Showing papers on "Porosome published in 2006"
TL;DR: It is concluded that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.
Abstract: During exocytosis a four-helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C-terminal end affected fusion triggering in vivo and led to two-step unfolding of the SNARE complex in vitro, indicating that the C-terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C-terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N-terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N- to C-terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.
287 citations
TL;DR: Present knowledge of fusion machines and fusion pores studied in vitro, in neurons, and in neuroendocrine cells are summarized and synthesized into some specific and detailed hypotheses for exocytosis.
Abstract: Exocytosis is initiated within a highly localized region of contact between two biological membranes. Small areas of these membranes draw close, molecules on the two surfaces interact, and structural transformations take place. Membrane fusion requires the action of proteins specialized for this task, and these proteins act as a fusion machine. At a critical point in this process, a fusion pore forms within the membrane contact site and then expands as the spherical vesicle merges with the flat target membrane. Hence, the operation of a fusion machine must be realized through the formation and expansion of a fusion pore. Delineating the relation between the fusion machine and the fusion pore thus emerges as a central goal in elucidating the mechanisms of membrane fusion. We summarize present knowledge of fusion machines and fusion pores studied in vitro, in neurons, and in neuroendocrine cells, and synthesize this knowledge into some specific and detailed hypotheses for exocytosis.
184 citations
TL;DR: It is concluded that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore.
139 citations
TL;DR: It is reported that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body and suggested that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.
Abstract: Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.
107 citations
TL;DR: It is proposed that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.
Abstract: Regulated exocytosis is thought to occur either by "full fusion," where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by "kiss-and-run," where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed "kiss-and-coat," which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.
82 citations
TL;DR: The molecular mechanism of SNARE‐induced membrane fusion was discovered and it was demonstrated that when t‐SNARE‐associated lipid membrane is exposed to v‐SNare‐associated vesicles in the presence of Ca2+, the SNARE proteins interact in a circular array to form conducting channels, thus establishing continuity between the opposing bilayers.
Abstract: Membrane fusion is a sine qua non process for cell physiology. It is critical for membrane biogenesis, intracellular traffic, and cell secretion. Although investigated for over a century, only in the last 15 years, the molecular machinery and mechanism of membrane fusion has been deciphered. The membrane fusion event elicits essentially three actors on stage: anionic phospholipids - phosphatidylinositols, phosphatidyl serines, specific membrane proteins, and the calcium ions, all participating in a well orchestrated symphony. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. Target membrane proteins, SNAP-25 and syntaxin (t- SNARE) and secretory vesicle-associated membrane protein (v-SNARE) or VAMPwere discovered in the 1990's and suggested to be the minimal fusion machinery. Subsequently, the molecular mechanism of SNARE-induced membrane fusion was discovered. It was demonstrated that when t-SNARE-associated lipid membrane is exposed to v-SNARE-associated vesicles in the presence of Ca(2+), the SNARE proteins interact in a circular array to form conducting channels, thus establishing continuity between the opposing bilayers. Further it was proved that SNAREs bring opposing bilayers close to within a distance of 2-3 Angstroms, allowing Ca(2+) to bridge them. The bridging of bilayers by Ca(2+) then leads to the expulsion of water between the bilayers at the contact site, allowing lipid mixing and membrane fusion. Calcium bridging of opposing bilayers leads to the release of water, both from the water shell of hydrated Ca(2+) ions, as well as the displacement of loosely coordinated water at the phosphate head groups in the lipid membrane. These discoveries provided for the first time, the molecular mechanism of SNARE-induced membrane fusion in cells. Some of the seminal discoveries are briefly discussed in this minireview.
66 citations
TL;DR: This study demonstrates that, irrespective of their arrangement, both forms of the SNARE complex can be disassembled in the presence of NSF-ATP.
Abstract: Full-length v-SNARE protein reconstituted in lipid vesicles, when exposed to t-SNARE-reconstituted lipid membrane, results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores and the establishment of continuity between the opposing bilayers. In contrast, when v-SNARE protein alone (without liposomes) is exposed to t-SNARE-reconstituted lipid membrane, they also self-assemble to form t-/v-SNARE complexes, although such complexes fail to possess the characteristic ring pattern, nor do they help in the establishment of continuity between the opposing bilayers. Hence, t-SNAREs and v-SNARE need to be membrane-associated to interact in a circular array to form conducting pores in the presence of calcium. This study demonstrates that, irrespective of their arrangement, both forms of the SNARE complex can be disassembled in the presence of NSF-ATP.
57 citations
TL;DR: At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane.
Abstract: Exocytosis is a highly ordered vesicle trafficking pathway that targets proteins to the plasma membrane for membrane addition or secretion. Research over the years has discovered many proteins that participate at various stages in the mammalian exocytotic pathway. At the early stage of exocytosis, co-atomer proteins and their respective adaptors and GTPases have been shown to play a role in the sorting and incorporation of proteins into secretory vesicles. At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane. There are multiple events that may occur between the budding of secretory vesicles from the Golgi and the fusion of these vesicles at the plasma membrane. The most obvious and best-known event is the transport of secretory vesicles from Golgi to the vicinity of the plasma membrane via microtubules and their associated motors. At the vicinity of the plasma membrane, however, it is not clear how vesicles finally dock and fuse with the plasma membrane. Identification of proteins involved in these events should provide important insights into the mechanisms of this little known stage of the exocytotic pathway. Currently, a protein complex, known as the sec6/8 or the exocyst complex, has been implicated to play a role at this late stage of exocytosis.
47 citations
TL;DR: In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion.
39 citations
TL;DR: This work studies the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles, and encounters a novel spontaneous vesicle enlargement mechanism called the engulfing lobe, which may explain how lipids added to a vesicular solution are quickly incorporated into the inner monolayer.
Abstract: Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.
34 citations
TL;DR: It is demonstrated that Syntaxin-1 co-localizes with cholesterol in solubilized synaptosomal membrane preparations, and is required for its stability in neuronal porosomes.
TL;DR: The discovery of the porosome in live cells, its morphology and dynamics at nanometer resolution and in real time, its isolation, its composition, and its structural and functional reconstitution in lipid membrane, are complete.
Abstract: The release of neurotransmitters at the nerve terminal for neurotransmission, release of insulin from β-cells of the endocrine pancreas for regulating blood glucose levels, the release of growth hormone from GH cells of the pituitary gland to regulate body growth, or the expulsion of zymogen from exocrine pancreas to digest food, are only a few examples of key physiological processes made possible by cell secretion. It comes as no surprise that defects in cell secretion are the cause for numerous diseases, and have been under intense investigation for over half century. Only in the last decade, the molecular machinery and mechanism of cell secretion has become clear. Cell secretion involves the docking and transient fusion of membrane-bound secretory vesicles at the base of plasma membrane structures called porosomes, and the regulated expulsion of intravesicular contents to the outside, by vesicle swelling. The discovery of the porosome in live cells, its morphology and dynamics at nanometer resolution and in real time, its isolation, its composition, and its structural and functional reconstitution in lipid membrane, are complete. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and the regulated expulsion of intravesicular contents during cell secretion, are also resolved. In this minireview, the monumental discovery of the porosome, a new cellular structure at the cell plasma membrane, is briefly discussed.
TL;DR: A new cellular structure ‘the porosome’ in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur, and a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states is provided.
Abstract: Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure ‘the porosome’ in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.
TL;DR: The porosme has been isolated, its composition determmined and it has been both structurally and functionally reconsituted n artificial lipid membrance, ultimately solving one of the most difficut, significant, and fundamental cellular process‐ cell seretion.
Abstract: Cell secretio has come age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be ‘fibrillar regions’ at teh cell plasama membrance, and electrophysological studies suggesting the presence of ‘fusion pores’ at the cell plasma membrane where secretion occurs. Finally, the fusion pore or ‘porosome’ has been discovered, and its morpholgy and dynamics determined at nm resolution and in real time in live secretory cells. The porosome has been isolated, its omposition determined at nm resolution and in real time in live secretory cells. The porosme has been isolated, its composition determmined and it has been jkboth structurally and functionally reconsituted n artificial lipid membrance. The discoversy of the porosome as the univeral secretory machinery in cell and the discovery of the molecular mechaninsm of vesicular content expulsion during cell secretin have fially enabled a clear understanding of this important cellular process. This review outlines the fascinating and exciting joumey leding to the dicovery of the porosme, ultimately solving one of the most difficut, significant, and fundamental cellular process- cell seretion.
TL;DR: The porosome is discovered as the universal secretory machinery, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined.
TL;DR: It is demonstrated for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required.
TL;DR: The aim of this chapter is to review the results obtained on pituitary cells, specialized to release a number of important hormones and to highlight that there are multiple mechanisms of exocytosis present in the same cell, and to address elementary properties of exoctosis, consisting of the interaction between a single vesicle and the plasma membrane.
Abstract: The elaborate intracellular membrane system of eukaryotic cells participates in vesicle trafficking and represents an important basis exploited in cell-to-cell signaling Communication between cells involves the release of neurotransmitters, hormones and other chemical messengers that are stored in secretory vesicles and granules A key event in the release of these primary messengers is exocytosis, consisting of fusion between the vesicle and the plasma membrane This leads to the formation of a fusion pore through which a diffusional continuum between the vesicle lumen and the extracellular space is established In the past, in vitro studies of biological membrane fusion considered this an almost impossible process, because large pressures had to be delivered to counteract the electrostatic repulsion owing to negatively charged membrane surfaces It is only a decade or so that the omnipresent fusion between biological membranes started to be understood in greater detail Since the SNARE hypothesis was proposed about a decade ago, several proteins have been identified to play a role in exocytosis, and attempts to define minimal molecular machinery for regulated exocytosis have been considered However, several studies provided evidence for multiple modes of exocytosis, and that exocytosis may not necessarily lead to the release of vesicle cargo The aim of this chapter is to review the results obtained on pituitary cells, specialized to release a number of important hormones and to highlight that there are multiple mechanisms of exocytosis present in the same cell Moreover, the goal is to address elementary properties of exocytosis, consisting of the interaction between a single vesicle and the plasma membrane These studies indicate that the long-thought concept of membrane fusion as an irreversible process will have to be changed Here we discuss an unusually regular reversible opening of the fusion pore termed “the pulsing pore”
01 Jan 2006
TL;DR: The current study was undertaken to further determine the structure of the porosome in resting pancreatic acinar cells and when co-isolated with zymogen granules, the secretory vesicles in exocrine pancreas in the rat, and demonstrates the presence of porosomes at the apical plasma membrane where secretion occurs.
Abstract: In the last decade, the discovery of a new cellular structure, the 'porosome' or fusion pore, and the discovery of SNAREinduced membrane fusion, and regulated expulsion of secretory products via secretory vesicle swelling, has finally provided us with an understanding of cell secretion at the molecular level. The current study was undertaken to further determine the structure of the porosome in resting pancreatic acinar cells and when co-isolated with zymogen granules, the secretory vesicles in exocrine pancreas in the rat. In agreement with earlier findings, our studies demonstrate the presence of porosomes at the apical plasma membrane where secretion occurs. Zymogen granules isolated from briefly stimulated pancreas using carbamylcholine, demonstrates as previously reported, the co-isolation of porosomes.
TL;DR: The discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane are discussed.
Abstract: Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a buildup of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes and vesicle swelling have also been resolved. These findings reveal the molecular machinery and mechanism of cell secretion. In this chapter, the discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane are discussed.
TL;DR: Tight control of the flux of membrane components through vesicle budding and fusion is essential to cellular organization, giving rise to subcellular compartments with distinct functions.
Abstract: Tight control of the flux of membrane components through vesicle budding and fusion is essential to cellular organization, giving rise to subcellular compartments with distinct functions. Cargo is generally selected for inclusion into vesicles by specific sorting signals, which are recognized by adaptor proteins, that link to protein components of vesicle coats. The membrane deformation required for vesicle formation is promoted by the coat complex in conjunction with accessory proteins, which may partially insert into the bilayer membrane. Specific targeting of vesicles is initially mediated through “tethering” molecules and then cemented through interactions between SNARE proteins, which also catalyze fusion. The essential mechanisms are conserved throughout the cell and from yeast to man.
Keywords:
Clathrin;
Coated Vesicle;
Endocytosis;
Membrane Fusion;
Rab Protein;
Secretory Pathway;
SNARE Protein