A region of the myosin rod important for interaction with paramyosin in Caenorhabditis elegans striated muscle.

The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.

Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

Cloning of an early immunodominant filarial antigen: a member of the Brugia malayi myosin heavy chain gene family.

Partial protective immunity to filariasis can be achieved in animals by vaccination with irradiated infective larvae. A Brugia malayi cDNA expression library was screened with serum pools from vaccinated and infected jirds to select clones that expressed potentially protective recombinant antigens that were preferentially recognized by sera from vaccinated animals. Bmmyo-2, the largest of a group of related clones, was studied in detail. Jirds produced strong antibody responses to the protein product of Bmmyo-2, Bmmhc-B, as early as 1 month after vaccination with irradiated larvae. Antibody responses to Bmmhc-B in infected jirds were weaker than those of vaccinated jirds, and they developed somewhat later. Antibodies produced to Bmmhc-B were reactive with a 200 kDa native B. malayi antigen by immunoblot and with muscle bands in the body wall of microfilarial by indirect immunofluorescence. Sequence analysis of the 1454 bp cDNA insert of Bmmyo-2 showed that it codes for a portion of the rod region of a B. malayi myosin heavy chain isoform. The deduced amino acid sequence of Bmmyo-2 is 74.6% identical with that of the corresponding region of Caenorhabditis elegans myosin heavy chain B, but only 64.6% identical with a recently described B. malayi myosin heavy chain, Bmmhc-A.

The Notch locus of Drosophila is required in epidermal cells for epidermal development.

The Notch locus of Drosophila plays an important role in cell fate decisions within the neurogenic ectoderm, a role thought to involve interactions at the cell surface. We have assayed the requirement for Notch gene expression in epidermal cells by two kinds of genetic mosaics. First, with gynandromorphs, we removed the wild-type gene long before the critical developmental events to produce large mutant clones. The genotype of cells in large clones was scored by means of an antibody to the Notch protein. Second, using mitotic recombination, we removed the gene at successively later times after completion of the mitotically active early cleavage stages, to produce small clones. These clones were detected by means of a linked mutation of cuticle pattern, armadillo. The results of both experiments demonstrate a requirement for Notch expression by epidermal cells, and thus argue against the model that the Notch product acts as a signal required only in the neuroblast to influence neighboring epidermal cells. The mitotic recombination experiment revealed that Notch product is required by epidermal cells subsequent to neuroblast delamination. This result implies that the Notch gene functions to maintain the determined state of epidermal cells, possibly by mediating cell surface interactions within the epidermis.

Local function of the Notch gene for embryonic ectodermal pathway choice in Drosophila.

Mutations at the Notch locus affect the fate of cells in the neurogenic region of the Drosophila embryo so that epidermal precursors become neuroblasts. We have analyzed the cellular requirements for wild-type Notch gene function by means of genetic mosaics, using a cuticle marker to distinguish hypodermal cell genotype. Cells that were genotypically Notch never gave rise to hypoderm within the neurogenic region of mosaic embryos. Mosaic dividing lines within the neurogenic region juxtapose N+ hypoderm with regions of neural hypertrophy. This autonomous action of Notch in hypodermal cells is consistent with a local function of the protein during neurogenesis. Comparison of clone distribution in Notch mosaics and controls suggests that islands of wild-type hypodermal cells fail to differentiate cuticle.