He, Bin et al. (2009) International Worm Meeting "The Distinct Roles of Self-assembly and GTP Hydrolysis in Regulating Dynamin's Association with Target Membranes and the Removal of Apoptotic Cells in C. elegans."

Leng, Xiaohong, Margolis, Moran, Zhou, Zheng, He, Bin, Etzion, Yael, Danino, Dganit, Yu, Xiaomeng

[International Worm Meeting, 2009]

Dynamin is a multi-domain GTPase that forms polymers along lipid surfaces and acts in multiple vesicular trafficking events. In addition to its well-known membrane fission function in endocytosis (1), C. elegans dynamin (DYN-1) is also known to promote regulate synaptic vesicle transport and cytokinesis (2,3). Recent studies found that DYN-1 is essential for the removal of apoptotic cells (3,4,5). In C. elegans, apoptotic cells are swiftly engulfed by surrounded tissues and degraded inside phagosomes. The swift engulfment and degradation of apoptotic cells rely on the efficient transport and fusion of intracellular vesicles to phagocytic cups and maturing phagosomes. DYN-1 is localized to the surfaces of extending pseudopods and nascent phagosomes in response to the signaling from phagocytic receptor CED-1, and promotes vesicle delivery to these regions. To understand the distinct roles of DYN-1''s self-assembly and GTP hydrolysis for the removal of apoptotic cells, we analyzed two classes of missense mutations of DYN-1. In vitro, Class I mutant DYN-1 fails to bind and/or hydrolyze GTP. In contrast, a Class II mutation specifically affects DYN-1''s self-assembly along lipid surfaces. Our genetic and cell biological studies in C. elegans generated multiple lines of evidence to indicate that DYN-1 undergoes self-assembly in vivo. Furthermore, we have found that self-assembly is essential for DYN-1''s association to the surfaces of pseudopods and phagosomes and the apical surface of intestinal cells, whereas the dissociation of DYN-1 from its target membranes is GTP-dependent. We propose that the self-assembly and GTP hydrolysis activities of DYN-1 play important and distinct roles in regulating DYN-1''s dynamic association to target membranes, an event known to be essential for DYN-1''s activity in apoptotic-cell removal and likely to be so for other cellular functions of DYN-1. References: 1.Grant, B. and D. Hirsh, Mol Biol Cell, 1999.10: 4311-26. 2.Clark, S.G., et al., Proc Natl Acad Sci USA, 1997. 94: 10438-43. 3.Thompson, H.M., et al., Current Biology, 2008.12: 2111-2117. 4.Yu, X., et al., Dev Cell, 2006. 10: 743-57. 5.Yu, X., et al., PLoS Biol, 2008. 6: e61. 6.Kinchen, J.M., et al., Nat Cell Biol, 2008. 10: 556-66.

Avinoam, Ori et al. (2011) International Worm Meeting "Conserved Eukaryotic Fusogens can Fuse Viral Envelopes to Cells."

Abutbul, Inbal, Sapir, Amir, Avinoam, Ori, Valansi, Clari, Danino, Dganit, White, Judith, Grunewald, Kay, Podbilewicz, Benjamin, Fridman, Karen, Zeev-Ben-Mordehai, Tzviya, Maurer, Ulrike

[International Worm Meeting, 2011]

Cell-cell fusion is a fundamental process that occurs during development of most eukaryotes. In C. elegans cell-cell fusion is necessary for fertilization and organogenesis of the hypodermis, pharynx, vulva, uterus, glands, and male tail. Moreover, cell fusion has been implicated in myogenesis, bone formation, placentation, neuronal function, stem-cell reprogramming and carcinogenesis. Despite the relative abundance of cell fusion events during development, the molecular mechanism of cell-fusion is unknown. The first family of proteins that mediate membrane fusion (i.e fusogens) was identified in C. elegans. Its first members, AFF-1 and EFF-1 (CeFFs) are essential for cell fusion and their expression is sufficient to fuse cells that normally do not fuse both in vivo and in cell-culture. Furthermore, CeFFs are required on both membranes in order for them to fuse. We have shown that FFs are functionally conserved within and beyond the nematode phylum and that divergent members from the nematode T. spiralis and the chordate B. floridae can mediate fusion of mammalian cells. We use CeFFs as prototypic fusogens to understand the principles of cell-cell fusion machineries. In order to study the molecular mechanism of FF proteins we generated pseudotyped Vesicular Stomatitis viruses that express CeFFs on the viral membrane, in place of the endogenous fusogenic glycoprotein. We found that CeFFs are able to rescue the infectious activity of the virus and mediate viral infection via virus-cell fusion. In addition, the viral mode of fusion and infection became dependent on the expression of FFs on the virus as well as on the target cell membrane. We investigated the structure of AFF-1 at nano-resolution using electron microscopy and tomography and found that it formed distinct supercomplexes resembling pentameric and hexameric flowers on pseudoviruses. Thus, the evolution, structure and function of FFs begin to unravel as they converge with glycoproteins of enveloped viruses as minimal fusogenic machineries capable of fusing membranes independently of other cellular co-factors.