Publications by authors named "Alex J B Kreutzberger"

22 Publications

  • Page 1 of 1

Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease.

J Virol 2021 Aug 18:JVI0097521. Epub 2021 Aug 18.

Department of Cell Biology, Harvard Medical School, 200 Longwood Av, Boston, MA 02115, USA.

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5-10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ∼5-fold or more decrease of half maximal effective concentrations (EC values) suggests a plausible treatment strategy based on the combined use of these inhibitors. Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include Apilimod, an inhibitor of PIKfyve kinase and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or with nafamostat mesylate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00975-21DOI Listing
August 2021

Inherited nuclear pore substructures template post-mitotic pore assembly.

Dev Cell 2021 Jun 14;56(12):1786-1803.e9. Epub 2021 Jun 14.

Department of Cell Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, 200 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. Electronic address:

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2021.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261643PMC
June 2021

Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease.

bioRxiv 2021 Aug 12. Epub 2021 Aug 12.

Department of Cell Biology, Harvard Medical School, 200 Longwood Av, Boston, MA 02115, USA.

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ~5-10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ~5-fold or more decrease of half maximal effective concentrations (EC values) suggests a plausible treatment strategy based on the combined use of these inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.06.01.446623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183009PMC
August 2021

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

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion.

Proc Natl Acad Sci U S A 2020 12 25;117(50):32105-32113. Epub 2020 Nov 25.

Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;

Cholesterol 25-hydroxylase () is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2012197117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749331PMC
December 2020

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

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

Unraveling the mechanisms of calcium-dependent secretion.

J Gen Physiol 2019 04 19;151(4):417-434. Epub 2019 Feb 19.

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

Ca-dependent secretion is a process by which important signaling molecules that are produced within a cell-including proteins and neurotransmitters-are expelled to the extracellular environment. The cellular mechanism that underlies secretion is referred to as exocytosis. Many years of work have revealed that exocytosis in neurons and neuroendocrine cells is tightly coupled to Ca and orchestrated by a series of protein-protein/protein-lipid interactions. Here, we highlight landmark discoveries that have informed our current understanding of the process. We focus principally on reductionist studies performed using powerful model secretory systems and cell-free reconstitution assays. In recent years, molecular cloning and genetics have implicated the involvement of a sizeable number of proteins in exocytosis. We expect reductionist approaches will be central to attempts to resolve their roles. The will continue to be an outlet for much of this work, befitting its tradition of publishing strongly mechanistic, basic research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1085/jgp.201812298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445591PMC
April 2019

Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion.

Science 2019 02;363(6426)

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Room 507 PCTB, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Enzymes that cut proteins inside membranes regulate diverse cellular events, including cell signaling, homeostasis, and host-pathogen interactions. Adaptations that enable catalysis in this exceptional environment are poorly understood. We visualized single molecules of multiple rhomboid intramembrane proteases and unrelated proteins in living cells (human and ) and planar lipid bilayers. Notably, only rhomboid proteins were able to diffuse above the Saffman-Delbrück viscosity limit of the membrane. Hydrophobic mismatch with the irregularly shaped rhomboid fold distorted surrounding lipids and propelled rhomboid diffusion. The rate of substrate processing in living cells scaled with rhomboid diffusivity. Thus, intramembrane proteolysis is naturally diffusion-limited, but cells mitigate this constraint by using the rhomboid fold to overcome the "speed limit" of membrane diffusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aao0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368390PMC
February 2019

Single-Molecule Analyses Reveal Rhomboid Proteins Are Strict and Functional Monomers in the Membrane.

Biophys J 2018 11 2;115(9):1755-1761. Epub 2018 Oct 2.

Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Intramembrane proteases hydrolyze peptide bonds within the membrane as a regulatory paradigm that is conserved across all forms of cellular life. Many of these enzymes are thought to be oligomeric, and that their resulting quaternary interactions form the basis of their regulation. However, technical limitations have precluded directly determining the oligomeric state of intramembrane proteases in any membrane. Using single-molecule photobleaching, we determined the quaternary structure of 10 different rhomboid proteins (the largest superfamily of intramembrane proteases) and six unrelated control proteins in parallel detergent micelle, planar supported lipid bilayer, and whole-cell systems. Bacterial, parasitic, insect, and human rhomboid proteases and inactive rhomboid pseudoproteases all proved to be monomeric in all membrane conditions but dimeric in detergent micelles. These analyses establish that rhomboid proteins are, as a strict family rule, structurally and functionally monomeric by nature and that rhomboid dimers are unphysiological.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2018.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224778PMC
November 2018

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

Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP.

Mol Biol Cell 2018 05 22;29(10):1238-1257. Epub 2018 Mar 22.

Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908.

In pancreatic β-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol's role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter-ference-induced depletion in cultured pancreatic β-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E17-08-0519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935073PMC
May 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

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

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

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

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
-->