Mutations in
unc-98 and
unc-96 lead to animals with reduced motility and a characteristic defect in muscle structure: M-lines and dense bodies are disrupted, and by polarized light microscopy birefringent needles are present. UNC-98 is a 37 kD protein consisting of four C2H2 Zn finger domains and several putative NLS and NES sequences (Mercer et al. MBC Vol.14, 2003). UNC-96 is a 48 kD protein with little similarity to known proteins. Immunofluorescence and GFP fusion studies demonstrate that UNC-98 localizes to focal adhesion structures and to the nucleus, while UNC-96 localizes to focal adhesions of C. elegans body wall muscle cells. Non-null mutations in
unc-15, the structural gene for paramyosin, also show needles by polarized light that contain paramyosin paracrystals (Waterston et al. JMB Vol.117, 1977). The paramyosin polypeptide exists mostly as a coiled-coil rod much like the rod portion of myosin heavy chain, and resides in the core of the thick filaments (Kagawa JMB Vol.207, 1989). A null mutation in
unc-15 leads to paralysis of the worm (Waterston et al. JMB Vol.117, 1977). Genetic evidence suggests that both UNC-98 and UNC-96 interact with paramyosin in vivo. Animals homozygous for non-null mutations in either
unc-98(
su130) and
unc-15(
e1215) or
unc-96(
sf18) and
unc-15(
e1215) are completely paralyzed and have a characteristic posture, a phenotype unseen in either mutant alone. Additionally, non-null
unc-15/+;
unc-98/+ and
unc-15/+;
unc-96/+ transheterozygotes, with only one wild-type copy of each gene, have more severe polarized light phenotypes than the
unc-98,
unc-96, or
unc-15 heterozygotes alone. Immunofluorescence data also suggests that these proteins interact. Anti-paramyosin stains the needles in
unc-98 and
unc-96 mutant muscle, while anti-UNC-98, anti-UNC-96 and anti-paramyosin colocalize in the needles of
unc-15 hypomorphs. Also, anti-UNC-98 and anti-UNC-96 have diffuse localization within muscles of the
unc-15 null. ELISAs demonstrate that UNC-98 and UNC-96 interact with paramyosin in vitro. Additionally, during a thick filament preparation UNC-98 appears in the 15K pellet, a fraction enriched for thick filaments.