A previous genetic screen for mutants with the Pat phenotype ( P aralyzed, A rrested elongation at T wo-fold) yielded a subset of mutants with severely disrupted sarcomere assembly in embryonic body-wall muscle cells (Williams, 1994). Mutants in this subset have defined the
pat-2 and
pat-3 genes which code for the a and b integrin subunits expressed in body-wall muscle cells, and identified several new genes likely to work in conjunction with
pat-2 and
pat-3 during muscle cell differentiation. Here we report that we have molecularly isolated
pat-4, one of these newly defined genes. We mapped
pat-4 to the left end of chromosome III and identified the gene C29F9.7 as a promising candidate. C29F9.7 is the C. elegans homologue of Integrin-Linked Kinase (ILK), a serine/threonine kinase. Overexpression of ILK in vertebrate tissue culture cells suggests that ILK plays a role cell cycle regulation, confers anchorage-independent cell growth, stimulates both focal adhesion and ECM assembly, upregulates the Wnt signaling pathway, and regulates GSK-3 and PKB/AKT activities. We rescued
pat-4 homozygous animals with the genomic cosmid clone DE10 and subsequently with a PCR product containing C29F9.7. We sequenced two
pat-4 mutant alleles and detected polymorphisms which either introduced a stop codon or altered a splice site. In addition, myosin organization is severely disrupted in
pat-4 embryos. These results demonstrate that
pat-4 codes for C. elegans ILK homologue, and ILK is therefore required for assembly of the myofilament lattice in body-wall muscle. We are taking several approaches to better understand the in vivo function of
pat-4. We are determining the localization of the PAT-4 protein by constructing a
pat-4::gfp fusion gene. We are also staining
pat-4 embryos with antibodies that recognize different components of dense bodies and M-lines. These structures form the contractile apparatus and link it to the underlying ECM. Our preliminary results indicate integrin polarization to the basal membrane is not affected by mutations in the
pat-4 gene. However, cytoskeletal organization is disrupted. These preliminary results support the hypothesis that
pat-4 functions as a downstream regulator of integrin-mediated signaling required for muscle assembly.