Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.
Actin depolymerizing factor (ADF)/cofilin is an essential enhancer of actin turnover. Multicellular organisms express multiple ADF/cofilin isoforms in different patterns of tissue distribution. However, the functional significance of different ADF/cofilin isoforms is not understood. The Caenorhabditis elegans unc-60 gene generates two ADF/cofilins,UNC-60A and UNC-60B, by alternative splicing. These two ADF/cofilin proteins have different effects on actin dynamics in vitro, but their functional difference in vivo remains unclear. Here, we demonstrate that the two isoforms are expressed in different tissues and are required for distinct morphogenetic processes. UNC-60A was ubiquitously expressed in most embryonic cells and enriched in adult gonads, intestine and oocytes. In contrast, UNC-60B was specifically expressed in the body wall muscle, vulva and spermatheca. RNA interference of UNC-60A caused embryonic lethality with variable defects in cytokinesis and developmental patterning. In severely affected embryos, a cleavage furrow was formed and progressed but reversed before completion of the cleavage. Also, in some affected embryos, positioning of the blastomeres became abnormal, which resulted in embryonic arrest. In contrast, an unc-60B-null mutant was homozygous viable, underwent normal early embryogenesis and caused disorganization of actin filaments specifically in body wall muscle. These results suggest that the ADF/cofilin isoforms play distinct roles in specific aspects of actin reorganization in vivo.
CsOH- or Ag(2)O-mediated cycloalkylation of (alkylidene)bisperoxides 3 and 1,n-dihaloalkanes (n = 3-8) provided the corresponding medium-sized 1,2,4,5-tetraoxacycloalkanes 4-8 in moderate yields. Subsequent evaluation of the antimalarial activity of the cyclic peroxides 4-8 in vitro and in vivo revealed that 1,2,6,7-tetraoxaspiro[7.11]nonadecane 4a has considerable potential as a new, inexpensive, and potent antimalarial drug.
Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolinactin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.
Ovulation in the nematode Caenorhabditis elegans is regulated by complex signal transduction pathways and cell-cell interactions. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells that provide contractile forces to push a mature oocyte into the spermatheca for fertilization. Although several genes that regulate sheath contraction have been characterized, basic components of the contractile apparatuses of the myoepithelial sheath have not been extensively studied. We identified major structural proteins of the contractile apparatuses of the myoepithelial sheath and characterized their nonstriated arrangement. Of interest, integrin and perlecan were found only at the dense bodies, whereas they localized to both dense bodies and M-lines in the striated body wall muscle. RNA interference of most of the myofibrillar components impaired ovulation in a soma-specific manner. Our results provide basic information that helps understanding the mechanism of sheath contraction during ovulation and establishing a new model to study morphogenesis of nonstriated muscle. Developmental Dynamics 236: 1093-1105, 2007.
To know whether genes involved in cell-cell communication typical of multicellular animals dramatically increased in concert with the Cambrian explosion, the rapid evolutionary burst in the major groups of animals, and whether these genes exist in the sponge lacking cell cohesiveness and coordination typical of eumetazoans, we have carried out cloning of the G-protein alpha subunit (Galpha) and the protein tyrosine kinase (PTK) cDNAs from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We obtained 13 Galpha and 20 PTK cDNAs. Generally animal gene families diverged first by gene duplication (subtype duplication) that gave rise to diverse subtypes with different primary functions, followed by further gene duplication in the same subtype (isoform duplication) that gave rise to isoform genes with virtually identical function. Phylogenetic trees of Galpha and PTK families including cDNAs from sponge and hydra revealed that most of the present-day subtypes had been established in the very early evolution of animals before the parazoan-eumetazoan split, the earliest branching among the extant animal phyla, by extensive subtype duplication: for PTK and Galpha families, 23 and 9 subtype duplications were observed in the early stage before the parazoan-eumetazoan split, respectively, and after that split, only 2 and 1 subtype duplications were found, respectively. After the separation from arthropods, vertebrates underwent frequent isoform duplications before the fish-tetrapod split. Furthermore, rapid amino acid changes appear to have occurred in concert with the extensive subtype duplication and isoform duplication. Thus the pattern of gene diversification during animal evolution might be characterized by bursts of gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually, and there might be no direct link between the Cambrian explosion and the extensive gene duplication that generated diverse functions (subtypes) of these families.
Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.
Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.
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