GERHARD WICHE, Ph.D.

Professor of Biochemistry and Molecular Cell Biology

phone     +43-1-4277 52852

Fax         +43-1-4277 52854

E-mail     gerhard.wiche@univie.ac.at

 

Role of cytolinkers in morphogenesis, signalling and disease

Groups:  G. Wiche

 

We are studying the structure, function and role in disease of the gigantic (>500 kDa) prototype cytolinker protein plectin and of related proteins using genetically altered mice and cell cultures combined with analyses of recombinant proteins on the molecular and atomic levels. Plectin and its isoforms are abundantly expressed in all types of mammalian tissues and cells. Due to the differential splicing of an unusually high number of first coding exons (Fig. 1), there exists a remarkable complexity of plectin gene transcripts, some with highly restricted tissue distribution. The various plectin isoforms interact with all major cytoskeletal filament networks and are major constituents of intracellular and plasma membrane-associated junctional complexes of the cytoskeleton. The concept that plectin isoforms play a crucial role in determining cell shape, control cytoskeleton dynamics during differentiation and cell cycle progression, and reinforce cells against mechanical stress, is supported by findings that link defects in plectin expression to human diseases characterized by skin blistering, muscular dystrophy and neurodegeneration. Plectin knock-out mice provide an important new tool for the study of plectin function in vivo and in vitro.

 

Transgenic and conditional knock-out (KO) mice: Models for human diseases (plectinopathies)

Plectin (null) KO mice survive only 2-3 days due to severe skin blistering. Like plectin-deficient patients these animals also show severe muscle disorders. However, due to their limited life span they have only a limited potential to serve as animal models for plectin-related human diseases (plectinopathies) and they are hardly usable for studying cell/tissue-specific functions of plectin isoforms in the context of the whole organism. Therefore, we have initiated studies to generate mice in which the expression of muscle or brain-specific plectin isoforms are suppressed in a cell lineage/tissue-specific manner, without affecting isoform expression in other tissues. Using the conditional Cre/loxP knock-out system we have already generated a mouse line that is deficient in the expression of a brain-specific exon 1C-encoded plectin isoform (Fig. 2). Another approach used by us to generate plectin deficiency in certain tissues/cells of mice without interference in their development, is the conditional plectin gene knock-out by crossing mouse lines carrying a floxed plectin gene with Cre-expressing transgenic mice.

 

Plectin (-/-) cell lines reveal novel functions of cytolinkers

In vitro cell cultures derived from plectin null and isoform-deficient mice have a great potential to provide novel insights into molecular mechanisms governing cytolinker functions in different cell types. Initial experiments with plectin (-/-) fibroblasts and astrocytes revealed an important new role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its originally proposed role in networking and mechanical stabilization of cells. The actin cytoskeleton, including focal adhesion contacts, is developed more extensively in plectin null compared to wild type cells. Also, it fails to show characteristic short term rearrangements in response to extracellular stimuli activating the rho/rac/cdc42 signalling cascades. As a consequence, cell motility, adherence, and shear stress resistance are altered, and morphogenic processes are delayed. Moreover, in plectin-deficient cells intermediate filaments have lost their connection to focal adhesion contacts. Results obtained with immortalized cultures of plectin-deficient keratinocytes, myoblasts, and endothelial cells point at interesting isoform/cell type-dependent regulation. According to a new concept emerging from these studies we propose that cytolinkers play an essential role as scaffolding platform of complex protein assemblies (transducisomes) required for cytoplasmic signalling.

 

Plectin: a cytolinker protein with many faces and binding partners

As a gigantic dumbbell-shaped homodimer comprising a long central a-helical coiled-coil rod domain flanked by N- and C-terminal globular domains, containing multiple structurally diverse subdomains, plectin provides a molecular backbone ideally suited to form flexible links between different cytoskeletal elements and to serve as docking site for regulatory proteins and protein machineries controlling cytoarchitecture and signalling. So far, close to 30 different protein species have been found to directly interact with plectin, including various subunit proteins of cytoskeletal filaments, transmembrane and plasma membrane or nuclear lamina-associated proteins, kinases and kinase-anchoring proteins. In mapping plectin's molecular interaction sites to distinct sequences we found that its highly conserved amino-terminal actin-binding domain (ABD), which is common to a large family of actin-binding proteins, also serves as docking site for cytoplasmic proteins other than actin, and thus is multifunctional. The versatility of this domain in plectin is further increased through tissue-specific expression of variants containing distinct versions of the ABD generated by differential splicing of transcripts.

To analyze the 3-D structure of proteins at the various interfaces of plectin and its interacting partners we have started to determine in solution and in the solid state the conformation of molecular subdomains involved in binding. Figure 3 shows crystals obtained after expression of the ABD version unique for skeletal muscle plectin in bacteria, and purification of the recombinant protein to homogeneity. Such crystals allowed structure analysis at 2.3 Å by X-ray diffraction. Recently, a similar approach led us to the successful analysis of the crystal structure of the integrin b4 subdomain that interacts with the multifunctional ABD of plectin. 3-D structural analysis of other plectin subdomain and binding protein interfaces are in progress.

 

Electron microscopy (EM) at the Vienna-BioCenter

The group is also in charge of the only EM facility available at the Vienna-BioCenter. This facility, comprising a Jeol 1210 transmission electron microscope and a well equipped EM-laboratory, allows state of the art technology applications, such as immunogold labeling of ultrathin cryosections (Fig. 4), high pressure freezing, freeze substitution & Lowicryl embedding. The unit is extensively used in several projects of the group, in particular for the phenotyping of transgenic and knock-out mice as well as the visualization of single molecules and protein complexes (e.g.rotary shadowing). In addition, the facility serves several other BioCenter-based groups, including some from the IMP.

 

List of Publications