BBMB Research Seminars
April 17, 2008
Leonid Chernomordik
National Institutes of Health
1414 Molecular Biology Buidling
4:10 p.m.
Membrane Fusion: Diverse and Conserved
Intracellular protein trafficking, cell invasion by enveloped viruses, and syncytia formation in development, all these processes share a common stage of membrane fusion. To identify the conserved and divergent mechanisms by which protein fusogens merge membrane bilayers, we explore fusion mediated by well characterized viral fusogens and by poorly characterized fusogens involved in developmental cell fusion and fusion stage of post-mitotic remodeling of intracellular membranes. To explore developmental fusion, we expressed candidate C. elegans fusogens EFF-1 and AFF-1 in heterologous cells. EFF-1 mediates fusion between most epithelial cells in C. elegans; and AFF-1 is responsible for fusion between the gonadal anchor cell and uterine cells. The fusogenic activity of EFF-1 and AFF-1 expressed at the surface of transfected cells indicates that both of these proteins are the actual fusogens rather than regulators of cell fusion reactions. While reconstituted developmental fusion shares the conserved fusion-through-hemifusion pathway with viral fusion, it radically differs from viral fusion in the basic arrangement of the fusion machinery. While in viral fusion only one of the two membranes carries protein fusogens, in EFF-1 mediated fusion both fusing membranes have to express EFF-1. This homotypic arrangement might be important for establishing defined boundaries between syncytia and surrounding mononucleated cells.
Most of the research on fusion mechanisms is focused on the fusogen-driven pathway that culminates in the fusion pore opening. However, in the case of cell-cell fusion, opening of small fusion pore(s) is only the beginning of the process that results in the loss of membrane junction between the cells and joins the cells into syncytium. We explored the later stages of cell fusion for cells expressing baculovirus fusion protein gp64 or influenza virus hemagglutinin. Confocal microscopy analysis of the morphology of the contact zones between gp64-expressing cells shows that at the micron scale fusion progresses through opening, expansion and merger of multiple pores or by an expansion of a single pore. Fusion pores in membrane junction colocalize with openings in the actin cortex. In contrast to early fusion stages controlled by protein fusogens, irreversible fusion pore expansion in syncytia requires cell metabolism. Effects of actin-modifying agents suggest that fusion pore(s) expansion in cell-cell fusion is not driven by actin cytoskeleton but requires local F-actin cortex dissociation. The specific mechanisms by which cell machinery expands early fusion intermediates generated by local interactions between protein fusogens and membranes will be discussed.
Post-mitotic reassembly of ER and nuclear envelope (NE) represents a still poorly understood example of intracellular fusion. We explored whether the minimal machinery responsible for ER and NE assembly requires transmembrane proteins distributed between both fusing membranes as expected within the classical design represented by ubiquitous SNARE machinery. Using the reconstitution system based on interphase extracts of Xenopus eggs, we found cytosolic proteins to be sufficient to mediate fusion between membrane vesicles (MVs) and liposomes with the lipid mixing rates similar to those observed in fusion between intact membrane vesicles. Membranes lacking transmembrane proteins fuse with MVs into ER structures and functional nuclear envelope in cytosol-, GTP- and active nuclear transport- dependent manner. These findings argue against the hypothesis that ER and NE assembly is driven by SNARE machinery and suggest that a massive and relatively promiscuous intracellular membrane remodeling in post-mitotic cells is supported by the peripheral membrane proteins provided by interphase cytosol.
Leonid V. Chernomordik, Ph.D, D.Sc.
I received my Ph.D. in Electrochemistry from the Frumkin Institute of Electrochemistry, Russian Academy of Sciences in 1979 and my Doctor of Sciences in Biophysics degree (the highest academic status degree in Russia, roughly equivalent to full professorship) from the Moscow State University in 1991. In 1991, after 15 years of research in the Frumkin Institute, I joined the National Institutes of Health, where I lead the Section on Membrane Biology in the Laboratory of Cellular and Molecular Biophysics, NICHD. I am interested in the biophysics and biochemistry of biological membranes and, in particular, in the mechanisms of membrane fusion in development, viral infections, exocytosis and most-mitotic remodeling of intracellular membranes. For diverse systems from model bilayers to several biological reactions we have dissected fusion into distinct mechanistic steps and established a conserved fusion stage – hemifusion- that involves merger of only contacting membrane leaflets while distal leaflets remain separate. Based on identification of hemifusion- and lipidic pore- fusion intermediates, the effects of membrane lipids, and theoretical analysis, we developed a 'stalk-pore' model of membrane fusion that describes fusion as a sequence of bending deformations of different leaflets of membranes to first connect the contacting membrane leaflets into a stalk-like hemifusion structure and, then, to break the distal leaflets to form a fusion pore. Based on the recent work of many groups, it appears that this stalk-pore pathway is shared by disparate fusion reactions mediated by different proteins. In our work on the mechanisms by which fusogenic proteins drive membrane fusion, we have proposed and substantiated a mechanism by which lateral interactions of fusion proteins can bend the membranes and, as a result, drive fusion pore expansion. It is my hope that identification of the molecular mechanisms of membrane remodeling will help in developing new ways of controlling cell fusion and intracellular fusion throughout normal development of many human organs and tissues and in many genetic and infectious diseases.
