C. elegans body wall muscle is precisely organized into A and I bands that consist of thick and thin filaments. Thin filaments are attached to Z-disc analogs (dense bodies) and thick filaments are organized around M-lines. Our lab has been studying two genes,
unc-98 and
unc-96 that, when mutant, have reduced motility and a nearly identical needle phenotype by polarized light microscopy. UNC-98 is a C2H2 Zn finger protein that localizes to M-lines, dense bodies and nuclei, and interacts with UNC-97(PINCH). We now report that
unc-96(F13C5.6) encodes a 418aa protein without recognizable domains that, by use of a full length
unc-96::GFP translational fusion, also localizes to M-lines and dense bodies but not to nuclei. Screening of a 2-hybrid bookshelf revealed that UNC-96 interacts with two conserved LIM domain proteins, F28F5.3 and F25H5.1. F28F5.3 and F25H5.1, like UNC-98, also interact with UNC-97. Promoter-GFP constructs reveal that these two genes are expressed in various muscle cells: pharyngeal, vulval, anal sphincter, anal depressor, and body wall. F25H5.1, a homolog of human FHL2, is expressed in adult body wall muscle, whereas F28F5.3 appears to be expressed in body wall muscle at the embryo and larval stages only. Antibodies to UNC-96 reveal that UNC-96 localizes to the M-line in body wall muscle, and also to the middle of the single sarcomere muscles of the pharyngeal terminal bulb and the anal-depressor muscles. We currently have 3 alleles of
unc-96; two of which appear to be protein nulls (
sf18,
su151) and one (
r291) which exhibits reduced protein by Western analysis. All mutants show a mild disruption of the organization of myosins A and B, actin, vinculin, and alpha-actinin, and more severe disorganization of UNC-89. We speculate that the reduced levels of particular components of dense bodies or M-lines(such as UNC-96) causes a breakdown of these protein assemblages and abnormal accumulations of proteins. In fact, the accumulations ("needles") within
unc-96 body wall muscle contain UNC-98 and paramyosin. Mutants for UNC-96 are vulnerable to both under and over expression of UNC-98, resulting in a detectable worsening of muscle structure by polarized light. Mutants also show an increased impairment of motility as adults vs. larvae. A lessened, needling phenotype is observed when the mutants (all alleles) are raised at 15 vs. 20 degrees.