Muscle-cell adhesion in C. elegans, and therefore locomotion, is dependent on a number of structures that link the contractile apparatus of the body-wall muscle to the cuticle (Francis and Waterston, 1985 & 1991). Body-wall muscle cells are positioned directly beneath the hypodermis and are organized into 4 quadrants that run along the anterior-posterior axis of the worm. The link between the muscle cells and the cuticle is mediated by a specialized basement membrane and by hypodermal cell-adhesion structures called fibrous organelles (Francis and Waterston, 1991). The fibrous organelles are restricted to the regions of the hypodermal syncitium adjacent to muscle and some mechanosensory neurons. Our molecular and genetic analysis of myotactin raises the possibility that myotactin is part of a link between the muscle or muscle basement membrane, and hypodermal fibrous organelles gene (Hresko et al. 1999). Myotactin is a large (4450 amino acids) putative transmembrane protein encoded by the
let-805 gene. The extracellular domain contains at least 32 fibronectin type III (FNIII) repeats and likely contributes to the structure of the basement membrane. In adult worms, myotactin colocalizes with fibrous organelle protein suggesting myotactin may also interact with fibrous organelles. These data suggest myotactin may directly or indirectly interact with both muscle and fibrous organelles. The phenotype of loss-of-function myotactin mutants is also consistent with a role for myotactin in linking muscle cells and fibrous organelles. In myotactin mutant embryos muscle cells detach from the fibrous organelle-associated intermediate filaments, although correctly localized early in embryogenesis, become delocalized at the three-fold stage. That is, the intermediate filaments are not restricted to regions of the hypodermis adjacent to muscle as in wild-type embryos of this stage To identify proteins that physically interact with myotactin, and may therefore also be part of a link between muscle and fibrous organelles, we performed a two-hybrid screen using the cytoplasmic tail of myotactin. We identified 10 clones in a screen of 1.5 x 104 clones, three of which are of particular interest . One is the cytoplasmic domain of myotactin itself. This result suggests myotactin may form multimers. A second molecule identified in the screen is the intermediate filament-A2 protein (
ifa-2; Dodemont et al. 1994;
mua-6; Hresko and Plenefisch, unpub results). An IFA-2::GFP fusion protein localizes to fibrous organelles in vivo suggesting MUA-6/intermediate filament protein could interact with myotactin. Furthermore, during the larval stages, the muscle cells of
mua-6 mutants detach from the hypodermis suggesting a role in muscle cell attachment The third protein of interest we identified is the G-protein b-subunit encoded by the
gpb-1 gene (van der Voorn et al., 1990). Zygotic loss of
gpb-1 gene function (
pk44 mutants) results in larval lethality (Zwaal et al, 1996) and an intermediate filament phenotype similar to that described for above for myotactin mutants. This suggests GPB-1/G-protein, like myotactin, may be invovled in maintaining the connection between muscle and fibrous organelles.