Leonid V Chernomordik

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Maryland, United States

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Publications (114)611.71 Total impact

  • Leonid V Chernomordik, Michael M Kozlov
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    ABSTRACT: In Drosophila myoblast fusion, the fusing cell invades another by actin-enriched protrusion. In this issue of Developmental Cell, Kim et al. (2015) examine the myoblast fusion mechanism from the perspective of the "receiving" cell and report that fusion depends on the ability of this cell to stiffen its actomyosin cortex. Copyright © 2015 Elsevier Inc. All rights reserved.
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    ABSTRACT: Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-to-cell fusion in development, tissue homeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. Here we explored fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of lysophosphatidylcholine (LPC) that reversibly blocks membrane merger. 16h later we removed LPC and observed cell fusion events that would normally develop within 16h to develop within 30-90 min. Thus, while osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour, in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus, dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes.
    Biochemical Journal 10/2014; DOI:10.1042/BJ20141233 · 4.78 Impact Factor
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    ABSTRACT: While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities, and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility.
    Human Molecular Genetics 04/2014; DOI:10.1093/hmg/ddu189 · 6.68 Impact Factor
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    ABSTRACT: Membranes of intracellular organelles are characterized by large curvatures with radii of the order of 10-30nm. While, generally, membrane curvature can be a consequence of any asymmetry between the membrane monolayers, generation of large curvatures requires the action of mechanisms based on specialized proteins. Here we discuss the three most relevant classes of such mechanisms with emphasis on the physical requirements for proteins to be effective in generation of membrane curvature. We provide new quantitative estimates of membrane bending by shallow hydrophobic insertions and compare the efficiency of the insertion mechanism with those of the protein scaffolding and crowding mechanisms.
    Current opinion in cell biology 04/2014; 29C:53-60. DOI:10.1016/j.ceb.2014.03.006 · 14.15 Impact Factor
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    ABSTRACT: Membranes of intracellular organelles are characterized by large curvatures with radii of the order of 10–30 nm. While, generally, membrane curvature can be a consequence of any asymmetry between the membrane monolayers, generation of large curvatures requires the action of mechanisms based on specialized proteins. Here we discuss the three most relevant classes of such mechanisms with emphasis on the physical requirements for proteins to be effective in generation of membrane curvature. We provide new quantitative estimates of membrane bending by shallow hydrophobic insertions and compare the efficiency of the insertion mechanism with those of the protein scaffolding and crowding mechanisms.
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    ABSTRACT: Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 μM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 μM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes.
    Biophysical Journal 05/2013; 104(9):1933-9. DOI:10.1016/j.bpj.2013.02.053 · 3.83 Impact Factor
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  • Santosh K. Verma, Eugenia Leikina, Leonid V. Chernomordik
    Biophysical Journal 01/2013; 104(2):91-. DOI:10.1016/j.bpj.2012.11.542 · 3.83 Impact Factor
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    ABSTRACT: Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Here, we accumulated murine myoblasts at the ready-to-fuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. Lifting the block allowed us to explore a largely synchronized fusion. We found that initial merger of two cell membranes detected as lipid mixing involved extracellular annexins A1 and A5 acting in a functionally redundant manner. Subsequent stages of myoblast fusion depended on dynamin activity, phosphatidylinositol(4,5)bisphosphate content, and cell metabolism. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification and in the identification of additional protein players controlling different fusion stages.
    The Journal of Cell Biology 12/2012; DOI:10.1083/jcb.201207012 · 9.69 Impact Factor
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    ABSTRACT: There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infections, yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at sub-neutralizing concentrations. Neutralization activity of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.
    Journal of Virology 10/2012; DOI:10.1128/JVI.02273-12 · 4.65 Impact Factor
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    ABSTRACT: The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.
    The EMBO Journal 12/2011; 31(3):767-79. DOI:10.1038/emboj.2011.439 · 10.75 Impact Factor
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    ABSTRACT: Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P(2), an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.
    Biochemical Journal 09/2011; 440(2):185-93. DOI:10.1042/BJ20111243 · 4.78 Impact Factor
  • Biophysical Journal 02/2011; 100(3). DOI:10.1016/j.bpj.2010.12.1992 · 3.83 Impact Factor
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    ABSTRACT: One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.
    Journal of Biological Chemistry 02/2011; 286(15):13226-34. DOI:10.1074/jbc.M110.181297 · 4.60 Impact Factor
  • Biophysical Journal 02/2011; 100(3). DOI:10.1016/j.bpj.2010.12.1991 · 3.83 Impact Factor
  • Leonid V Chernomordik, Michael M Kozlov
    Current Topics in Membranes 01/2011; 68:xv-xviii. · 1.77 Impact Factor
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    Michael M Kozlov, Harvey T McMahon, Leonid V Chernomordik
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    ABSTRACT: Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cells and release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and in most cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling.
    Trends in Biochemical Sciences 12/2010; 35(12):699-706. DOI:10.1016/j.tibs.2010.06.003 · 13.52 Impact Factor
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    ABSTRACT: Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.
    PLoS Pathogens 10/2010; 6(10):e1001131. DOI:10.1371/journal.ppat.1001131 · 8.06 Impact Factor
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    Sung-Tae Yang, Elena Zaitseva, Leonid V Chernomordik, Kamran Melikov
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    ABSTRACT: Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.
    Biophysical Journal 10/2010; 99(8):2525-33. DOI:10.1016/j.bpj.2010.08.029 · 3.83 Impact Factor
  • Sung-Tae Yang, Leonid Chernomordik, Kamran Melikov
    Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.3692 · 3.83 Impact Factor

Publication Stats

7k Citations
611.71 Total Impact Points


  • 2008–2014
    • Eunice Kennedy Shriver National Institute of Child Health and Human Development
      Maryland, United States
  • 2011
    • Hanbat National University
      • Program in Applied Chemistry and Biotechnology
      Taiden, Daejeon, South Korea
  • 2010
    • University of California, Santa Cruz
      • Department of Molecular Cell & Developmental Biology
      Santa Cruz, CA, United States
  • 1993–2010
    • National Institutes of Health
      • • Section on Membrane Biology
      • • Laboratory of Cellular and Molecular Biology
      베서스다, Maryland, United States
  • 1993–2007
    • National Institute of Child Health and Human Development
      Maryland, United States
  • 2005
    • Université Montpellier 2 Sciences et Techniques
      Montpelhièr, Languedoc-Roussillon, France
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2002
    • Drexel University
      Philadelphia, Pennsylvania, United States
  • 1998–2002
    • Tel Aviv University
      • Department of Physiology and Pharmacology
      Tel Aviv, Tel Aviv, Israel
  • 1997–1999
    • Rush Medical College
      Chicago, Illinois, United States
  • 1987–1997
    • A. N. Frumkin Institute of Physical Chemistry and Electrochemistry
      Moskva, Moscow, Russia
  • 1980–1984
    • Russian Academy of Sciences
      • Institute of Chemistry
      Moskva, Moscow, Russia