Vesicular PtdIns(3,4,5) P 3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion

Regulation of nuclear envelope (NE) dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion may also be conserved in the formation of both pronuclear and zygote NEs in the fertilised egg. Here, we determine that Class I PI3-kinases are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation and around male and female pronuclei prior to membrane fusion. We illustrate that Class I PI3-kinase activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, by coincidence amplified-FRET monitored by fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P3 occurring during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of NE membranes.</jats:p

Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation

The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer's and Parkinson's and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.</p

Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation

The endoplasmic reticulum (ER) is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, Type 2 diabetes, cancer, Alzheimer’s and Parkinson’s diseases and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. Much evidence suggests that ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids in ER morphology, we developed a sea urchin oocyte model which is a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol, phosphatidylethanolamine and phosphatidylcholine by microinjection of enzymes or lipid delivery by fusion with liposomes and evaluated shape changes with two- and three-dimensional confocal imaging and three-dimensional electron microscopy techniques. Decreases and increases in the levels of lipids such as diacylglycerol or phosphatidylethanolamine characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules respectively. The effects of endogenous alterations of diacylglycerol were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that shaping proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to future studies directed at understanding the precise spatial and compositional requirements of lipid species in interactions leading to alterations of organelle shaping

Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism

Thinking Through Improvisation: Do General Improvisation Studies Belong in a Liberal Arts Curriculum?

Thinking Through Improvisation&nbsp;implies two meanings: 1) carefully examining all that improvisation encompasses including how it is practiced, and 2) using improvisation to generate ideas or performances. Using a First Year Seminar course I taught for 20 years, I illustrate how a general course in improvisation can introduce students to improvisation as a way of thinking in diverse fields and can strengthen liberal arts skills in critical and creative thinking. Interdisciplinary and multicultural approaches are readily incorporated as are a range of activities including writing, critical reading, performance, and creative problem solving. Risk taking, trust, creativity, adaptability, teamwork, respect for knowledge, abstract and practical thinking and the joy of creative discovery are explored through discussion and practice of improvisation. Scientific explanations of improvisation are compared to subjective experiences of improvisational performance. These activities lay a groundwork for creative explorations of the discipline-oriented curriculum in the range of fields subsequently encountered by liberal arts students. &nbsp

36. Male germ line specific histones of sea urchins and sea stars

Volume: 166Start Page: 475End Page: 48

Synthesis and absorption spectra of 2,4,6-trihlorodiphenyl ether

The repeat of Schwab\u27s work confirmed his experimental results for the reaction of the pseudo-hypochlorite of 2,4,6-trichlorophenol with benzene and anhydrous alumnium chloride