Emery-Dreifuss-muscle dystrophy: the roles of lamin A/C and emerin

Project leaders: Marie-Christine Dabauvalle in collaboration with Clemens Müller-Reible (Institute of Human Genetics, Biocenter)

Staff: Eleonora Krüger (since 02/97), Beate Reichart (09/00-08/02), Isabell Motsch (since 04/00), Ruth Klafke (09/02-05/03), Martin Gareiß (since 10/02), Sebastian Kandert (since 09/04).

Objectives: The project aims at an understanding of the function of the nuclear proteins lamin A/C and emerin in the pathogenesis of Emery-Dreifuss-muscle dystrophy (EDMD). Although emerin and lamin A/C are expressed ubiquitously, pathological effects are tissue-specific and essentially restricted to sceletal and heart muscle.

Approach: Using cell lines derived from different species we have analyzed the distribution and function of emerin during the early steps of nuclear envelope reassembly (1). Using biopsies from patients suffering from EDMD and over 30 primary cell lines etablished from these patients we are studying the effects of emerin mutations (X-EDMD patient; X-linked recessive disorder) as well as lamin mutations (AD-EDMD patient; autosomal dominant disorder) on nuclear structure and function. We are further analyzing the properties of mutant lamin or emerin proteins, including assembly and interactions with known lamin binding proteins in vitro and in vivo, and search for new interaction partners. In a second approach we are studying the expression of emerin during embryogenesis of Xenopus laevis.

Progress: Our data obtained after microinjection of antibodies to emerin and immunocytochemistry suggest that emerin plays a specific role during the early steps of nuclear reassembly at the end of mitosis. Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections from EDMD patients were studied by immunocytochemistry and electron microscopy. As compared to control cells, the cellular levels of A-type lamins were reduced in an AD-EDMD patient carrying the point mutation R377H in the lamin A/C gene. In contrast, the amount of emerin and lamin B appeared unaltered. By electron microscopy, we identified nuclei with striking morphological alterations. The peripheral chromatin appeared more condensed and was no longer associated with the inner nuclear membrane. Immunofluorescence microscopy revealed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was redistributed into the cytoplasm where it occurred in association with the ER (Fig. 1). By immunofluorescence analysis we observed in muscle cells derived from EDMD patients, but not in control cells, a marked intranuclear redistribution of the phosphorylated form of RNA polymerase II. From our results we conclude that wild-type lamin A is required not only for the retention of LBR at the inner nuclear membrane but also for a correct spatial organization of transcriptionally active loci in muscle cells (3).

We have sequenced Xenopus emerin and raised specific monoclonal antibodies. We were able to demonstrate by immunoblotting experiments that emerin is absent from oocytes and from early developmental stages and becomes first detectable at stage 43. Interestingly, the expression of lamin A follows a similar temporal pattern. Results obtained by whole mount in situ hybridization show that emerin-mRNA is first detectable in the heart and head regions of stage 28 embryos (5).

Significance: Regeneration of an injured muscle requires that satellite cells reenter the cell cycle and start to proliferate. Our data indicate a role of emerin in the reorganization of the nuclear envelope at the end of mitosis. Hence it is reasonable to conclude that the proliferation competence of emerin-deficient satellite cells is reduced or impaired following muscle degeneration. In addition, the observed spatial rearrangement of chromatin in cells with a point mutation in the lamin A/C gene may affect transcription of muscle-specific genes which could explain the tissue-specific symptoms.

Future projects: For a better understanding of the functions of emerin and lamin A for the organization of the cell nucleus we will study the dynamics and interactions of these proteins by expressing GFP-tagged fusion proteins. We want to investigate their role in nuclear envelope assembly, nuclear structure and chromatin organization by employing Xenopus laevis as a model system, which offers both in vivo (oocytes, embryos) and in vitro approaches (cell free extract prepared from eggs, in vitro assembly of nuclei). Combined with in vitro binding assays (overlay and pull down experiments) this model system should lead to a better understanding of the role of emerin and lamin A for nuclear organization and should help to identify novel binding partners.

The important question as to whether missense mutations in lamins lead to altered gene expression will be studied by using DNA microarray analyses, comparative 2-D protein gel electrophoresis and proteomic analyses with wild type and patient cells. We will use primary cells lines from AD-EDMD patients (lymphoblasts, myoblasts and fibroblasts) our, and cell lines which can be first mutated (e.g. by RNA silencing) and then induced to differentiate in vitro such as myoblasts cell lines C2C12 (murine muscle myoblasts).

Fig. 1: Mislocalization of lamin B receptor (LBR) in cells with mutation R377H of the lamin A/C gene

Fibroblasts (a,b,e) and myoblasts (c,d,f) from a healthy donor (a,c) or EDMD patient (b,d,e,f) were double-labelled with a mAb directed against lamin B (a-d), lamin A/C (e,f) and with guinea pig antibodies recognizing LBR (a´-f´) and analyzed by confocal microscopy. Note the colocalization of lamin B and LBR at the nuclear envelope in control cells (a´´,c´´). In contrast, in patient cells LBR is partly displaced to the cytoplasm (b´´,d´´,e’’, f’’).

Collaborations: Prof Dr. Georg Krohne, Division of Electron Microscopy, Biocenter, Würzburg; Dr. Albert Sickman, Protein Mass Spectrometry and Functional Proteomics Group, Rudolf-Virchow-Center for Experimental Biomedicine, Würzburg; Prof. Dr. Hanswalter Zentgraf, Applied Tumorvirology, Electron Microscopy Group and Dr. Christine Dreger, Department of Cell Biology, German Cancer Research, Heidelberg; Prof. Dr. Heinz Reichmann, Neurological University Clinic, Dresden; Prof. Dr. Juliet Ellis, Randall Centre for the Molecular Mechanism of Cell Function, King’s College London, UK.

Publications:

Current external funding: DFG (SFB 581, TP B7) until 12/2003; BMBF (MD-NET) since 05/03.