Plectin Isoforms and Cytoskeleton Dynamics

As a prototype cytolinker protein, plectin has all the molecular features of a multifunctional cross-linking and organizing element of the cytoskeleton. It displays a multi-domain structure, versatile binding activities, and strategic subcellular localizations for strengthening cells against mechani-cal stress; it forms cross-links between different cytoskeletal networks and it is present at cell-matrix and cell-cell contacts. Furthermore, evidence is emerging for an important new role of plectin as a scaffolding platform for the docking and assembly of protein complexes involved in signaling. Plectin is expressed in form of several isoforms generated by differential splicing, and there is increasing evidence that some of these isoforms have distinct specific functions. Based on results from the previous project, we now propose to study the role of four of these isoforms in cytoskeleton dynamics. These isoforms are particularly attractive because their subcellular localization or molecular structure strongly suggest distinct functions. Isoform 1c, colocalizing with microtubules, represents most probably the long-sought specific linker between the inter-mediate filament and the microtubule networks, isoform 1f predominates at adherens junctions, and isoform 1b at mitochondria; the rod-less isoform is expected to behave atypically. Our focus will be on i) plectin (isoform) 1c and its role in microtubule dynamics and organelle transport, ii) plectin 1f as a mediator of actin and intermediate filament interactions with adherens junctions, iii) functions of rod-less plectin isoforms, and iv) identification of mitochondrial binding proteins and functional analysis of plectin 1b. Our experimental approach will rely to a large extent on primary or immortalized cell cultures derived from plectin null or isoform- deficient mice and their normal (wild type) counterparts, including keratinocytes, fibroblasts, and myocytes, as well as primary melanocytes and hippocampal neurons. Most of the methodology to be used has been well established in our lab, such as the generation of cell lines and primary cultures, expression of wild type and mutant versions of proteins in cultured cells for microscopic analyses, and in bacteria or the baculovirus system for biochemical analyses. In addition, video microscopy will be used for live imaging of cytoskeletal filament dynamics and movement of organelles, such as melanosomes and mitochondria. The proposed research project is expected to shed light on the roles of specific plectin isoforms in regulating distinct aspects of cytoskeleton dynamics and may help to explain related defects or abnormalities arising in pathological situations.

As a prototype cytolinker protein, plectin has all the molecular features of a multifunctional cross-linking and organizing element of the cytoskeleton. It displays a multi-domain structure, versatile binding activities, and strategic subcellular localizations for strengthening cells against mechanical stress. Furthermore, an important new role of plectin as a scaffolding platform for the docking and assembly of protein complexes involved in signaling has emerged. Plectin is expressed in form of several isoforms generated by differential splicing. The major objective of this project was to study specific roles of distinct plectin isoforms in organization and dynamics of the cytoskeleton, and their impact on cytoarchitecture and signaling. Our studies relied strongly on a series of conditional and isoform-deficient knock out mouse lines that we had previously generated and on primary cell cultured derived from them. We could clearly establish that 4 isoforms of plectin (1f, 1d, 1b, and 1) are the major organizers of the myofiber cytoskeleton with important implications not only for plectin-related but also other common types of muscular dystrophy. Furthermore, using live imaging techniques, we were able to directly visualize plectin(1f)-mediated targeting and anchorage of the IF cytoskeleton at adhesion complexes of fibroblasts with consequences for cell migration as well as stress-induced signaling. Furthermore, plectin 1a was found to regulate the anchorage of keratin filaments at hemidesmosomes with implications for skin blistering. Another milestone was reached with plectin 1b`s identification as the first cytoskeleton-mitochondrion linker protein. Other studies revealed interesting links between plectin-controlled astrocyte cytoarchitecture and Alexander disease (a brain disorder leading to the death of juveniles), and between plectin IF networking and nitrosylation/oxidation-inflicted cytoskeleton damage in vascular system (endothelial) cells. Further, we could show that plectin isoforms directly affect IF network assembly/disassembly kinetics with consequences for spreading and even division of cells, and that plectin contributes to mechanical properties of living cells. Lastly, we obtained very interesting results implying plectin, specifically isoform 1c, in microtubule dynamics and organelle transport, adding yet another skill to the portfolio of this multitalented protein. Studies performed under this project led to the publication of 21 original papers and one book chapter. This work provides a solid basis for new (follow-up) research projects that will continue and extend these studies to gain deeper insights into the molecular mechanisms underlying plectin`s multiple functions and ultimately to better understand the pathogenesis of the various diseases caused by plectin insufficiencies.