Destabilizing microtubules - a novel role of plectin 1C

Plectin (>500 kDa) is one of the largest and most versatile cytolinker proteins. It binds to all types of intermediate filament (IF) networks and exhibits the features expected for a multifunctional networking and organizing element of the cytoskeleton, including a multi-domain structure, diverse binding activities, and unusual isoform diversity. Apart from IFs, plectin is involved in structural as well as dynamic aspects of the actin filament system, and thus is emerging as a central player in many cellular processes requiring restructuring and reorganization of the cytoskeleton. Plectin`s interaction with the microtubule (MT) network has remained largely unexplored. Based on pilot experiments and observations from previous studies from us and others, we have strong evidence that plectin, specifically its isoform P1c, binds to and destabilizes MTs, affecting their dynamic behavior. In light of the many cellular functions that rely on a dynamically well balanced MT network system, these findings could have important implications for the pleiotropic phenotypes caused by plectin deficiency, which affects skin, brain, skeletal muscle and heart. The major objectives of this proposal are to i) validate P1c as a bona fide MT destabilizer, ii) characterize the molecular mechanism(s) of IF-plectin-MT crosstalk in keratinocytes and neurons, iii) test the hypothesis that plectin (and IFs) participate in the regulation of MTs, and iv) assess the physiological significance of such regulation. To reach these goals our studies will be focused on the role of P1c in MT dynamics and cellular processes known to be MT-dependent, such as cell polarization and vesicular transport involved in glucose uptake and synaptic transmission; another focus will be nerve conduction, a process hypothetically linked to MT dynamics. Most of the studies will be carried out ex vivo using primary cell cultures (keratinocytes and neurons), isolated intact neurons, and immortalized (p53-/-) P1c-deficient cell lines, all derived from an already available P1c knockout mouse line. The project will rely to a large extent on live imaging video microscopy, complemented with in vitro approaches using recombinant proteins. Keratinocytes and neurons will also be used for the identification of MT-associated proteins (MAPs) interacting with P1c. By analyzing the role of IF network- associated plectin in MT-dependent cell functions in an overlapping way in these two cell systems, we expect to gain novel insights into how the IF system influences MT-dependent cell dynamics, and how this crosstalk may contribute to the intricate disease pathogenesis associated with plectin deficiencies.

The ubiquitously expressed cytolinker protein plectin, which was first identified some 30 years ago in our laboratory, turned out to play a decisive role in cytoskeleton organization of and its dynamics. Loss-of-function mutations in the plectin gene cause multi-systemic diseases affecting primarily the skin, striated muscle, the nervous system, and the vasculature. In previous studies we have shown that the proper functioning of one of the three major cytoskeletal filament systems, the intermediate filaments (IFs), in a wide variety of cells is crucially dependent on IF consolidation through plectin-mediated cross-linking and site-specific anchorage. On the other hand, we also showed that the generally more dynamic actomyosin network, which exerts contractile forces on cells, was regulated by plectin in a way favoring its dynamics. The major objective of this project was to analyze whether plectin in partnership with IFs affects also microtubules (MTs), the other highly dynamic cytoskeleton component, and if so what consequences this has for the various cellular functions that are dependent on the dynamic state of MTs. As P1c, one of the several isoforms of plectin that are differentially expressed in various tissues and cells, specifically binds to tubulin (the subunit protein of MTs) and at the same time represents the major isoform expressed in epithelial and brain tissues, we focused on the role of this isoform in epidermal keratinocytes and primary dorsal root ganglia (DRG) sensory neurons, both of which have a high MT content. Additional studies were performed with skeletal muscle cells (myocytes). The most important tools for our studies were isoform P1c-deficient and total plectin-deficient (plectin-null) knock out mice that together with normal littermate mice served as source for primary cell cultures to be studied ex vivo. Comprising experimental approaches on the molecular and cellular levels, our studies demonstrated that in both cell systems studied (keratinocytes and DRG neurons), P1c deficiency leads to increased MT stability coupled with decreased MT dynamics. Experiments performed with immortalized plectin-deficient myocytes supported this notion. Using ex vivo combined with biochemical in vitro experiments, the mechanism underlying MT destabilization could be traced to IF-bound plectin antagonizing the MT-stabilizing function of MT-associated proteins. In keratinocytes, plectin deficiency and the associated deregulation of MTs led to disturbances in multiple physiological processes and functions of cells, including alterations in shape and directional migration, increased focal adhesion turnover rates, elevated glucose uptake, formation of aberrant mitotic spindles, and alterations in cellular growth rates. In neurons, P1c deficiency led to diminished neurite branching, increased sensitivity towards oxidative stress, impaired synaptic vesicles transport, and, again, higher uptake of glucose. On the organismal level, P1c-deficient mice showed impaired pain reception and loss of memory. The outcome of this project not only established plectin as an essential element in the orchestration and proper execution of MT-dependent cellular processes, but also suggested the reduction of MT dynamics as the mechanism behind neuropathies of patients suffering from plectin disorders.