A Genetic Analysis of Intermediate Filament Domain Functions in C. elegans

University Of Toledo, Toledo OH

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Abstract

DESCRIPTION (provided by applicant): A simple in vivo model for investigating intermediate filament (IF) organization and function has been developed in the model organism Caenorhabditis elegans. Congenital or acquired disruptions of IF proteins in humans including cytoplasmic IF (cIFs) and nuclear lamins, and the cIF-containing junctions is associated with a wide variety diseases and developmental abnormalities, including skin blistering diseases, skeletal and cardiac myopathies, and neuronal dysfunction. However the contributions of specific IF domains to their function and interaction with intermediate filament associated proteins (IFAPs) is, with the exception of the rod domain's importance to IF assembly, still poorly understood. In C. elegans, a stress resistant network of IFs is required for the transmission of muscular force to the exoskeleton. Mutations in genes that disrupt this network have been isolated, and shown to encode IFs, IFAPs and hemidesmosome(HD) associated proteins. One of these genes encodes IFA-2, one of four epidermally expressed cIFs. Null mutations in this gene reveal that it is specifically required for the structural integrity of the muscle-cuticle force transduction pathway in postembryonic development. Preliminary studies also suggest that the head domain is not essential for IFA-2 function, and that IFA-2 complexes with HD proteins. Our long- term objective is to understand the cell and molecular biology of IF function during growth and development, IFA-2 provides an intriguing and convenient entry to this understanding. To define the domains of the IFA-2 protein required for its function, constructs containing deletion derivatives of ifa-2 fused to GFP will be introduced into a) ifa-2 (null) animals b) wild type animals. The effects of the truncations will be assayed by direct phenotypic observation, by examining the localization of the IFA- 2 ::GFP fusion protein, and by examining the localization of HD markers. To identify the proteins that mediate interactions between IFA-2 and IFs/HDs, a yeast two-hybrid screen will be used. Finally, to identify genes that are required for the localization of IFA-2 to HDs a genetic screen for mutations that disrupt IFA-2 localization to HDs will be carried out. PUBLIC HEALTH RELEVANCE: Defects in intermediate filament (IF) proteins that normally form stress resistant networks in cells, and contribute to their architectural integrity and cellular function, are linked to a number of human disease conditions, including congenital defects. By studying the roles of specific IF protein domains in the interactions of IFs with their normal cellular partners, a better understanding of the etiology of IF associated diseases will emerge, with the potential for developing therapies to alleviate the disease conditions. This work also expands understanding of the basic biology of intermediate filaments, and their roles in normal and aberrant development.