C. elegans embryonic body-wall muscle cells form strong attachments to the basement membrane once they migrate to their final position beneath the hypodermis. These attachment structures, the dense bodies and M-lines, organize the assembly of the myofilament lattice and anchor it to the hypodermis. Dense bodies and M-lines contain the transmembrane protein integrin, and are analogs of focal adhesions, the attachments formed by vertebrate cells in culture that have been used extensively to investigate integrin's adhesive and cell signaling functions. We are using genetic approaches to investigate dense body and M-line assembly in C. elegans. Our current focus is upon the subset of pat genes (Paralyzed Arrested elongation at Two-fold) for which corresponding mutants have defects in dense body and/or M-line assembly. Two examples are
pat-2 and
pat-3, which code for integrin a and b subunits present in dense bodies and M-lines. A third is
pat-4, which our lab has recently shown codes for integrin-linked kinase, an integrin-associated signaling molecule that also present in dense bodies and M-lines. Here we report our progress on a fourth gene in this subset,
pat-6.
pat-6 maps genetically to the right end of chromosome IV. Using a candidate gene approach we have rescued
pat-6 mutants with a PCR fragment containing the predicted gene T21D12.4. RNAi experiments using dsRNA corresponding to T21D12.4 produced broods of 100% Pat embryos, further evidence that T21D12.4 is
pat-6. We sequenced the two existing
pat-6 alleles and found that both are likely to be null mutations:
st561 and
st570 introduce stop codons into the first and third exons, respectively, of the T21D12.4 gene. Taken together, these results confirm that
pat-6 corresponds to T21D12.4. The PAT-6 protein has significant homology (50% similarity at the amino acid level) to the human CGI-56 protein. Both proteins contain a pair of CH domains, which are thought to bind to actin filaments. We expressed GFP-tagged PAT-6 in transgenic animals and found that it can completely rescue
pat-6 mutants, and is localized to muscle attachment sites and touch neurons. Immunostaining experiments with antibodies to perlecan, integrin and vinculin suggest that the assembly of these proteins into nascent dense bodies and M-lines is independent of PAT-6. We are now investigating PAT-6 localization in mutants lacking other dense body and M-line proteins. Preliminary results suggest that PAT-6 functions downstream of integrin in the assembly pathway. To investigate the function of the PAT-6 CH domains, we have constructed transgenic strains expressing truncated variants of this protein. Mutant PAT-6 protein missing the second CH domain can localize to dense bodies and M-lines in a wild-type background, but is not able to rescue the muscle development defects of
pat-6 null animals. In conclusion, our analysis of
pat-6 has identified a new protein involved in the assembly of a focal adhesion analog in C. elegans.