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.

Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) 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 before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P3 that occurs 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 nuclear envelope membranes.

Principle of duality in phospholipids: regulators of membrane morphology and dynamics.

To suggest and develop intelligent strategies to comprehend the regulation of organelle formation, a deeper mechanistic interpretation requires more than just the involvement of proteins. Our approaches link the formation of endomembranes with both signalling and membrane physical properties. Hitherto, membrane morphology, local physical structure and signalling have not been well integrated. Our studies derive from a cross-disciplinary approach undertaken to determine the molecular mechanisms of nuclear envelope assembly in echinoderm and mammalian cells. Our findings have led to the demonstration of a direct role for phosphoinositides and their derivatives in nuclear membrane formation. We have shown that phosphoinositides and their derivatives, as well as acting as second messengers, are modulators of membrane morphology, and their modifying enzymes regulate nuclear envelope formation. In addition, we have shown that echinoderm eggs can be exploited as a milieu to directly study the roles of phospholipids in maintaining organelle shape. The use of the echinoderm egg is a significant step forward in obtaining direct information about membrane physical properties in situ rather than using simpler models which do not provide a complete mechanistic insight into the role of phospholipids in membrane dynamics.

Effects of phosphoinositides and their derivatives on membrane morphology and function.

Currently, one of the fundamental problems in the study of membrane function and morphology is that the roles of proteins and lipids are usually investigated separately. In most cases proteins are predominant, with lipids taking a subsidiary role. This polarised view is in part due to the more straightforward and familiar techniques used to investigate proteins. Here, we summarise how phospholipids can be studied in cells with new tools that can acutely (rapidly and specifically) modify phospholipid composition of membranes in subcellular compartments. We point out some of the important physical effects that phosphoinositides in particular can have in altering membrane bilayer morphology, and provide specific examples to illustrate the roles that these phospholipids may play in maintaining the geometry of endomembranes.

Phosphatidylinositol metabolism and membrane fusion.

Membrane fusion underlies many cellular events, including secretion, exocytosis, endocytosis, organelle reconstitution, transport from endoplasmic reticulum to Golgi and nuclear envelope formation. A large number of investigations into membrane fusion indicate various roles for individual members of the phosphoinositide class of membrane lipids. We first review the phosphoinositides as membrane recognition sites and their regulatory functions in membrane fusion. We then consider how modulation of phosphoinositides and their products may affect the structure and dynamics of natural membranes facilitating fusion. These diverse roles underscore the importance of these phospholipids in the fusion of biological membranes.

Lipid quantification and structure determination of nuclear envelope precursor membranes in the sea urchin.

Nuclear envelope assembly is a fundamental cellular process normally taking place once in every cell cycle in eukaryotes. The timing of fusion of nuclear membrane precursors to form the complete double membrane surrounding the chromosomes is tightly controlled, but much remains unclear concerning its regulation. Small amounts of material available and the high background of irrelevant cellular membranes have limited detailed analysis. We have employed several sensitive and high-resolution techniques to analyze the nuclear membrane structure, composition, and dynamics using purified membrane fractions and a cell-free system that results in nuclear envelope formation. We discuss the application of cholesterol and phospholipid colorimetric assays, fluorescent filipin labeling, electrospray ionization tandem mass spectrometry coupled to HPLC (HPLC-ESI/MS/MS), electron microscopy (EM), and solid-state nuclear magnetic resonance (NMR) spectroscopy. Colorimetric assays determine the amounts of inorganic phosphates from phospholipids and cholesterol/ cholesteryl esters present in membrane-containing fractions. Filipin staining of natural membranes allows the localization and relative quantification of cholesterol. HPLC-ESI/MS/MS determines the quantitative composition of membrane phospholipid species from small amounts of membranes. Cryosectioning of cryoprotected sperm cells facilitates EM verification of membrane domains existing in vivo. Deuterium solid-state NMR provides information about membrane rigidity and lipid-phase behavior. The sensitivity, quantification, and structural determinations provided by these techniques should prove useful in studying membrane dynamics in a variety of systems exhibiting membrane fusion.

PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.

Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes.

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.

A sea urchin cell-free system to study male pronuclear assembly and activation.

Typically sperm nuclei are genetically inert and contain extremely compacted chromatin. Following fertilization, the first steps in their conversion to somatic nuclei (male pronuclei) which will support further development involve chromatin decondensation and the formation of a new nuclear envelope. We have studied the reactivation of sea urchin sperm nuclei in a cell-free system derived from homogenates of activated sea urchin egg cytoplasm. The cell-free system has provided several novel insights including requirements for sperm-specific histone phosphorylation on N- and C-terminal extensions and disassembly of the sperm nuclear lamina for decondensation, the utilization of remnant regions of the sperm nuclear envelope to direct polarized binding and fusion of egg membranes to form the new nuclear envelope, and a role for phosphoinositide metabolism in initiation of membrane fusion through binding of a minor membrane fraction enriched in PtdIns (4,5)P2, PLCγ and SFK1 which locally produces a fusigenic lipid, diacylglycerol.

The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

Quantification of exocytosis kinetics by DIC image analysis of cortical lawns.

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62-74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.

A structural role for lipids in organelle shaping.

The importance of proteins in shaping the membranes that define the perimeters of organelles is well documented. By forming cross-links, motors, or scaffolds or by inserting into membranes, proteins can harness energy to deform membranes, particularly when high degrees of curvature are necessitated-as in small membrane vesicles, tubules of the endoplasmic reticulum, the edges of endoplasmic reticulum sheets or Golgi apparatus cisternae, and membrane fusion intermediates (stalks). Here we propose that membrane lipids displaying negative curvature act in concert with membrane proteins to contribute to the alteration and maintenance of bending in biological membranes. We emphasize recent data from studies of sea urchin eggs and embryos and suggest how novel approaches can lead to future directions for investigating the roles of such lipids in vivo.

Dynamics of PLCγ and Src family kinase 1 interactions during nuclear envelope formation revealed by FRET-FLIM.

The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.

Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.

The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.

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 (PLCgamma), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P(2)) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.

Nuclear envelope formation: mind the gaps.

During mitosis in metazoans, the nuclear envelope (NE) breaks down at prophase and reassembles at telophase. The regulation of NE assembly is essential to correct cell functioning. The complex issue of the regulation of NE formation remains to be solved. It is still uncertain that a single mechanism depicts NE formation during mitosis. The aim of this review is to address some of the cytological, biophysical, and molecular aspects of models of NE formation. Our emphasis is on the role of lipids and their modifying enzymes in envelope assembly. We consider how the NE can be used as a model in characterizing membrane dynamics during membrane fusion. Fusion mechanisms that give insight into the formation of the double membrane of the envelope are summarized. We speculate on the possible roles of phosphoinositides in membrane fusion and NE formation.

Nuclear envelope formation in vitro: a sea urchin egg cell-free system.

The formation of the nuclear envelope (NE) typically occurs once during every mitotic cycle in somatic cells, and also around the sperm nucleus following fertilization. Much of our understanding of NE assembly has been derived from systems modeling the latter event in vitro. In these systems, demembranated sperm nuclei are combined with fertilized egg cytoplasmic extracts and an ATP-regenerating system and in a multistep process they form the functional double bilayer of the NE. Using a system that we developed from sea urchin gametes, we have demonstrated that NE assembly is regulated by membrane vesicles in a spatial and temporal fashion, emphasizing the roles of phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), diacylglycerols (DAG), and lipid-modifying enzymes in NE assembly.

Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides.

BACKGROUND: The cytoplasm of eukaryotic cells is a highly dynamic compartment where membranes readily undergo fission and fusion to reorganize the cytoplasmic architecture, and to import, export and transport various cargos within the cell. The double membrane of the nuclear envelope that surrounds the nucleus, segregates the chromosomes from cytoplasm and regulates nucleocytoplasmic transport through pores. Many details of its formation are still unclear. At fertilization the sperm devoid of nuclear envelope pores enters the egg. Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope. Remnants are conserved from annelid to mammalian sperm. METHODOLOGY/PRINCIPAL FINDINGS: Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei. CONCLUSIONS/SIGNIFICANCE: We report nuclear envelope remnants are relatively fluid membranes rich in sterols, devoid of sphingomyelin, and highly enriched in polyphosphoinositides and polyunsaturated phospholipids. The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides. Based on their atypical biophysical characteristics and phospholipid composition, we suggest a possible role for nuclear envelope remnants in membrane fusion leading to nuclear envelope assembly.