Worm Breeder's Gazette 13(2): 80 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The protein talin is found in a variety of vertebrate cells. In skeletal muscle it is located at myotendonous junctions and costamers. In non-muscle cells it is found at focal adhesions, sites where the actin cytoskeleton associates with the transmembrane protein integrin. It binds relatively weakly to the proteins integrin and actin, and relatively strongly to the protein vinculin in vitro. Its cellular location and biochemistry suggests that it is one link in a chain of proteins that function to attach actin to the membrane.
The dense bodies found in C. elegans body wall muscle are sites where actin filaments are linked to the cell membrane. Dense bodies are known to contain the proteins integrin, vinculin, alpha-actinin and actin. They are, therefore, good models for the actin-membrane attachments found in vertebrate cells. We and others have been characterizing the proteins found in the dense body. Mutations exist that eliminate integrin and vinculin. Hresko et al. (M. Hresko, personal communication) have examined the organization of several muscle proteins in these mutants. Their data shows that integrin can assemble in the absence of vinculin, but that integrin is necessary for the localization of vinculin to the dense body. This data suggests a model in which the dense body assembles at the membrane beginning with integrin. We intend to examine how talin fits into this model.
Michael Hengartner in the laboratory of R. Horvitz recovered a putative C. elegans talin cDNA in a search for unc-69 cDNAs. The clone was subsequently found to be chimeric, and the talin homology unrelated to unc-69 .This clone was kindly given to us for further analysis of the talin homologous region. It was hybridized to the YAC grids from the physical mapping project and found to map to the clone Y71G12 .This clone derives from the far left of LG I. There are no known muscle affecting genes in this part of the map.
We have recovered additional, uncorrupted talin cDNAs. We have nearly completed sequencing 2.7 kb of the longest clone. The derived amino acid sequence is 40% identical to mouse talin over a stretch of 375 residues. On a Northern blot it hybridizes to a message of approximately 7 kb which is large enough to encode a protein of approximately 200 kd, the size of vertebrate talin. We have made a translational fusion between the putative talin cDNA and E. coli maltose binding protein, and purified the resulting fusion protein on an amylose column. Rabbit antibodies were raised against the fusion protein. When the resulting antiserum was used against Western blots of C. elegans protein extracts it recognized a polypeptide of 200 kd. Collectively, these data support the supposition that we are working on a homolog of vertebrate talin. We have not yet determined its organization in embryos or adults in situ. We anticipate the an examination of talin in wild type C. elegans, and in the mutants described above, will allow us to determine the relevance of the in vitro biochemistry of the vertebrate protein to the function of C. elegans talin in vivo.