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protein-driven expansion of fusion pores in viral

fusion pores and developmental cell fusion

Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology Eugenia Leikina, DVM, Senior Research Assistant Helene Delanoe, PhD, Postdoctoral Fellow Kamran Melikov, PhD, Postdoctoral Fellow Aditya Mittal, PhD, Postdoctoral Fellow Corinne Ramos, PhD, Postdoctoral Fellow Dawson Brown, BS, Postbaccalaureate Fellow Fay Trimor, BS, Postbaccalaureate Fellow Elena Zaitseva, PhD, Contractor Benjamin Podbilewicz, PhD, Guest Researcher^a

Protein-mediated fusion of two membranes into one is involved in all the following processes: fertilization and syncytia formation in bones, muscles, and placenta; exocytosis; and enveloped virus infections. Most of what we know about fusion derives from studying how well-characterized viral fusion proteins (e.g., influenza virus hemagglutinin or HA) open fusion pores connecting aqueous compartments. Our recent work has focused on the later stages of an expansion of these pores leading, in the case of cell fusion, to the complete loss of the membranes that separate two cells and, in the case of viruses, to delivery of the relatively bulky viral genome into the host cell. Our studies on HA-mediated fusion indicate that low pH–activated HAs not only initiate fusion by catalyzing early fusion intermediates but also provide the driving force for the entire fusion reaction by bending the contacting membranes out of their initial shape. This latter function, driving the expansion of a fusion pore, is common to many fusion proteins and represents the most demanding part of their job. Our work is aimed at exploring the mechanisms by which activated fusion proteins control progression of diverse fusion reactions toward their completion. Involvement of fusion proteins located outside the contact zone in fusion pore expansion Leikina, Mittal, Cho,^b Melikov, Chernomordik; in collaboration with Kozlov To formulate a possible mechanism by which proteins generate the fusion force, we compared fusion with another type of membrane remodeling, namely, fission of one membrane into two. Based on the literature, it appeared that proteins driving membrane merger in fusion do so in radically different ways from those driving fission. Fission is known and likely to be mediated by proteins that are not located between merging membranes. In contrast, fusion has been generally believed to result from the local action of only those fusion proteins that are located in the contact zone between the membranes and interact directly with the target membrane. However, the role of the fusion proteins outside the contact zone has never been tested. We assessed the role of these “outsider� proteins in HA-mediated fusion between red blood cells and either HA-expressing cells or viral particles. HA cells and bound RBCs establish extended contact zones (CZs) with areas on the order of tens of square microns that are characterized by a relatively constant intermembrane distance of about 13 nm that is close to the height of the HA ectodomain. Within this system, readily distinguishable pools of insider and outsider HAs facilitate characterization of their relative fusogenic activity. To inhibit or enhance selectively the actions of HA outsiders, antibodies that bind to HA and proteases that cleave it were conjugated to polystyrene microspheres (20 nm, 100 nm, and 2 micron diameter) too large to enter the CZ. We also allowed HA outsiders to interact with additional red blood cells. We found the HA outsiders to be necessary and sufficient for fusion. Interferance with the activity of the HA outsiders inhibited fusion. Selective conversion of HA outsiders alone into a fusion-competent conformation was sufficient to achieve fusion. The mechanisms remain to be understood by which fusion proteins that, at the time of the activation, are located outside the CZ influence fusion. The functional role of HA outsiders in our experiments might indicate that, as in the case of vacuolar fusion, most HA-mediated fusion events develop along the circumference rather than in the central region of the CZ. If so, HA outsiders located near the periphery of the CZ can be important for fusion. Alternatively, HA outsiders might not need to be in the immediate proximity of the fusion site and might drive opening and expansion of a fusion pore by generating tension in membrane bilayer. Our discovery of a functional role of fusion proteins located outside the CZ goes against the accepted view that fusion is a highly localized phenomenon driven by only a few fusion proteins in the CZ that directly interact with the target membrane. At the same time, our results rationalize the interesting reports that the entry of many viruses and exocytotic fusion are inhibited by macromolecules that are added to predocked membranes and are, apparently, too large to enter the tight contact zone rapidly. Our finding also strengthens an attractive hypothesis, namely, that the oppositely directed processes of membrane fusion and fission work according to a common principle: the proteins drive membrane remodeling from outside the zone of the actual membrane rearrangement. Chernomordik LV, Kozlov MM. Protein-lipid interplay in fusion and fission of biological membranes. Annu Rev Biochem 2003;72:175-207. Leikina E, Mittal A, Cho MS, Melikov K, Kozlov MM, Chernomordik LV. Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion. J Biol Chem 2004;279:26526-26532. Mechanistic dissection of tissue-specific eff-1–mediated cell fusion in C. elegans Mittal, Podbilewicz, Chernomordik; in collaboration with Hall, Gattegno, Kolotuev, Nguyen, Shemer, Suissa, Valansi To study the mechanisms of cell fusion during development of multicellular organisms, we have focused on the relatively well-characterized fusion in C. elegans. Normal development of C. elegans involves numerous cell fusions, with nearly a third of all the nuclei in the adult located in syncytia. Genetic screens for mutations that inhibit cell fusion within epithelia of C. elegans led to identification of the gene eff-1 (epithelial fusion failure) that encodes type-I membrane proteins EFF-1, which are expressed as cells become fusion-competent (Mohler et al., Dev Cell 2002;2:355-362). EFF-1, which was earlier found to be required for cell fusion, is now demonstrated to be sufficient. To dissect the pathway of cell fusion during embryonic development of C. elegans, we developed a new system that simultaneously records, measures, and analyzes individually fusing epidermal cells in live embryos. In contrast to studies on simpler fusion systems that investigate maximum pore sizes of a few nanometers (microfusion), we measured the kinetics of large expanding gaps of the order of hundreds of nanometers/microns (macrofusion) resulting from single cell-cell fusions critical for animal development. We have found that, at these scales, each fusion event follows sigmoidal kinetics in wild-type and idf-1 mutant embryos that have Irregular Dorsal Fusion. From the sigmoids, we can define lag and macrofusion times as the kinetic parameters for each pair of fusing cells. We found that incubations at 9ºC block microfusion but not embryonic elongation, and idf-1 mutations either block early cell fusion steps or slow macrofusion rates (Gattegno et al., submitted). Dissection of cell fusion in a living animal allows us to address the intriguing questions of when, where, and at what rate cells fuse within tissues. We show that each cell pair within the skin of the C. elegans embryo decides to fuse, or not to fuse, with characteristic kinetic parameters. The functional dissection of eff-1 activity reveals that it acts both in the initiation and expansion of membrane fusion. The emerging research strategy of combining genetic, ultrastructural, and kinetic analyses will, it is hoped, be applicable to different examples of developmental fusion. Shemer G, Suissa M, Kolotuev I, Nguyen KCQ, Hall DH, Podbilewicz B. EFF-1 is sufficient to initiate and execute tissue-specific cell fusion in C. elegans. Curr Biol 2004;14:1587-1591. ^aAssociate Professor, on sabbatical from Technion-Israel Institute of Technology, Haifa, Israel ^bMyoung-Soon Cho, biologist, now at Laboratory of Cell Biology, NHLBI, Bethesda, MD COLLABORATORS Tamar Gattegno, MSc, Technion-Israel Institute of Technology, Haifa, Israel David H. Hall, PhD, Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY Irina Kolotuev, MSc, Technion-Israel Institute of Technology, Haifa, Israel Michael Kozlov, PhD, Associate Professor, Tel Aviv University, Israel Ken C.Q. Nguyen, MSc, Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY Gidi Shemer, PhD, Technion-Israel Institute of Technology, Haifa, Israel Meital Suissa, MSc, Technion-Israel Institute of Technology, Haifa, Israel Clari Valansi, MSc, Technion-Israel Institute of Technology, Haifa, Israel For further information, contact lchern@helix.nih.gov