Membrane fusion is a common stage of enveloped virus infections, exocytosis, and cell fusion in development. Recently we have extended our work on the fusion mechanisms from a relatively well characterized fusion reaction mediated by influenza virus hemagglutinin (HA) to less explored developmental cell fusion. In our work on HA-mediated fusion we studied fusion between protein-free liposomes and HA-expressing cells and viral particles. We identified distinct fusion intermediates preceding lipid mixing and dissimilar in their stability and propensity to complete fusion. The properties of these intermediates depend on liposome composition and, apparently, on surface density of HAs. To explore the pathway of developmental fusion, we focused on a transmembrane protein EFF-1 of C. elegans shown previously to be essential for fusion. We show that expression of EFF-1 drives hemifusion and fusion. Interestingly, syncytiogenesis requires EFF-1 in both fusing cells. Thus, while the first example of a developmental fusogen activity, EFF-1-mediated fusion, shares hemifusion steps with pathways of viral and intracellular fusion, it differs from these reactions in the homotypic organization of the fusion machinery. The analysis of the mechanisms by which fusion proteins as diverse as HA and EFF-1 drive fusion will hopefully bring new insights into mechanisms of ubiquitous fusion reaction.? ? 1. The only established protein fusogens up to date were the proteins that mediate viral and intracellular fusion. The candidate fusogens for the diverse and fundamental cell-cell fusion processes were characterized mainly at the genetic level and in the in vivo context. ? In C. elegans hermaphrodites, 300 out of a total of 959 somatic nuclei reside in syncytial cells that originate through programmed and stereotyped cell-cell fusions in living embryos and larvae. The transmembrane protein EFF-1 was identified using genetic screens as a C. elegans candidate developmental fusogen, that is required and sufficient to mediate fusion in the context of living animal. However, it has remained possible that EFF1 mediates fusion by regulating an un-identified fusogen present at the surface of C. elegans cells. Potential membrane fusion proteins must meet several gold standards to be defined as fusogens: first, genetics and in vitro biochemical assays must demonstrate the protein is necessary for membrane fusion events; second, cell biological approaches must show the protein is expressed and active at the fusion site; third, expression of the protein in heterologous cells must be sufficient to induce cell-cell fusion. While there are many candidate fusogens, only intracellular SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), membrane glycoproteins from different enveloped viruses and type I proteins from non-enveloped reoviruses have passed these three tests. ? ? To find out whether EFF1 is a bone fide fusogen or a regulator of fusion driven by another yet un-identified EFF-1 fusogen, we expressed three EFF-1 isoforms in heterologous Sf9 insect cells chosen for these studies because they normally do not form syncytia and they have been used for cell fusion studies induced by viral fusogens. We have shown that the C. elegans transmembrane proteins EFF-1, expressed at the surface of insect cells, initiate cell fusion and produce multinucleate syncytia and, thus, established EFF-1 as the first developmental fusogen. We found that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing and identified as a key intermediate in viral and intracellular fusion. ? ? Importantly, syncytiogenesis requires EFF-1 to be expressed in both fusing cells. To test whether this mechanism applies also in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion needs EFF-1 in both cells. Thus, while the first example of a developmental fusogen activity, EFF-1-mediated fusion, shares hemifusion steps with viral and intracellular fusion, it differs from these reactions in the homotypic organization of the fusion machinery. While viral fusogens are located at one of the fusing membranes, EFF-1 is required in both fusion partners. EFF-1 is also distinct from SNARE-dependent intracellular fusion, where two fusing membranes carry different but complementary sets of protein fusogens. Homotypic machinery may provide a better control of a developmental reaction than required for heterotypic virus-host cell fusion during infection. For example, homotypic fusion may prevent fusion with cells at the edges of a multinucleated cell, allowing a better control of syncytia size and shape. This mechanistic aspect of cell fusion during syncytia formation is critical for the normal development of many organs in nematodes and in the formation of diverse tissues in mammalian organs as diverse as muscles, bones, placenta and eye. We anticipate that EFF-1 expression in other heterologous systems may be used to fuse cells with potential applications for gene therapy and manipulation of stem cell fates.? ? 2. Influenza HA mediated fusion is one of the best-characterized membrane fusion reactions. To invade the cell, influenza virus binds to sialic acid receptors at the cell surface and delivers viral RNA into the cytosol by fusing the viral envelope and the membrane of acidified endosome. The structure of the initial neutral-pH conformation of HA and the restructuring of HA at acidic pH have been characterized in detail through different experimental approaches. The pathway of HA-mediated fusion has been studied in different experimental systems. However, the mechanisms that couple low-pH-dependent restructuring of HA and membrane rearrangement remain elusive. To explore early intermediates in HA-mediated membrane fusion and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine with different hydrocarbon chains. We found that the composition of the target membrane affects the stability of fusion intermediates at a stage prior to lipid mixing. A more fusogenic target membrane effectively blocks non-productive release of the conformational energy of HA, and even for the same liposome composition, Based on identified phenotypes of partial lipid mixing and partial inactivation, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. The two types of distinct intermediates observed in HA-cell/liposome fusion, and the effects of the target membrane on their properties, might be explained by the heterogeneity of the cell membrane and, in particular, by heterogeneous distribution of HA molecules at the cell surface. The relationship between fusion and inactivation patterns and local HA density was confirmed by the lack of either partial lipid mixing or partial inactivation phenotypes in liposome fusion to viral particles with a homogeneously high density of HA. The diversity of the fusion intermediates emerging from this study emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.

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U.S. National Inst/Child Hlth/Human Dev
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