The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.

BACKGROUND: Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform\u27s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. METHODOLOGY/PRINCIPAL FINDINGS: Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. CONCLUSIONS/SIGNIFICANCE: Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However, prohibiting phosphorylation of candidate 14-3-3-binding sites does not impact localization of the fusogen. Hypodermal membrane fusion activity persists when 14-3-3 expression levels are reduced

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

Moho depth across the Trans-European Suture Zone from P-and S-receiver functions

The Mohorovicic discontinuity, Moho for short, which marks the boundary between crust and mantle, is the main first-order structure within the lithosphere. Geodynamics and tectonic evolution determine its depth level and properties. Here, we present a map of the Moho in central Europe across the Teisseyre-Tornquist Zone, a region for which a number of previous studies are available. Our results are based on homogeneous and consistent processing of P- and S-receiver functions for the largest passive seismological data set in this region yet, consisting of more than 40 000 receiver functions from almost 500 station. Besides, we also provide new results for the crustal Vp/Vs ratio for the whole area. Our results are in good agreement with previous, more localized receiver function studies, as well as with the interpretation of seismic profiles, while at the same time resolving a higher level of detail than previous maps covering the area, for example regarding the Eifel Plume region, Rhine Graben and northern Alps. The close correspondence with the seismic data regarding crustal structure also increases confidence in use of the data in crustal corrections and the imaging of deeper structure, for which no independent seismic information is available. In addition to the pronounced, stepwise transition from crustal thicknesses of 30km in Phanerozoic Europe to more than 45 beneath the East European Craton, we can distinguish other terrane boundaries based on Moho depth as well as average crustal Vp/Vsratio and Moho phase amplitudes. The terranes with distinct crustal properties span a wide range of ages, from Palaeoproterozoic in Lithuania to Cenozoic in the Alps, reflecting the complex tectonic history of Europe. Crustal thickness and properties in the study area are also markedly influenced by tectonic overprinting, for example the formation of the Central European Basin System, and the European Cenozoic Rift System. In the areas affected by Cenozoic rifting and volcanism, thinning of the crust corresponds to lithospheric updoming reported in recent surface wave and S-receiver function studies, as expected for thermally induced deformation. The same correlation applies for crustal thickening, not only across the Trans-European Suture Zone, but also within the southern part of the Bohemian Massif. A high Poisson’s ratio of 0.27 is obtained for the craton, which is consistent with a thick mafic lower crust. In contrast, we typically find Poisson’s ratios around 0.25 for Phanerozoic Europe outside of deep sedimentary basins. Mapping of the thickness of the shallowest crustal layer, that is low-velocity sediments or weathered rock, indicates values in excess of 6km for the most pronounced basins in the study area, while thicknesses of less than 4km are found within the craton, central Germany and most of the Czech Republic.Peer reviewe