共查询到20条相似文献,搜索用时 15 毫秒
1.
Zhen Zhang 《Biophysical journal》2010,98(11):2524-2534
A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca2+-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca2+-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca2+-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape. 相似文献
2.
Fabrice Dumas Richard D. Byrne Ben Vincent Tina M. C. Hobday Dominic L. Poccia Banafshé Larijani 《PloS one》2010,5(8)
Membrane fusion plays a central role in many cell processes from vesicular
transport to nuclear envelope reconstitution at mitosis but the mechanisms that
underlie fusion of natural membranes are not well understood. Studies with
synthetic membranes and theoretical considerations indicate that accumulation of
lipids characterised by negative curvature such as diacylglycerol (DAG)
facilitate fusion. However, the specific role of lipids in membrane fusion of
natural membranes is not well established. Nuclear envelope (NE) assembly was
used as a model for membrane fusion. A natural membrane population highly
enriched in the enzyme and substrate needed to produce DAG has been isolated and
is required for fusions leading to nuclear envelope formation, although it
contributes only a small amount of the membrane eventually incorporated into the
NE. It was postulated to initiate and regulate membrane fusion. Here we use a
multidisciplinary approach including subcellular membrane purification,
fluorescence spectroscopy and Förster resonance energy transfer
(FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate
that initiation of vesicle fusion arises from two unique sites where these
vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic
reticulum-derived membranes that make up the bulk of the NE to ultimately
enclose the chromatin. We show how initiation of multiple vesicle fusions can be
controlled by localised production of DAG and propagated bidirectionally.
Phospholipase C (PLCγ), GTP hydrolysis and
(phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are
required for the latter process. We discuss the general implications of membrane
fusion regulation and spatial control utilising such a mechanism. 相似文献
3.
Pradip?K. Tarafdar Hirak Chakraborty Michael?J. Bruno Barry?R. Lentz 《Biophysical journal》2015,109(9):1863-1872
Although the importance of a SNARE complex in neurotransmitter release is widely accepted, there exist different views on how the complex promotes fusion. One hypothesis is that the SNARE complex’s ability to bring membranes into contact is sufficient for fusion, another points to possible roles of juxtamembrane regions (JMRs) and transmembrane domains (TMDs) in catalyzing lipid rearrangement, and another notes the complex’s presumed ability to bend membranes near the point of contact. Here, we performed experiments with highly curved vesicles brought into contact using low concentrations of polyethylene glycol (PEG) to investigate the influence of the synaptobrevin (SB) TMD with an attached JMR (SB-JMR-TMD) on the rates of stalk and pore formation during vesicle fusion. SB-JMR-TMD enhanced the rates of stalk and fusion pore (FP) formation in a sharply sigmoidal fashion. We observed an optimal influence at an average of three peptides per vesicle, but only with phosphatidylserine (PS)-containing vesicles. Approximately three SB-JMR-TMDs per vesicle optimally ordered the bilayer interior and excluded water in a similar sigmoidal fashion. The catalytic influences of hexadecane and SB-JMR-TMD on fusion kinetics showed little in common, suggesting different mechanisms. Both kinetic and membrane structure measurements support the hypotheses that SB-JMR-TMD 1) catalyzes initial intermediate formation as a result of its basic JMR disrupting ordered interbilayer water and permitting closer interbilayer approach, and 2) catalyzes pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state. 相似文献
4.
Prabhodh S. Abbineni Joseph S. Briguglio Edwin R. Chapman Ronald W. Holz Daniel Axelrod 《Molecular biology of the cell》2022,33(6)
Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10−10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis. 相似文献
5.
Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins. 相似文献
6.
High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion
Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. 相似文献
7.
Arun Anantharam Bibiana Onoa Robert H. Edwards Ronald W. Holz Daniel Axelrod 《The Journal of cell biology》2010,188(3):415-428
Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin. 相似文献
8.
The freeze-fracture, freeze-etch technique can be employed to reveal new details of the process of fusion of two unit membranes For this study, mucocyst discharge in Tetrahymena pyriformis provides a model system with certain general implications The undischarged mature mucocyst is a saclike, membrane-bound, secretory vesicle containing crystalline material The organelle tip finds its way toward a special site, a rosette of 150 Å diameter particles within the plasma membrane. To match this site, the mucocyst membrane forms an annulus of 110 Å diameter particles, above whose inner edge the rosette particles sit. Discharge of some mucocysts is triggered by fixation. As discharge proceeds, the organelle becomes spherical and its content changes from crystalline to amorphous. The cytoplasm between the two matching membrane sites is squeezed away and the membranes fuse Steps in membrane reorganization can be reconstructed from changes in rosette appearance in the fracture faces. First, a depression in the rosette—the fusion pocket—forms. The rosette particles spread at the lip as the pocket deepens and enlarges from 60 to 200 nm. The annulus particles then become visible at the lip, indicating completed fusion of the A fracture faces of mucocyst and plasma membranes The remaining B faces of the two membranes have opposite polarities When the content of the mucocyst is released, the edges of these faces join so that the unit membrane runs uninterruptedly around the lip and into the pocket. 相似文献
9.
Inbal Abutbul-Ionita Jarungjit Rujiviphat Iftach Nir G. Angus McQuibban Dganit Danino 《The Journal of biological chemistry》2012,287(44):36634-36638
Cellular membrane remodeling events such as mitochondrial dynamics, vesicle budding, and cell division rely on the large GTPases of the dynamin superfamily. Dynamins have long been characterized as fission molecules; however, how they mediate membrane fusion is largely unknown. Here we have characterized by cryo-electron microscopy and in vitro liposome fusion assays how the mitochondrial dynamin Mgm1 may mediate membrane fusion. Using cryo-EM, we first demonstrate that the Mgm1 complex is able to tether opposing membranes to a gap of ∼15 nm, the size of mitochondrial cristae folds. We further show that the Mgm1 oligomer undergoes a dramatic GTP-dependent conformational change suggesting that s-Mgm1 interactions could overcome repelling forces at fusion sites and that ultrastructural changes could promote the fusion of opposing membranes. Together our findings provide mechanistic details of the two known in vivo functions of Mgm1, membrane fusion and cristae maintenance, and more generally shed light onto how dynamins may function as fusion proteins. 相似文献
10.
Churchward MA Rogasevskaia T Brandman DM Khosravani H Nava P Atkinson JK Coorssen JR 《Biophysical journal》2008,94(10):3976-3986
The Ca2+-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, αT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary “spontaneous” lipid rearrangements that must occur during native membrane fusion—Ca2+-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore. 相似文献
11.
Walter Stühmer 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2015,370(1672)
Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å3 for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes. 相似文献
12.
Niharika Mehrotra Justin Nichols Rajesh Ramachandran 《Molecular biology of the cell》2014,25(6):879-890
The self-assembling GTPase dynamin catalyzes endocytic vesicle scission via membrane insertion of its pleckstrin homology (PH) domain. However, the molecular mechanisms underlying PH domain–dependent membrane fission remain obscure. Membrane-curvature–sensing and membrane-curvature–generating properties have been attributed, but it remains to be seen whether the PH domain is involved in either process independent of dynamin self-assembly. Here, using multiple fluorescence spectroscopic and microscopic techniques, we demonstrate that the isolated PH domain does not act to bend membranes but instead senses high membrane curvature through hydrophobic insertion into the membrane bilayer. Furthermore, we use a complementary set of short- and long-distance Förster resonance energy transfer approaches to distinguish PH-domain orientation from proximity at the membrane surface in full-length dynamin. We reveal, in addition to the GTP-sensitive “hydrophobic mode,” the presence of an alternate, GTP-insensitive “electrostatic mode” of PH domain–membrane interactions that retains dynamin on the membrane surface during the GTP hydrolysis cycle. Stabilization of this alternate orientation produces dramatic variations in the morphology of membrane-bound dynamin spirals, indicating that the PH domain regulates membrane fission through the control of dynamin polymer dynamics. 相似文献
13.
Alexander M. Walter Katrin Wiederhold Dieter Bruns Dirk Fasshauer Jakob B. S?rensen 《The Journal of cell biology》2010,188(3):401-413
Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)–dependent exocytosis pathway at an intermediate “cocked” state, from which fusion can be triggered by Ca2+. It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin–SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca2+-triggered C-terminal assembly and membrane fusion. 相似文献
14.
Tabares L Alés E Lindau M Alvarez de Toledo G 《The Journal of biological chemistry》2001,276(43):39974-39979
Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis. 相似文献
15.
Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(4):723-731
BackgroundUnderstanding the effects of graphene-based nanomaterials on lipid membranes is critical to determine their environmental impact and their efficiency in the biomedical context. Graphene has been reported to favourably interact with biological and model lipid membranes.MethodsWe report on a systematic coarse-grained molecular dynamics study of the interaction modes of graphene nanometric flakes with POPC/cholesterol liposome membranes. We have simulated graphene layers with a variety of sizes and oxidation degrees, and we have analyzed the trajectories, the interaction modes, and the energetics of the observed phenomena.ResultsThree interaction modes are reported. Graphene can be transiently adsorbed onto the liposome membrane and/or inserted in its hydrophobic region. Inserted nanosheets prefer a perpendicular orientation, and tilt in order to maximize the contact with phospholipid tails while avoiding the contact with cholesterol molecules. When placed between two liposomes, graphene facilitates their fusion in a single vesicle.ConclusionsGraphene can be temporary adsorbed on the liposome before insertion. Bilayer curvature has an influence on the orientation of inserted graphene particles. Cholesterol molecules are depleted from the surrounding of graphene particles. Graphene layers may catalyse membrane fusion by bypassing the energy barrier required in stalk formation.General significanceNanometric graphene layers can be adsorbed/inserted in lipid-based membranes in different manners and affect the cholesterol distribution in the membrane, implying important consequences on the structure and functionality of biological cell membranes, and on the bioaccumulation of graphene in living organisms. The graphene-mediated mechanism opens new possibilities for vesicle fusion in the experimental context. 相似文献
17.
Role of calcium in triggering the release of transmitters at the neuromuscular junction 总被引:2,自引:0,他引:2
C J Duncan 《Cell calcium》1983,4(3):171-193
Calcium ions have a key role in triggering the release of packaged transmitter at the amphibian neuromuscular junction and of the chromaffin granules at the adrenal medulla. It is suggested that (i) proteins on the vesicle and plasma membranes are of particular importance in promoting membrane fusion and exocytosis (ii) they may be divalent cation-stimulated ATPases, which form the calcium-binding sites or have a specific calcium-binding protein in close molecular apposition (iii) these ATPases in synaptic vesicles and chromaffin granules also generate a protonmotive force which is associated with the uptake of transmitter (iv) the osmotic properties of the vesicle may be important during fission, but it is not suggested that chemiosmotic effects are involved in Ca2+-triggered fusion (v) the action of calcium is markedly co-operative (vi) the adrenal medullary cell and the n.m.j. may differ in the Ca2+-binding site; there is evidence for the involvement of calmodulin in granule-plasmalemma fusion in the chromaffin cells, but not at present (surprisingly) for a role of this Ca2+-binding protein at the n.m.j. (vii) exocytosis requires MgATP (viii) phosphorylation of the ATPase may well be involved; phosphorylation via cAMP does not seem to be involved in fusion in either system (ix) the ATPase may undergo configurational changes during exocytosis and is markedly sensitive to the physical state of its phospholipid environment and to the oxidation of its -SH groups. 相似文献
18.
We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals. 相似文献
19.
Katharina?M. Scherer Jan-Hendrik Spille Hans-Georg Sahl Fabian Grein Ulrich Kubitscheck 《Biophysical journal》2015,108(5):1114-1124
The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane. 相似文献
20.
Kunding AH Mortensen MW Christensen SM Bhatia VK Makarov I Metzler R Stamou D 《Biophysical journal》2011,(11):2693-2703
The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle–vesicle pairs of different diameter (30–200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30–60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2–10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes. 相似文献