[Show abstract][Hide abstract] ABSTRACT: High-throughput techniques are needed to analyze individual virions to understand how viral heterogeneity translates into pathogenesis since in bulk analysis the individual characteristics of virions are lost. Individual Dengue virions (DENV) undergo a maturation that involves a proteolytic cleavage of prM precursor into virion-associated M protein. Here, using a new nanoparticle-based technology, “flow virometry”, we compared the maturation of individual DENV produced by BHK-21 and LoVo cells. The latter lacks the furin-protease that mediates prM cleavage. We found that prM is present on about 50% of DENV particles produced in BHK-21 cells and about 85% of DENV virions produced in LoVo, indicating an increase in the fraction of not fully matured virions. Flow virometry allows us to quantify the number of fully mature particles in DENV preparations and proves to be a useful method for studying heterogeneity of the surface proteins of various viruses.
[Show abstract][Hide abstract] ABSTRACT: Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1-/-). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1-/- myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma.
[Show abstract][Hide abstract] ABSTRACT: Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature peptide entry involves endocytosis. In contrast, at a concentration ≥10μM R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that similar entry pathway is induced at 1-2μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and sub- physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10 to 20μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration. This Ca(2+) signaling correlated with the efficiency of the peptide entry. Preloading cells with the Ca(2+) chelator BAPTA inhibited both Ca(2+) spikes and peptide entry, suggesting that an increase in intracellular Ca(2+) precedes and is required for peptide entry. One of the hallmarks of Ca(2+) signaling is a transient cell surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered novel mechanistic link between calcium signaling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug delivery vehicles.
Copyright 2015 The Author(s).
Preview · Article · Aug 2015 · Biochemical Journal
[Show abstract][Hide abstract] 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.
Full-text · Article · Oct 2014 · Biochemical Journal
[Show abstract][Hide abstract] 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.
Full-text · Article · Apr 2014 · Human Molecular Genetics
[Show abstract][Hide abstract] 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.
Full-text · Article · Apr 2014 · Current opinion in cell biology
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Preview · Article · May 2013 · Biophysical Journal
[Show abstract][Hide abstract] 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.
Full-text · Article · Dec 2012 · The Journal of Cell Biology
[Show abstract][Hide abstract] 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.
Full-text · Article · Oct 2012 · Journal of Virology
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Feb 2011 · Journal of Biological Chemistry