Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for successful completion of cytokinesis. In addition, both PIP₂and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP₂to yield diacylglycerol (DAG) and inositol trisphosphate (IP₃), which in turn induces calcium (Ca²⁺⁾release from the ER. Several studies suggest PIP₂must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP₂regulates cytokinesis remains to be elucidated.</p> <p>Results</p> <p>Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH₃(edelfosine) on cytokinesis in crane-fly and <it>Drosophila </it>spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH₃produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH₃.</p> <p>Conclusion</p> <p>We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP₂hydrolysis acts via Ca²⁺to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.</p

The Sac1 Lipid Phosphatase Regulates Cell Shape Change and the JNK Cascade during Dorsal Closure in Drosophila

AbstractThe Sac1 lipid phosphatase dephosphorylates several phosphatidylinositol (PtdIns) phosphates and, in yeast, regulates a diverse range of cellular processes including organization of the actin cytoskeleton and secretion [1]. We have identified mutations in the gene encoding Drosophila Sac1. sac1 mutants die as embryos with defects in dorsal closure (DC). DC involves the migration of the epidermis to close a hole in the dorsal surface of the embryo occupied by the amnioserosa. It requires cell shape change in both the epidermis and amnioserosa and activation of a Jun N-terminal kinase (JNK) MAPK cascade in the leading edge cells of the epidermis [2]. Loss of Sac1 leads to the improper activation of two key events in DC: cell shape change in the amnioserosa and JNK signaling. sac1 interacts genetically with other participants in these two events, and our data suggest that loss of Sac1 leads to upregulation of one or more signals controlling DC. This study is the first report of a role for Sac1 in the development of a multicellular organism

Genetic dissection of the phosphoinositide cycle in Drosophila photoreceptors.

Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2-, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and TbR332H), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified Drosophila PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (dPIP5K18 )

Megakaryocyte NLRP3 hyperactivation induces mild anemia and potentiates inflammatory response in mice

BackgroundThe NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been described in both immune cells and platelets, but its role in the megakaryocyte (MK) lineage remains elusive.ObjectiveThe aim of this study was to explore the role of NLRP3 inflammasome in megakaryocytes and platelets.MethodsWe generated Nlrp3A350V/+/Gp1ba-CreKI/+ mice carrying a mutation genetically similar to the one observed in human Muckle–Wells syndrome, which leads to hyperactivity of NLRP3 specifically in MK and platelets.ResultsPlatelets from the mutant mice expressed elevated levels of both precursor and active form of caspase-1, suggesting hyperactivity of NLRP3 inflammasome. Nlrp3A350V/+/Gp1ba-CreKI/+ mice developed normally and had normal platelet counts. Expression of major platelet receptors, platelet aggregation, platelet deposition on collagen under shear, and deep vein thrombosis were unchanged. Nlrp3A350V/+/Gp1ba-CreKI/+ mice had mild anemia, reduced Ter119+ cells in the bone marrow, and splenomegaly. A mild increase in MK TGF-β1 might be involved in the anemic phenotype. Intraperitoneal injection of zymosan in Nlrp3A350V/+/Gp1ba-CreKI/+ mice induced increased neutrophil egression and elevated levels of a set of proinflammatory cytokines, alongside IL-10 and G-CSF, in the peritoneal fluid as compared with control animals.ConclusionMK/platelet NLRP3 inflammasome promotes the acute inflammatory response and its hyperactivation in mice leads to mild anemia and increased extramedullary erythropoiesis

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility

Deficiency in the Multicopy Sycp3-Like X-Linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation

Slx and Slxl1 are genes present in multiple copies on the mouse X chromosome. Using transgenically-delivered small interfering RNAs to disrupt their function, we show that Slx and Slxl1 are important for normal sperm differentiation and male fertility

Lipid metabolic perturbation is an early-onset phenotype in adult spinster mutants: a Drosophila model for lysosomal storage disorders

Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations in spinster (spin) mutants, a Drosophila model of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels of ceramidase and altering these lipids in spin mutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral to spin function in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed in spin mutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes in spin mutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD