Volker Kiessling | University of Virginia (original) (raw)
Papers by Volker Kiessling
Biophysical Journal, Nov 1, 2017
Little attention has been given to how the asymmetric lipid distribution of the plasma membrane m... more Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.
Biophysical Journal, May 1, 2013
Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstituti... more Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of~20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism.
Biophysical Journal, Jul 1, 2015
Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by ... more 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 SNAREmediated 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 a-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.
Proceedings of the National Academy of Sciences of the United States of America, Aug 4, 2014
Biophysical Journal, 2010
We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typical... more We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes could induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures (Biophys.J. 91:3313-26 [2006]). The lipid requirements for this transbilayer coupling in asymmetric cholesterol-rich two-phase lipid bilayers were further investigated and found to be roughly correlated with their chain-melting phase transition temperatures (Biochemistry 47: 2190-8 [2008]). Bilayers containing brain PC (bPC), brain sphingomyelin (bSM) and cholesterol in the outer leaflet and bPC, bPE, POPS and Chol in the inner leaflet, form stable asymmetric twophase bilayers that are thought to mimic mammalian plasma membranes. In the current work we have studied the calcium-dependent binding and lipid targeting of C2 domains of the presynaptic fusion calcium sensor protein synaptotagmin on asymmetric two-phase bilayers. C2A domains favor disordered over ordered inner leaflet lipid domains. Domain preference does not depend on the PS
Biophysical Journal, Sep 1, 2017
Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and e... more Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and enhances evoked release. Complexin binds to the fully assembled four-helical neuronal SNARE core complex as revealed in competing molecular models derived from x-ray crystallography. Presently, it is unclear how complexin binding to the postfusion complex accounts for its effects upon spontaneous and evoked release in vivo. Using a combination of spectroscopic and imaging methods, we characterize in molecular detail how complexin binds to the 1:1 plasma membrane t-SNARE complex of syntaxin-1a and SNAP-25 while simultaneously binding the lipid bilayer at both its N-and C-terminal ends. These interactions are cooperative, and binding to the prefusion acceptor t-SNARE complex is stronger than to the postfusion core complex. This complexin interaction reduces the affinity of synaptobrevin-2 for the 1:1 complex, thereby retarding SNARE assembly and vesicle docking in vitro. The results provide the basis for molecular models that account for the observed clamping effect of complexin beginning with the acceptor t-SNARE complex and the subsequent activation of the clamped complex by Ca 2þ and synaptotagmin.
Biophysical Journal, 2015
Biophysical Journal, 2015
Fusion between the viral envelope and target cell membrane is a crucial step for infection of inf... more Fusion between the viral envelope and target cell membrane is a crucial step for infection of influenza and other enveloped viruses. The influenza envelope protein hemagglutinin provides a necessary driving force for fusion. However, both viral and target membrane composition can also have a significant impact on infectivity. Here, we examine the effect of sterol composition on viral membrane fusion kinetics.
Biophysical Journal, 2010
In this study, effects of cholesterol on viscoelastic properties of the plasma membrane are inves... more In this study, effects of cholesterol on viscoelastic properties of the plasma membrane are investigated. We use optical tweezers to extract nanotubes (tethers) from the plasma membrane of human embryonic kidney (HEK) cells. We obtain time-resolved tether force measurements under cholesterol depleted and cholesterol enriched conditions. Using these data, elastic and viscous parameters of the plasma membrane are quantified and correlated to the changes in the membrane cholesterol level.
Biophysical Journal, Feb 1, 2011
Journal of Biological Chemistry, Apr 1, 2010
We suggest that subscribers photocopy these corrections and insert the photocopies in the origina... more We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
Journal of Structural Biology, Oct 1, 2009
Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been pr... more Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre-and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.
bioRxiv (Cold Spring Harbor Laboratory), Aug 26, 2022
The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their ... more The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A compelling body of work also suggests Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. Using fluorescence lifetime imaging with an order sensitive dye, FLIPPER-TR, and by measuring pseudoviral membrane thickness via cryo electron microscopy (cryoEM), Serinc5 was found to increase membrane heterogeneity, skewing the distribution towards a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a TIRF-based single particle fusion assay, we found that incorporation of exogenous phosphatidylethanolamine (PE) into the viral membrane rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane-lipid chain order, rigidity, line tension, and lateral pressure-which decrease accessibility of fusion intermediates and disfavor
Biophysical Journal, Nov 1, 2010
The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles w... more The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in~20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and~20 ms, respectively.
Nature Communications, Apr 26, 2016
Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. ... more Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of k B T to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.
Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca 2+ sensor for syn... more Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca 2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca 2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.
Biophysical Journal, 2015
Journal of Physical Chemistry B, Dec 15, 2010
We utilized epifluorescence microscopy to investigate the morphological changes in labelled lipid... more We utilized epifluorescence microscopy to investigate the morphological changes in labelled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from POPG, POPC, POPE and mixtures thereof as well as from E. coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents like calcium by using the Langmuir Blodgett/Langmuir Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups, and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.
Biophysical Journal, Nov 1, 2006
Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholi... more Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l o) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l o phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipidanchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain .90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l o phase domains. We show here that l o phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l o and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.
Biochimica Et Biophysica Acta - Biomembranes, 2009
Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are o... more Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.
Biophysical Journal, Nov 1, 2017
Little attention has been given to how the asymmetric lipid distribution of the plasma membrane m... more Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.
Biophysical Journal, May 1, 2013
Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstituti... more Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of~20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism.
Biophysical Journal, Jul 1, 2015
Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by ... more 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 SNAREmediated 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 a-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.
Proceedings of the National Academy of Sciences of the United States of America, Aug 4, 2014
Biophysical Journal, 2010
We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typical... more We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes could induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures (Biophys.J. 91:3313-26 [2006]). The lipid requirements for this transbilayer coupling in asymmetric cholesterol-rich two-phase lipid bilayers were further investigated and found to be roughly correlated with their chain-melting phase transition temperatures (Biochemistry 47: 2190-8 [2008]). Bilayers containing brain PC (bPC), brain sphingomyelin (bSM) and cholesterol in the outer leaflet and bPC, bPE, POPS and Chol in the inner leaflet, form stable asymmetric twophase bilayers that are thought to mimic mammalian plasma membranes. In the current work we have studied the calcium-dependent binding and lipid targeting of C2 domains of the presynaptic fusion calcium sensor protein synaptotagmin on asymmetric two-phase bilayers. C2A domains favor disordered over ordered inner leaflet lipid domains. Domain preference does not depend on the PS
Biophysical Journal, Sep 1, 2017
Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and e... more Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and enhances evoked release. Complexin binds to the fully assembled four-helical neuronal SNARE core complex as revealed in competing molecular models derived from x-ray crystallography. Presently, it is unclear how complexin binding to the postfusion complex accounts for its effects upon spontaneous and evoked release in vivo. Using a combination of spectroscopic and imaging methods, we characterize in molecular detail how complexin binds to the 1:1 plasma membrane t-SNARE complex of syntaxin-1a and SNAP-25 while simultaneously binding the lipid bilayer at both its N-and C-terminal ends. These interactions are cooperative, and binding to the prefusion acceptor t-SNARE complex is stronger than to the postfusion core complex. This complexin interaction reduces the affinity of synaptobrevin-2 for the 1:1 complex, thereby retarding SNARE assembly and vesicle docking in vitro. The results provide the basis for molecular models that account for the observed clamping effect of complexin beginning with the acceptor t-SNARE complex and the subsequent activation of the clamped complex by Ca 2þ and synaptotagmin.
Biophysical Journal, 2015
Biophysical Journal, 2015
Fusion between the viral envelope and target cell membrane is a crucial step for infection of inf... more Fusion between the viral envelope and target cell membrane is a crucial step for infection of influenza and other enveloped viruses. The influenza envelope protein hemagglutinin provides a necessary driving force for fusion. However, both viral and target membrane composition can also have a significant impact on infectivity. Here, we examine the effect of sterol composition on viral membrane fusion kinetics.
Biophysical Journal, 2010
In this study, effects of cholesterol on viscoelastic properties of the plasma membrane are inves... more In this study, effects of cholesterol on viscoelastic properties of the plasma membrane are investigated. We use optical tweezers to extract nanotubes (tethers) from the plasma membrane of human embryonic kidney (HEK) cells. We obtain time-resolved tether force measurements under cholesterol depleted and cholesterol enriched conditions. Using these data, elastic and viscous parameters of the plasma membrane are quantified and correlated to the changes in the membrane cholesterol level.
Biophysical Journal, Feb 1, 2011
Journal of Biological Chemistry, Apr 1, 2010
We suggest that subscribers photocopy these corrections and insert the photocopies in the origina... more We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
Journal of Structural Biology, Oct 1, 2009
Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been pr... more Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre-and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.
bioRxiv (Cold Spring Harbor Laboratory), Aug 26, 2022
The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their ... more The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A compelling body of work also suggests Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. Using fluorescence lifetime imaging with an order sensitive dye, FLIPPER-TR, and by measuring pseudoviral membrane thickness via cryo electron microscopy (cryoEM), Serinc5 was found to increase membrane heterogeneity, skewing the distribution towards a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a TIRF-based single particle fusion assay, we found that incorporation of exogenous phosphatidylethanolamine (PE) into the viral membrane rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane-lipid chain order, rigidity, line tension, and lateral pressure-which decrease accessibility of fusion intermediates and disfavor
Biophysical Journal, Nov 1, 2010
The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles w... more The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in~20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and~20 ms, respectively.
Nature Communications, Apr 26, 2016
Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. ... more Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of k B T to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.
Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca 2+ sensor for syn... more Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca 2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca 2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.
Biophysical Journal, 2015
Journal of Physical Chemistry B, Dec 15, 2010
We utilized epifluorescence microscopy to investigate the morphological changes in labelled lipid... more We utilized epifluorescence microscopy to investigate the morphological changes in labelled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from POPG, POPC, POPE and mixtures thereof as well as from E. coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents like calcium by using the Langmuir Blodgett/Langmuir Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups, and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.
Biophysical Journal, Nov 1, 2006
Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholi... more Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l o) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l o phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipidanchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain .90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l o phase domains. We show here that l o phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l o and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.
Biochimica Et Biophysica Acta - Biomembranes, 2009
Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are o... more Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.