Publications by authors named "Volker Kiessling"

44 Publications

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Nat Commun 2021 02 3;12(1):761. Epub 2021 Feb 3.

Department of Chemistry, University of Virginia, Charlottesville, VA, USA.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca 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 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 fusion probability and kinetics 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 model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21090-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859215PMC
February 2021

Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry.

Nat Struct Mol Biol 2021 02 18;28(2):181-189. Epub 2021 Jan 18.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA.

Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-020-00548-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992113PMC
February 2021

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Elife 2021 Jan 7;10. Epub 2021 Jan 7.

Department of Pharmacology, University of Virginia, Charlottesville, United States.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.64787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806264PMC
January 2021

Distinct insulin granule subpopulations implicated in the secretory pathology of diabetes types 1 and 2.

Elife 2020 11 9;9. Epub 2020 Nov 9.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, United States.

Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of β-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.62506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738183PMC
November 2020

HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy.

J Biol Chem 2020 11 11;295(45):15183-15195. Epub 2020 Aug 11.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia, USA; Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA. Electronic address:

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.014466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650252PMC
November 2020

Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos.

J Neurochem 2020 09 9;154(6):598-617. Epub 2020 Mar 9.

Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.14986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426247PMC
September 2020

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties.

Nat Commun 2019 08 29;10(1):3904. Epub 2019 Aug 29.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11873-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715626PMC
August 2019

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

Nat Struct Mol Biol 2018 10 5;25(10):911-917. Epub 2018 Oct 5.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-018-0130-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176490PMC
October 2018

Quaternary structure of the small amino acid transporter OprG from .

J Biol Chem 2018 11 20;293(44):17267-17277. Epub 2018 Sep 20.

From the Department of Molecular Physiology and Biological Physics, Center for Cell and Membrane Physiology and

is an opportunistic human pathogen that causes nosocomial infections. The outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded β-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel-loop region might be crucial for its activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.004461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222097PMC
November 2018

Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life.

Glycobiology 2018 02;28(2):108-121

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 480 Ray C. Hunt Dr., Charlottesville, VA 22908, USA.

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/glycob/cwx096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192386PMC
February 2018

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability.

Biophys J 2017 Nov 13;113(9):1912-1915. Epub 2017 Oct 13.

Department of Molecular Physiology and Biological Physics, Center for Cell and Membrane Physiology at the University of Virginia, Charlottesville, Virginia. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2017.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685784PMC
November 2017

HIV virions sense plasma membrane heterogeneity for cell entry.

Sci Adv 2017 06 28;3(6):e1700338. Epub 2017 Jun 28.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.

It has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of HIV. However, it remains largely unknown why virions prefer cholesterol-rich heterogeneous membranes to uniformly fluid membranes for membrane fusion. Using giant plasma membrane vesicles containing cholesterol-rich ordered and cholesterol-poor fluid lipid domains, we demonstrate that the HIV receptor CD4 is substantially sequestered into ordered domains, whereas the co-receptor CCR5 localizes preferentially at ordered/disordered domain boundaries. We also show that HIV does not fuse from within ordered regions of the plasma membrane but rather at their boundaries. Ordered/disordered lipid domain coexistence is not required for HIV attachment but is a prerequisite for successful fusion. We propose that HIV virions sense and exploit membrane discontinuities to gain entry into cells. This study provides surprising answers to the long-standing question about the roles of cholesterol and ordered lipid domains in cell entry of HIV and perhaps other enveloped viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.1700338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489272PMC
June 2017

Reconstitution of calcium-mediated exocytosis of dense-core vesicles.

Sci Adv 2017 07 19;3(7):e1603208. Epub 2017 Jul 19.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.

Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble -ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.1603208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517108PMC
July 2017

Complexin Binding to Membranes and Acceptor t-SNAREs Explains Its Clamping Effect on Fusion.

Biophys J 2017 Sep 26;113(6):1235-1250. Epub 2017 Apr 26.

Department of Chemistry, University of Virginia, Charlottesville, Virginia; Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, Virginia. Electronic address:

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 and synaptotagmin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2017.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607037PMC
September 2017

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion.

Front Mol Neurosci 2017 16;10:72. Epub 2017 Mar 16.

Center for Membrane and Cell Physiology, University of VirginiaCharlottesville, VA, USA; Department of Molecular Physiology and Biological Physics, University of VirginiaCharlottesville, VA, USA.

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2017.00072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352703PMC
March 2017

A quantized mechanism for activation of pannexin channels.

Nat Commun 2017 01 30;8:14324. Epub 2017 Jan 30.

Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms14324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290276PMC
January 2017

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

Sci Rep 2016 09 8;6:32866. Epub 2016 Sep 8.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep32866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015116PMC
September 2016

The role of cholesterol in membrane fusion.

Chem Phys Lipids 2016 09 11;199:136-143. Epub 2016 May 11.

Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion?-Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chemphyslip.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972649PMC
September 2016

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

Biophys J 2016 May 10;110(10):2147-50. Epub 2016 May 10.

Center for Membrane and Cell Physiology and Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, Virginia. Electronic address:

Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2016.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881159PMC
May 2016

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

Nat Commun 2016 Apr 26;7:11401. Epub 2016 Apr 26.

Center for Membrane and Cell Physiology, Department of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800886, Charlottesville, Virginia 22908, USA.

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 kBT 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853434PMC
April 2016

High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion.

Biophys J 2015 Jul;109(2):319-29

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2015.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621810PMC
July 2015

Supported lipid bilayers as models for studying membrane domains.

Curr Top Membr 2015 11;75:1-23. Epub 2015 Apr 11.

Department of Molecular Physiology and Biological Physics, Center for Membrane Biology, University of Virginia, Charlottesville, VA, USA.

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.ctm.2015.03.001DOI Listing
March 2016

Reconstituting SNARE-mediated membrane fusion at the single liposome level.

Methods Cell Biol 2015 8;128:339-63. Epub 2015 Apr 8.

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.

Successful reconstitutions of SNARE-mediated intracellular membrane fusion have been achieved in bulk fusion assays since 1998 and in single liposome fusion assays since 2004. Especially in neuronal presynaptic SNARE-mediated exocytosis, fusion is controlled by numerous accessory proteins, of which some functions have also been reconstituted in vitro. The development of and results obtained with two fundamentally different single liposome fusion assays, namely liposome-to-supported membrane and liposome-to-liposome, are reviewed. Both assays distinguish between liposome docking and fusion steps of the overall fusion reaction and both assays are capable of resolving hemi-and full-fusion intermediates and end states. They have opened new windows for elucidating the mechanisms of these fundamentally important cellular reactions with unprecedented time and molecular resolution. Although many of the molecular actors in this process have been discovered, we have only scratched the surface of looking at their fascinating plays, interactions, and choreographies that lead to vesicle traffic as well as neurotransmitter and hormone release in the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.mcb.2015.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443872PMC
March 2016

HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains.

Nat Chem Biol 2015 Jun 27;11(6):424-31. Epub 2015 Apr 27.

1] Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA. [2] Center for Membrane Biology, University of Virginia, Charlottesville, Virginia, USA.

Lipid rafts in plasma membranes have emerged as possible platforms for the entry of HIV and other viruses into cells. However, little is known about how lipid phase heterogeneity contributes to viral entry because of the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T cell membranes and found that raft-like liquid-ordered (Lo-phase) lipid domains were necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion were low in homogeneous liquid-disordered (Ld-phase) and Lo-phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo-Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld-phase vesicles proceeded only to hemifusion. Thus, we propose that edges but not areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.1800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433777PMC
June 2015

Variable cooperativity in SNARE-mediated membrane fusion.

Proc Natl Acad Sci U S A 2014 Aug 4;111(33):12037-42. Epub 2014 Aug 4.

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; and

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1407435111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143004PMC
August 2014

The juxtamembrane linker of full-length synaptotagmin 1 controls oligomerization and calcium-dependent membrane binding.

J Biol Chem 2014 Aug 27;289(32):22161-71. Epub 2014 Jun 27.

From the Departments of Chemistry and the Center for Membrane Biology, University of Virginia, Charlottesville, Virginia 22904

Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca(2+)-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.569327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139229PMC
August 2014

Regulation of Rac1 translocation and activation by membrane domains and their boundaries.

J Cell Sci 2014 Jun 2;127(Pt 11):2565-76. Epub 2014 Apr 2.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA

The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo-ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.149088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038948PMC
June 2014

Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate.

Proc Natl Acad Sci U S A 2013 Nov 11;110(48):19384-9. Epub 2013 Nov 11.

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908.

The assembly of the three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure, little is known about the prefusion state of individual membrane-bound SNAREs and how they change conformation from the unzippered prefusion to the zippered postfusion state in a membrane environment. We have solved the solution NMR structure of micelle-bound syntaxin-1A in its prefusion conformation. In addition to the transmembrane helix, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the "0-layer" residue Gln226. This unexpected structural order of the N- and C-terminal halves of the uncomplexed SNARE motif suggests the formation of partially zippered SNARE complex intermediates, with the 0-layer serving as a proofreading site for correct SNARE assembly. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices of syntaxin interact with lipid bilayers and that association with the other target-membrane SNARE SNAP-25 lifts the SNARE motif off the membrane as a critical prerequisite for SNARE complex assembly and membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1314699110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845119PMC
November 2013

Rapid fusion of synaptic vesicles with reconstituted target SNARE membranes.

Biophys J 2013 May;104(9):1950-8

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2013.03.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647153PMC
May 2013

Transbilayer coupling of lipid dynamics.

Authors:
Volker Kiessling

Biophys J 2012 Dec 18;103(12):2409-10. Epub 2012 Dec 18.

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2012.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525845PMC
December 2012
-->