wang, S.-Y., Maric, H.M., Kralovics, R., Bennett, K.L., Müller, A.C., Mechtler, K., Menche, J., Bradner, J.E., Winter, G.E., Klavins, K., Casanova, E., Bock, C., Zuber, J., Kubicek, S.: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation. Nature Genetics. 51, 990--998 (2019).
The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
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Peters, S., Schlegel, J., Becam, J., Avota, E., Sauer, M., Schubert-Unkmeir, A.: Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels. Infect. Immun. 87, e00410-19-- (2019).
Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Hines, R.M., Maric, H.M., Hines, D.J., Modgil, A., Panzanelli, P., Nakamura, Y., Nathanson, A.J., Cross, A., Deeb, T., Brandon, N.J., Davies, P., Fritschy, J.-M., Schindelin, H., Moss, S.J.: Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin. Nature Communications. 9, 3130-- (2018).
Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.
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Bleem, A., Christiansen, G., Madsen, D.J., Maric, H., Strømgaard, K., Bryers, J.D., Daggett, V., Meyer, R.L., Otzen, D.E.: Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms. Journal of Molecular Biology. 430, 3751--3763 (2018).
Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Büchel, G., Carstensen, A., Mak, K.-Y., Roeschert, I., Leen, E., Sumara, O., Hofstetter, J., Herold, S., Kalb, J., Baluapuri, A., Poon, E., Kwok, C., Chesler, L., Maric, H.M., Rickman, D.S., Wolf, E., Bayliss, R., Walz, S., Eilers, M.: Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle. Cell Reports. 21, 3483--3497 (2017).
Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.
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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.
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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.
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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>
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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
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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.
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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-- (2016).
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.
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Cheng, J., Sahani, S., Hausrat, T. J., Yang, J.-W., Ji, H., Schmarowski, N., Endle, H., Liu, X., Li, Y., Böttche, R., Radyushkin, K., Maric, H. M., Hoerder-Suabedissen, A., Molnár, Z., Prouvot, P.-H., Trimbuch, T., Ninnemann, O., Huai, J., Fan, W., Visentin, B., Sabbadini, R., Strømgaard, K., Stroh, A., Luhmann, H. J., Kneussel, M., Nitsch, R., Vogt, J.: Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling. Neuron. 92, 126--142 (2016).
Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Maric, H.M., Kasaragod, V.B., Haugaard-Kedström, L., Hausrat, T.J., Kneussel, M., Schindelin, H., Strømgaard, K.: Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors. Angewandte Chemie International Edition. 54, 490--494 (2015).
Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.
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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
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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.
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Terpitz, U.: Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung. Naturwissenschaftliche Rundschau. 68, 465-466 (2015).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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>
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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.
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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)
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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.
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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.
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Maric, H.M., Kasaragod, V.B., Hausrat, T.J., Kneussel, M., Tretter, V., Strømgaard, K., Schindelin, H.: Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin. Nature Communications. 5, 5767-- (2014).
γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Holm, T., Klein, T., Löschberger, A., Klamp, T., Wiebusch, G., van de Linde, S., Sauer, M.:
