[
Development & Evolution Meeting,
2006]
Proper conversion of the contractile force generated by sliding myofilaments within muscle into movement of a single nematode requires attachment of actin to structures known as dense bodies. These structures in C. elegans muscle are both homologous and analogous to Z-lines found in mammalian muscle cells. The dense body projects inward from the sarcolemma towards the cytoplasm and anchors thin filaments that participate in an organized lattice, the sarcomere. This lattice also contains myosin thick filaments, which are anchored to the sarcolemma by the M-line.
Dense bodies play a pivotal role in the development of muscle cells. Evidence supporting this conclusion comes from examination of mutants with loss of core structural proteins. Loss of these proteins results in either lethality, or animals with reduced movement. While most muscle mutants have been uncovered through mutational screens focusing on these phenotypes, we propose that additional sarcomeric proteins exist for which there is a less severe or entirely different mutant phenotype.
In order to search for these proteins, we used a combination of SAGE expression data (McKay et al. 2003) and predicted protein information to uncover a small number of potential candidates.
One of these, the LIM domain protein C28H8.6, has a high level of homology to the C-terminal half of human paxillin, a core component of focal adhesions. This multi-LIM domain protein appears to have an important role in C. elegans muscle. First, a full-length C28H8.6::GFP functional fusion localizes to dense bodies. Secondly, the homozygous gene knockout of C28H8.6 results in uncoordinated animals arrested at the L1 stage of development. Further analysis is underway to characterize this newly discovered muscle protein.
[
International Worm Meeting,
2007]
Attachment of actin and myosin filaments to dense bodies and M-lines respectively is necessary to convert the force generated by sliding myofilaments into movement of the worm. Not surprisingly, worm muscle attachment complexes also contain many of the same protein components as vertebrate focal adhesion complexes, which rely on anchoring of actin filaments for movement of migrating cells over the extracellular matrix. One of the major focal adhesion components, paxillin, has to date not been identified in the worm as a full-length protein. Here, we describe work that demonstrates such a protein is present in the worm, plays an important role in body wall muscle, and is homologous to full length paxillin in humans and other species. In order to identify novel genes affecting C. elegans body wall muscle, we used tissue specific SAGE and microarray expression data (McKay et al. 2003; Moerman lab, unpublished; Miller III lab, unpublished) to compile a list of genes with enriched expression in body wall muscle cells. One of these, the LIM domain protein C28H8.6 is highly muscle enriched and has a high level of homology to the C-terminal half of human paxillin. We have also found that the predicted gene directly upstream, C28H8.13, contains homology to the N-terminal half of paxillin. We believe that these two predicted genes constitute one coding sequence hereby termed cePaxillin. Our initial analysis has shown that cePaxillin plays a significant role in C. elegans muscle. First, we have found that a GFP translational fusion containing the 4 LIM domains of cePaxillin localizes to dense bodies and M-lines, but not the nucleus where other body wall muscle LIM proteins have been found. Secondly, a homozygous gene knockout of cePaxillin provided by the C. elegans Knockout Consortium results in uncoordinated animals arrested at the L1 developmental stage. These developmentally arrested worms do not die immediately, but live for a normal lifespan while displaying mild disorganization of the myofilament lattice in their muscle cells. Lastly, yeast two-hybrid data has shown interactions between cePaxillin and other body wall muscle proteins. A genome wide yeast two-hybrid screen (Li et al. 2004) found an interaction between the LIM domain region of cePaxillin and the dense body protein ALP-1, and our own preliminary studies have shown that cePaxillin binds to the dense body protein UIG-1 and the dense body/M-line protein UNC-95. We will continue to characterize the role that cePaxillin plays in body wall muscle, and where it fits in the sarcomere assembly pathway.