Mutants altering coordinate synthesis of specific myosins during nematode muscle development.

Abstract

Mutations in the unc-52 gene on linkage group II retard the construction of body-wall muscle sarcomeres during larval development in the nematode Caenorhabditis elegans. Unc-52 mutants show decreased accumulation of myosin heavy chains relative to other polypeptides during larval development, correlating with the structural retardation. Pulse radiolabeling experiments show that decreased synthesis of specific body-wall myosin heavy chains that are encoded by the unc-54 gene on linkage group I is responsible for the defective myosin accumulation. In the wild type, a constant ratio of the synthesis of the unc-54-coded myosin B to myosin A, about 2:1, is maintained during the larval stages in which the synthesis of both myosins increases exponentially and rapid sarcomere growth and addition ensues. During the first 26 hr of larval development, before any structural or behavioral effects of unc-52 mutations are apparent, the synthesis of myosin heavy chains is also normal. By 38 hr, decreased synthesis of myosin B is detected in the unc-52 mutant SU200, when sarcomere growth slows considerably. The effects of mutation in the une-52 locus are trans acting upon the synthesis of unc-54-coded myosin in a specific set of muscle cells during a defined period of larval development.The nematode Caenorhabditis elegans synthesizes at least three forms of myosin heavy chains (1). The A form, with a Mr of 210,000 in wild-type and mutant nematodes, is found in both body-wall and pharyngeal muscles. The B form, which also has a Mr of 210,000 in wild-type nematodes, is structurally altered with a Mr of 203,000 in strain E675 arising from mutation in the unc-54 gene on linkage group I. B myosin heavy chains are located only in body-wall muscles. The C form, with a Mr of 206,000 in wild-type and mutant nematodes, is found only in pharyngeal muscles. The A and B myosin heavy chains have different peptide structures and are the products of at least two structural genes (2-6). The A and B chains assemble into myosin molecules that are homodimeric; no hybrid myosin has been detected (3, 7). In body-wall muscles, these two forms of myosin molecules, which may be termed A and B on the basis of their heavy-chain composition, are located within the same cells and in the same sarcomeres (8). A and B myosin heavy chains and myosin molecules are synthesized at a constant ratio of about 1:2 throughout larval development and early adult maturation. The relative amounts of A and B myosins that accumulate are identical to these synthetic ratios, suggesting that synthesis is the primary determinant of the relative accumulation rates of the two myosins in nematode body-wall muscle cells (9).Two lines of evidence suggest that the synthesis of myosin heavy chains in C. elegans is regulated by specific genetic control mechanisms. The unique location of B myosin in the body-wall muscle cells and of C myosin in the pharynx-implies differential gene expression between the two types of muscle cell. The differential synthesis of A and B myosins su...

The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans.

“…No true thick filaments are observed in its body wall muscles, but 32-nm-wide myosin-containing stubs and 15nm-wide "lucent" tubes have been identified (7). CB669 and HE200 are uric-52 mutants that exhibit decreased levels of myosin heavy chain B synthesis and accumulation concomitant with retarded myofibrillar synthesis in larval and early adult maturation (9,25). CB1407 partially suppresses specific unc-15 and unc-54 myofibrillar-defective phenotypes (15) and shows a twofold increase in myosin heavy chain A (14,24).…”

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

“…Young-adult worms were hand picked, placed in SDS sample buffer, heated at 95˚C for 10 minutes and, after brief cooling, run in 7.5% SDS-PAGE to minimize protein degradation (Miller et al, 1983;Zengel and Epstein, 1980). Mouse monoclonal anti-FLAG M2-peroxidase (HRP) antibody (Sigma), Hsp90 mouse monoclonal antibody AC88 (Stressgen), rabbit polyclonal anti-C. elegans UNC-45 antibody (gift of Thorsten Hoppe, University of Cologne, Germany), mouse monoclonal anti-C. elegans MHC A (myosin heavy chain A) antibody (mAb 5-6), mouse monoclonal anti-C. elegans MHC B (myosin heavy chain B) antibody (mAb 28.2) and mouse monoclonal anti-C. elegans MHC D (myosin heavy chain D) antibody (mAb 5-17) (Miller et al, 1983) were employed for specific immunoblotting (Barral et al, 1998;Landsverk et al, 2007).…”

The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis

“…In C. elegans, RNF-5 localizes to the dense bodies and the M-line of the body wall muscles, where it regulates the levels of the LIM domain protein UNC-95. In C. elegans , UNC-95 has been genetically associated with uncoordinated movement [21] and shown to be important for formation of muscle attachment sites (M-lines and Z-lines) associated with the downstream process of sarcomere organization [20] . In a mammalian cell culture system, RNF5 has been shown to ubiquitinate and regulate the localization of the protein paxillin [22] , a critical component of focal adhesion, involved in cell adhesion and motility.…”