Intermediate filaments (IFs) are a major structural component of striated muscle. To study the organization and functions of muscle IFs, we have constructed two types of mutant forms of the IF protein vimentin. C-terminal truncations of vimentin co- assemble with and disrupt IFs. The expression of such "disruptive" mutants in the rat myogenic cell line L6 will be used to study the effects of disrupting IF organization during the course of muscle differentiation in vitro. A combination of light and election microscopy will be used to determine whether an intact IF system is required for myoblast fusion, the establishment of sarcomeric organization, the formation and stability of costameres (i.e. regions of interaction between Z- lines and the plasma membrane), or the lateral alignment of myofibrils into myofibers. To complement these in vitro studies, we will examine the effects of disruptive vimentin mutants on skeletal muscle organization in the developing Xenopus tadpole. In the context of the swimming tadpole, functions of IFs related to mechanical integration of muscle and muscle components may become more obvious than in cultured cells. To identify proteins involved in muscle IF organization, we have made mutants forms of vimentin that do not co-assemble with endogenous IFs. These soluble, "non-assemblying" mutants retain either the C- and/or N-terminal domains of the vimentin molecule intact. It is through these domains that IF proteins are thought to intact with other molecules. Non-asssemblying mutants are therefore expected to interact with, and perhaps sequester, specific IF-associated proteins involved in the interaction between IFs and the Z-disc, the plasma membrane, microtubules and mitochondria. We will determine whether the expression of non- assemblying vimentin mutants affects IF organization or cellular differentiation in muscle and myogenic cells. We will then determine whether specific cellular proteins co-isolate with the exogenous, non-assemblying, mutant proteins. The study of these proteins should extend our understanding of how the changing organization of IFs during myogenesis is controlled. The result of our studies should provide a clear definition of the functions of IFs in striated muscle and, through the identification of IF-associated proteins, a better understanding of the molecular architecture of muscle.
