The reaction of an abundant 106-kDa polypeptide with a specific monoclonal antibody has been localized in intestinal and muscle cells of the nematode Caenorhabditis elegans. This protein was first detected in 4-6 cells of the clonal E lineage of 100-cell embryos. This lineage is committed to the intestinal cell fate. The antigen continued to be expressed in the differentiating gut and then appeared in early differentiating body wall muscle cells of 400- to 500-cell embryos. Molecular cloning and sequencing showed that the largest cDNA clone contained 3274 bp and encoded a sequence of 1005 amino acids. The predicted polypeptide of 112,799 MW contains separate domains for the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Their enzymatic activities had been shown previously to be highest in embryos and L1 larvae (Khan, F. R., and McFadden, B. A. (1980). FEBS Lett. 115, 312-314; Khan, F. R., and McFadden, B. A. (1982). Exp. Parasitol. 54, 48-54; Wadsworth, W. G., and Riddle, D. L. (1989). Dev. Biol. 132, 167-173). The domain-specific sequences were shown to be contiguous in genomic DNA and are separated by an intron of 68 bp. A single polypeptide and both enzymatic activities are precipitated by the antibody, and peptide fragments resulting from limited proteolytic digestion contained amino acid sequences which overlap the predicted junctional region. The physical localization of the gene correlates with a small region of the left arm of Linkage Group V to which multiple embryonic lethal mutations have been mapped.

Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin-binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rhokinase may regulate myosin II phosphorylation. ß

The mRNA and the bifunctional protein for the two glyoxylate cycle (GC) enzymes, isocitrate lyase and malate synthase, are expressed in a tissue- and stage-specific pattern in Caenorhabditis elegans. Since expression of the two enzymes for the carbon-conserving glyoxylate cycle is regulated by the availability of carbon sources in microorganisms, we have studied the bifunctional GCP gene expression under fasting conditions and in certain mutants of C. elegans in order to understand possible mechanisms regulating its expression during nematode development. The GCP mRNA and protein levels were elevated in early larvae which were never fed, a result consistent with previous enzyme activity measurements (Khan, F.R., & McFadden, B.A. (1982) Exp. Parasitol. 54, 48-54]. However, larvae of later stages also expressed higher levels of GCP mRNA and protein when they were shifted from normal to fasting growing conditions. The GCP expression appeared to be regulated primarily at the transcriptional level throughout development. Although the expression of both the GCP gene and lin-14 peaks at about the same time during development and are induced by fasting, the regulation of the GCP gene is independent of the heterochronic lin-14 control mechanism of postembryonic lineages, as demonstrated by the fact that there was no significant change of the GCP at both mRNA and protein levels in the heterochronic lin-4 (lf) and lin-14 (gf) mutants compared to the wild type.

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.

ABSTRACT. Thick filaments are stable assemblies of myosin that are characteristic of specific muscle types from both vertebrates and invertebrates. In general, their structure and assembly require remarkably precise determination of lengths and diameters, structural differentiation and nonequivalence of myosins, a high degree of inelasticity and rigidity, and dynamic regulation of assembly and disassembly in response to both extracellular and intracellular signals. Directed assembly of myosin in which additional proteins function in key roles, therefore, is more likely to be significant than the simple self assembly of myosin into thick filaments. The nematode Caenorhabditis elegans permits a wide spectrum of biochemical, genetic, molecular and structural approaches to be applied to the experimental testing of this hypothesis. Biochemical analysis of C. elegans thick filaments reveals that paramyosin, a homologue of the myosin rod that is the unique product of a single genetic locus, exists as two populations which differ by post-translational modification. The major paramyosin species interacts with the two genetically specified myosin heavy chain isoforms. The minor paramyosin species is organized within the cores of the thick filaments, where it is associated stoichiometrically with three recently identified proteins P20, P28 and P30. These proteins have now been characterized molecularly and contain unique, novel amino acid sequences. Structural analysis of the core shows that seven paramyosin sub filaments are crosslinked by additional internal proteins into a highly rigid tubule. P20, P28 and P30 are proposed to couple the paramyosin subfilaments together into the core tubule during filament assembly. Mutants that affect paramyosin assembly are being characterized for alterations in the core proteins. A fourth protein has been identified recently as the product of the unc-45 gene. Computational analysis of this gene's DNAsuggests that the predicted protein may exhibit protein phosphatase and chaperone activities. Genetic analysis shows that three classes of specific unc-45 mutant proteins differentially interact with the two myosins during thick filament assembly. The unc-45 protein is proposed to be a myosin assemblase, a protein catalyst of thick filament assembly.The assembly of myosin into thick filaments has served as a paradigm for molecular self-assembly generally (25) and for molecular mechanisms of myofibril assembly more specifically (12). The precise construction of the resulting structures and their organization into highly ordered arrays within muscle cells contrasts greatly with the results of cell-free experiments using purified components (21). The paradigm of self-assembly ( Fig.