RESUMO
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.
Assuntos
Bicamadas Lipídicas/química , Modelos Biológicos , Membrana Nuclear/química , Fosfolipídeos/química , Animais , Forma do Núcleo Celular , Equinodermos , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Humanos , Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Membrana Nuclear/metabolismo , Óvulo/química , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismoRESUMO
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.
Assuntos
Membrana Celular/fisiologia , Fosfatidilinositóis/fisiologia , Animais , Membrana Celular/ultraestrutura , Diglicerídeos/fisiologia , Humanos , Fusão de Membrana , Lipídeos de Membrana/química , Membrana Nuclear/químicaRESUMO
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.
Assuntos
Membrana Nuclear/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/metabolismo , Espermatozoides/citologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Lytechinus , Masculino , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/química , Fosforilação , Espermatozoides/metabolismo , Strongylocentrotus purpuratus , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Membrana Nuclear/química , Fosfatidilinositóis/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Sistema Livre de Células , Diglicerídeos/fisiologia , Feminino , Masculino , Fusão de Membrana/fisiologia , Lipídeos de Membrana/análise , Membrana Nuclear/fisiologia , Óvulo/fisiologia , Paracentrotus , Fosfatidilinositóis/análise , Fosfoinositídeo Fosfolipase C , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , EspermatozoidesRESUMO
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.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Fusão de Membrana , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Membrana Nuclear/metabolismo , Mapeamento de Interação de Proteínas , Animais , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Masculino , Microscopia de Fluorescência/métodos , Óvulo/metabolismo , Ligação Proteica , Ouriços-do-MarRESUMO
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.
Assuntos
Lytechinus/enzimologia , Membrana Nuclear/enzimologia , Fosfolipase C gama/metabolismo , Quinases da Família src/metabolismo , Animais , Ativação Enzimática/fisiologia , Masculino , Fosforilação/fisiologiaRESUMO
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.
Assuntos
Diglicerídeos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membrana Nuclear/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diacilglicerol Quinase/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Mamíferos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Microinjeções , Mitose/efeitos dos fármacos , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/farmacologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/ultraestrutura , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/administração & dosagem , Monoéster Fosfórico Hidrolases/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Ouriços-do-Mar/citologia , Ouriços-do-Mar/efeitos dos fármacos , Ouriços-do-Mar/embriologia , Receptor de Lamina BRESUMO
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.
Assuntos
Fusão de Membrana , Fosfatidilinositóis/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Diglicerídeos/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Transferência Ressonante de Energia de Fluorescência , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Lytechinus/citologia , Masculino , Fusão de Membrana/efeitos dos fármacos , Microscopia de Fluorescência , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Pirrolidinonas/farmacologiaRESUMO
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.
Assuntos
Fusão de Membrana/fisiologia , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Modelos Químicos , Membrana Nuclear/ultraestruturaRESUMO
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.
Assuntos
Sistema Livre de Células/metabolismo , Membrana Nuclear/metabolismo , Óvulo/metabolismo , Ouriços-do-Mar/metabolismo , Animais , Colesterol/metabolismo , Feminino , Espectroscopia de Ressonância Magnética , Masculino , Membrana Nuclear/fisiologia , Óvulo/fisiologia , Fosfatidilcolinas/metabolismo , Ouriços-do-Mar/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
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.
Assuntos
Membrana Nuclear/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Esteróis/metabolismo , Animais , Sistema Livre de Células , Feminino , Fertilização/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana/antagonistas & inibidores , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Substrato Quinase C Rico em Alanina Miristoilada , Ouriços-do-Mar , Espermatozoides/metabolismo , Esteróis/químicaRESUMO
Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.