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Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion-like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of [beta]-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.

Ca2+/S100A1(sub 2) is a Ca2+ -binding protein that activates the myosin-associated giant protein kinase twitchin with a high specificity. The S100A1(sub 2)-binding region of twitchin is present in the autoregulatory sequence that is involved in the intrasteric autoinhibition of twitchin. The activation of twitchin is Ca2+ dependent and increases in the presence of low amounts of Zn2+. S100A1(sub 2) is a possible activator of the entire family of giant protein kinases that participate in muscle contraction and cytoskeletal structure.

Normal muscular development in the nematode Caenorhabditis elegans is regulated by the presence of 6,632-amino acid polypeptide. The gene coding for this protein, designated as unc-89, produces a polypeptide that contains immunoglobulin and phosphorylation domains. The protein determines the formation of M-lines settles in the center of the A-band to simulate M-line growth. The presence of diverse sequences points to its activity as signal inducer and assembly protein.

The crystal structure of twitchin kinase, which has a catalytic core and a 60 residue carboxy-terminal end, gives proof for the intrasteric model of protein kinase modulation with the autoregulatory step which employs varied properties of the enzyme's active site to control enzyme activity. All members of myosin light-chain kinase, calmodulin-dependent protein kinase II subfamilies and other enzymes also utilize this mechanism.

Null mutations of the Caenorhabditis elegans unc-22 gene, when associated with hindered movement and a distortion in the muscle structure, give rise to a distinct body surface twitch. Earlier sequence analysis of the unc-22 had revealed that the encoded polypeptide termed twitching is comprised of a bone protein kinase domain, several variants of both the immunoglobulin-like domain and a fibronectin type III-like domain. The present experiment provides an explanation for the weak phenotype of a transposon insertion allele after additional information on the DNA sequence was revealed.

A study examined two self-sterile mutations that dictate the spermatogenesis-defective gene spe-17 influences unc-22, and they do not complement each other for Unc-22 and spermatogenesis faults. These mutations are deficiencies (hcDfl and hDfl3) that influence more than one transcription unit. The sequencing of spe-17 DNAs revealed that the deduced 142 amino acid protein sequence is extremely charged and rich in serine and threonine, but exhibits no major homology to any previously identified protein sequence.

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for 'dual oxidase' because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed crosslinking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Research was conducted to examine the hypothesis that one of the gene products of Caenorhabditis elegans, UNC-60B, is required for proper assembly of actin into myofibrils and that its normal function is to regulate a dynamic state of actin filaments. Results demonstrate for the first time an isoform-specific requirement of ADF/cofilin for the assembly of a differentiated actin cytoskeleton.