Body wall muscle is fundamental for nematode locomotion. Among the more than several hundred proteins required for sarcomere assembly, maintenance and function, there are muscle specific giant proteins (>700 kDa). C. elegans has 3 giants; twitchin, UNC-89, and TTN-1. UNC-89 consists of 53 Ig domains, 2 Fn domains, 2 kinase domains, and KSP repeat sequence. UNC-89 is localized at M-lines, the structure in the middle of the sarcomere where thick filaments are cross-linked. Mutations in unc-89 result in sarcomere disorganization (including absence of M-lines) and reduced locomotion. To understand how UNC-89 is positioned and how it carries out its functions, we are conducting a systematic identification of interacting molecules by using the yeast two-hybrid (Y2H) system, and have identified a number of interesting interactors (Gieseler et al. WormBook).
By using 1/3IK-Ig53-Fn2 region of UNC-89 as bait to screen a Y2H library, we identified PPTR-1 and PPTR-2. PPTR-1 and PPTR-2 are protein phosphatase 2A (PP2A) regulatory subunit B subunits (B56). A deletion mutant of pptr-1, tm3103, shows sarcomere disorganization. Anti-PPTR-1 antibodies localize to I-bands. Since antibodies generated to a highly conserved region of human B56 that is likely to react with both PPTR-1 and PPTR-2 stain I-bands and M-lines, we hypothesize that PPTR-2 may localize to M-lines. We also characterized muscle functions of other PP2A regulatory subunits. Antibodies to SUR-6 (B subunit)(provided by Kevin O'connell) localize to I-bands. A ts mutant of sur-6, or550, shows sarcomere disorganization at the restrictive temperature. Mutants of rsa-1 (B’’ subunit) display a characteristic myosin heavy chain A (MHC A) staining pattern. Normally, MHC A is located at the center of A-bands, localized as a straight single line. In rsa-1 mutants, MHC A localizes as two parallel lines with the M-line in the center. Since some unc-54 (MHC B) mutants (s74) show a similar split MHC A staining pattern, an RSA-1-associated PP2A complex might regulate phosphorylation of myosin heavy chains. The PP2A A subunit (PAA-1) and C subunit (LET-92) are also expressed in muscle. We addressed their muscle function by RNAi feeding from the L1 stage. Worms fed bacteria expressing paa-1 or let-92 dsRNA show muscle detachment from the basement membrane, resulting in folded “jack-knife” appearance. Taken together, protein dephosphorylation performed by PP2A might be crucial for many aspects of sarcomere assembly/maintenance.