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1.
SNARE‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle
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Massimo D'Agostino Herre Jelger Risselada Laura J Endter Véronique Comte‐Miserez Andreas Mayer 《The EMBO journal》2018,37(19)
Constitutive membrane fusion within eukaryotic cells is thought to be controlled at its initial steps, membrane tethering and SNARE complex assembly, and to rapidly proceed from there to full fusion. Although theory predicts that fusion pore expansion faces a major energy barrier and might hence be a rate‐limiting and regulated step, corresponding states with non‐expanding pores are difficult to assay and have remained elusive. Here, we show that vacuoles in living yeast are connected by a metastable, non‐expanding, nanoscopic fusion pore. This is their default state, from which full fusion is regulated. Molecular dynamics simulations suggest that SNAREs and the SM protein‐containing HOPS complex stabilize this pore against re‐closure. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long‐lived, physiologically relevant and regulated state of SNARE‐dependent membrane fusion. 相似文献
2.
Plonsky I Kingsley DH Rashtian A Blank PS Zimmerberg J 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(6):377-386
Background information. Protein‐mediated merger of biological membranes, membrane fusion, is an important process. To investigate the role of fusogenic proteins in the initial size and dynamics of the fusion pore (a narrow aqueous pathway, which widens to finalize membrane fusion), two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the HA (haemagglutinin) of influenza X31. Results. The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human RBCs (red blood cells) upon acidification of the medium. A high‐time‐resolution electrophysiological study of fusion pore conductance revealed fundamental differences in (i) the initial pore conductance; pores created by HA were smaller than those created by GP64; (ii) the ability of pores to flicker; only HA‐mediated pores flickered; and (iii) the time required for pore formation; HA‐mediated pores took much longer to form after acidification. Conclusion. HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein. 相似文献
3.
Flaviviruses comprise a number of important human pathogens including yellow fever, dengue, West Nile, Japanese encephalitis
and tick-borne encephalitis viruses. They are small enveloped viruses that enter cells by receptor-mediated endocytosis and
release their nucleocapsid into the cytoplasm by fusing their membrane with the endosomal membrane. The fusion event is triggered
by the acidic pH in the endosome and is mediated by the major envelope protein E. Based on the atomic structures of the pre-
and post-fusion conformations of E, a fusion model has been proposed that includes several steps leading from the metastable
assembly of E at the virion surface to membrane merger and fusion pore formation trough conversion of E into a stable trimeric
post-fusion conformation. Using recombinant subviral particles of tick-borne encephalitis virus as a model, we have defined
individual steps of the molecular processes underlying the flavivirus fusion mechanisms. This includes the identification
of a conserved histidine as being part of the pH sensor in the fusion protein that responds to the acidic pH and thus initiates
the structural transitions driving fusion. 相似文献
4.
Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems. 相似文献
5.
Rabies virus-induced membrane fusion pathway 总被引:1,自引:0,他引:1
Gaudin Y 《The Journal of cell biology》2000,150(3):601-612
Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers. 相似文献
6.
David J. Schibli 《Molecular membrane biology》2013,30(6):361-371
Recent crystal structures of Flavivirus and Alphavirus fusion proteins (class II) confirm two major principles of protein machineries that mediate the merger of two opposing lipid bilayers. First, the fusion protein can bridge both membranes tethered by two membrane anchors. Second, refolding or domain rearrangement steps lead to the positioning of both anchors into close proximity at the same end of an elongated structure. Although these two steps are in principle sufficient to pull two opposing membranes together and initiate membrane fusion, accumulating evidence suggests that the process requires the concerted action of a number of fusion proteins at and outside the contact sites. This review will focus on the structures of viral class I and class II fusion proteins and their similarities in facilitating membrane fusion. 相似文献
7.
Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition
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SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets. 相似文献
8.
The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 micro s. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties. 相似文献
9.
膜的融合是一个基本的生命过程,在生物的生长发育中有着重要作用。通过融合,两套独立的双层脂分子合二为一,完成一定的生物功能。膜融合分子机制的关键在于其主要成分:融合蛋白。Ⅰ、Ⅱ类病毒融合蛋白形成“发夹”,胞内囊泡与目标膜各提供的融合蛋白形成“类亮氨酸拉链”,这些结构将独立的膜拉近,继而促使膜合为一体。细胞与细胞间融合蛋白的作用机制目前还未明确,在各种膜融合中,脂双层的变化可能是类似的,但介导融合的分子机制应该是不同的。目前,对于膜融合很多方面的理解还停留在假说阶段。理解了膜融合的过程和分子机制不仅将极大地促进生物学的发展,更重要是将为相关的疾病治疗打下坚实的基础。 相似文献
10.
The protein coat in membrane fusion: lessons from fission 总被引:2,自引:0,他引:2
Multiple cell biological processes involve two opposite rearrangements of membrane configuration, referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force which drives sequential stages of the fusion reaction from early local intermediates to an expanding fusion pore remains unknown. Fusion proceeds via stages, which are analogous but in the opposite direction to that of membrane budding-off and fission driven by protein coats. On the basis of this analogy, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. To relieve internal stresses, the fusion protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that 'bystander' proteins (i.e. fusion proteins outside the contact zone) generate the driving force for fusion offers a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions. 相似文献
11.
Cell-cell fusion is a crucial and highly regulated event in the genesis of both form and function of many tissues. One particular type of cell fusion, myoblast fusion, is a key cellular process that shapes the formation and repair of muscle. Despite its importance for human health, the mechanisms underlying this process are still not well understood. The purpose of this review is to highlight the recent literature pertaining to myoblast fusion and to focus on a comparison of these studies across several model systems, particularly the fly, zebrafish and mouse. Advances in technical analysis and imaging have allowed identification of new fusion genes and propelled further characterization of previously identified genes in each of these systems. Among the cellular steps identified as critical for myoblast fusion are migration, recognition, adhesion, membrane alignment and membrane pore formation and resolution. Importantly, striking new evidence indicates that orthologous genes govern several of these steps across these species. Taken together, comparisons across three model systems are illuminating a once elusive process, providing exciting new insights and a useful framework of genes and mechanisms. 相似文献
12.
《Molecular membrane biology》2013,30(3):97-112
This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of x-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, β-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk’ intermediates. In addition, the importance of the formation of specific, ‘higher-order’ fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides. 相似文献
13.
Virus membrane fusion 总被引:3,自引:0,他引:3
Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations. 相似文献
14.
《Cell calcium》2017
Regulated exocytosis can be split into a sequence of steps ending with the formation and the dilation of a fusion pore, a neck-like connection between the vesicle and the plasma membrane. Each of these steps is precisely controlled to achieve the optimal spatial and temporal profile of the release of signalling molecules. At the level of the fusion pore, tuning of the exocytosis can be achieved by preventing its formation, by stabilizing the unproductive narrow fusion pore, by altering the speed of fusion pore expansion and by completely closing the fusion pore. The molecular structure and dynamics of fusion pores have become a major focus of cell research, especially as a promising target for therapeutic strategies. Electrophysiological, optical and electrochemical methods have been used extensively to illuminate how cells regulate secretion at the level of a single fusion pore. Here, we describe recent advances in the structure and mechanisms of the initial fusion pore formation and the progress in therapeutic strategies with the focus on exocytosis. 相似文献
15.
Alenka Gu?ek Jernej Jorga?evski Urszula Górska Bo?tjan Rituper Marko Kreft Robert Zorec 《Channels (Austin, Tex.)》2015,9(2):96-101
In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition. 相似文献
16.
The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel-like, narrow structures after formation and proceed through semi-stable states ('fusion pore flickering'), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow-secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long-lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant. 相似文献
17.
Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing. 相似文献
18.
《Channels (Austin, Tex.)》2013,7(2):96-101
In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition. 相似文献
19.
The mechanism of sperm-egg fusion in mammals is a research area that has greatly benefited from the use of gene deletion technology. Because fertilization is internal in mammals and the gametes (particularly the eggs) are sparse in number, in vitro studies have considerable limitations. Using gene deletions, a few cell surface proteins in both gametes have been identified as essential for gamete fusion. Ongoing studies are directed at analysis of the function of these proteins and the search for additional proteins that may be involved in this process. So far, no mammalian proteins have been found that also function in sperm-egg fusion of non-mammalian species or in other types of cell-cell fusion. 相似文献
20.
Comprehensive kinetic analysis of influenza hemagglutinin-mediated membrane fusion: role of sialate binding
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The data of Danieli et al. (J. Cell Biol. 133:559-569, 1996) and Blumenthal et al. (J. Cell Biol. 135:63-71, 1996) for fusion between hemagglutinin (HA)-expressing cells and fluorescently labeled erythrocytes has been analyzed using a recently published comprehensive mass action kinetic model for HA-mediated fusion. This model includes the measurable steps in the fusion process, i.e., first pore formation, lipid mixing, and content mixing of aqueous fluorescent markers. It contains two core parameters of the fusion site architecture. The first is the minimum number of aggregated HAs needed to sustain subsequent fusion intermediates. The second is the minimal number of those HAs within the fusogenic aggregate that must undergo a slow "essential" conformational change needed to initiate bilayer destabilization. Because the kinetic model has several parameters, each data set was exhaustively fitted to obtain all best fits. Although each of the data sets required particular parameter ranges for best fits, a consensus subset of these parameter ranges could fit all of the data. Thus, this comprehensive model subsumes the available mass action kinetic data for the fusion of HA-expressing cells with erythrocytes, despite the differences in assays and experimental design, which necessitated transforming fluorescence dequenching intensities to equivalent cumulative waiting time distributions. We find that HAs bound to sialates on glycophorin can participate in fusion as members of the fusogenic aggregate, but they cannot undergo the essential conformational change that initiates bilayer destabilization, thus solving a long-standing debate. Also, the similarity in rate constants for lipid mixing and content mixing found here for HA-mediated fusion and by Lee and Lentz (Proc. Natl. Acad. Sci. U.S.A. 95:9274-9279, 1998) for PEG-induced fusion of phosphatidylcholine liposomes supports the idea that subsequent to stable fusion pore formation, the evolution of fusion intermediates is determined more by the lipids than by the proteins. 相似文献