Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. In this study, we explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. Our aim was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. Our results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies.

Germ cell survival in C. elegans and C. remanei is affected when the DEAD box RNA helicases VBH-1 or Cre-VBH-1 are silenced.

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH-1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH-1 homolog in this gonochoristic species. We found that Cre-VBH-1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH-1 and Cre-VBH-1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre-vbh-1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh-1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre-vbh-1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH-1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one.

Distinct developmental roles for direct and indirect talin-mediated linkage to actin.

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is essential to connect extracellular matrix-bound integrins to the cytoskeleton. Talin can connect to the cytoskeleton either directly, through its actin-binding motifs, or indirectly, by recruiting other actin-binding proteins. Talin's carboxy-terminal end contains a well-characterized actin-binding domain (ABD). We tested the role of the C-terminal ABD of talin in integrin function in Drosophila. We found that introduction of mutations that reduced actin binding in vitro into the isolated C-terminal Talin-ABD impaired actin binding in vivo. Moreover, when engineered into full-length talin, these mutations disrupted a subset of integrin-mediated adhesion-dependent developmental events. Specifically, morphogenetic processes that involve dynamic, short-term integrin-mediated adhesion were particularly sensitive to impaired function of the C-terminal Talin-ABD. We propose that during development talin connects integrins to the cytoskeleton in distinct ways in different types of integrin-mediated adhesion: directly in transient adhesions and indirectly in stable long-lasting adhesions. Our results provide insight into how a similar array of molecular components can contribute to diverse adhesive processes throughout development.

Myosin Va is proteolysed in rat cerebellar granule neurons after excitotoxic injury.

Cerebellar granule neurons when exposed to glutamate die through an excitotoxic mechanism induced by overactivation of glutamate receptors. This kind of cell death is mediated by an overload of intracellular calcium involving calpain activation, a Ca2+ -dependent intracellular cysteine protease, among other intracellular responses. On the other hand, class V myosins are proteins that move cargo along actin filaments and one of its members, myosin Va, is involved in vesicles transport. Here we studied the effect of excitotoxicity on myosin Va in cultured cerebellar granule neurons. Western blot analysis of control cultures shows a band corresponding to myosin Va as well as an 80 kDa band corresponding to its proteolytic product by calpain. When cells are exposed to glutamate (500 microM), kainate (100 microM) or NMDA (150 microM) during 3-24 h, the proteolytic processing of myosin Va is markedly increased. This proteolysis is inhibited by leupeptin (100 microM) and calpain inhibitor I (50 microM). These inhibitors also significantly improve the morphological appearance of the neurons possibly through the preservation of the cytoskeleton integrity. Our results suggest that myosin Va is a target for calpain I during an excitotoxic injury and could lead to a new area of research to address the participation of molecular motors in neurotoxicity.