MEMBRANE FUSION MEDIATED BY VIRAL AND DEVELOPMENTAL PROTEIN FUSOGENS
, Head, Section on Membrane Biology
Evgenia Leikina, DVM, Senior Research Assistant
Kamran Melikov, PhD, Staff Scientist
Elena Zaitseva, PhD, Research Fellow
Elvira Rafikova, PhD, Visiting Fellow
Jean-Philippe Richard, PhD, Visiting Fellow
Sung-Tae Yang, PhD, Visiting Fellow
Andrew Chen, BS, Postbaccalaureate Fellow
Anna Gabrielian, Special Volunteer
Membrane trafficking, viral infections, and embryonic development involve membrane fusion. While some viral fusogens are well characterized, most fusogens remain unknown. Recently, we explored fusogens that mediate cell-cell fusion in C. elegans development. Even though most of the fusion events involve fusogen EFF-1, we identified an additional and structurally similar fusogen, AFF-1, that is responsible for the fusion of uterine cells to the gonadal anchor cell, establishing a uterine-vulval tube. AFF-1 is required and sufficient for this fusion in vivo and, when expressed in exogenous cells, fuses them. EFF-1 and AFF-1 are the founding members of a family of nematode fusogens.
In a cell-free system, we also explored post-mitotic nuclear envelope reassembly. Liposomes pre-incubated with frog egg cytosolic extract bound to chromatin and fused at the liposome’s surface in a GTP-dependent manner, suggesting that vesicle targeting to the chromatin and fusion initiation in nuclear assembly do not require transmembrane proteins.
Diverse fusions converge to a pathway that includes the conserved early intermediate hemifusion stalk. Our most recent analysis suggests that stalk formation involves point-like protrusions. This pre-stalk intermediate has energy that is lower than that of stalk and therefore does not limit fusion. The point-like protrusion completes the fusion-through-hemifusion model of membrane merger.
Identification of a protein that fuses anchor cell and uterine cells in C. elegans development
Cell fusion is fundamental for reproduction and organogenesis. Most cell fusion events in C. elegans are mediated by the EFF-1 fusogen that we explored in our earlier work. However, fusion between the anchor cell and the utse (uterus seam cell) syncytium that establishes a continuous uterine-vulval tube, a passage for eggs to exit the vulva, proceeds normally in eff-1 mutants. By isolating mutants in which the anchor cell fails to fuse, we identified aff-1. Ectopic expression of AFF-1 results in the fusion of cells that normally do not fuse in C. elegans. The necessity of AFF-1 for specific fusion events, combined with AFF-1’s ability to promote ectopic cell fusions, suggests that this protein acts directly in the cell fusion process. Our findings indicate that EFF-1–expressing cells do not fuse with AFF-1–expressing cells. To extend our findings further, we expressed the AFF-1 protein in heterologous cultures of Sf9 insect cells that do not usually undergo cell fusion. Western blot analysis detected a single band corresponding to AFF-1 protein. Surface biotinylation and immunofluorescence revealed that the protein is distributed both in intracellular compartments and at the surface of Sf9-transfected cells. Using confocal and fluorescence microscopy, we demonstrated that transfection with aff-1 generated multinucleate cells containing two to six nuclei. We detected 20 percent multinucleation in cells transfected with aff-1 (Sf9-AFF-1 cells) compared with only 3 percent in cells transfected with an empty vector. AFF-1–induced multinucleation results from cell-cell fusion rather than from a failure of cell division as evidenced by the experiments with an inhibitor of cell division. The inhibitor blocked cell division at the transition between G1 and S phases but did not affect multinucleation supporting a mechanism of multinucleation independent from failure in cytokinesis in the presence of karyokinesis. Comparison between the levels of expression and fusogenic activities of AFF-1 and EFF-1 revealed that, at similar surface densities, AFF-1 is a much more potent fusogen than EFF-1. The ability of AFF-1 to fuse heterologous cells confirmed that the protein is an actual fusogen rather than a regulator of fusion reaction.
AFF-1 and EFF-1 differ in their expression patterns but demonstrate a striking conservation in the position and number of all 16 cysteines in their ectodomains. Despite clear homologues in other nematodes, AFF-1 and EFF-1 exhibit only minor similarity to proteins from other vertebrates and invertebrates. In summary, EFF-1 and AFF-1 are the founding members of a family of fusogens in C. elegans and probably in other nematodes. The discovery of a new developmental fusogen in C. elegans implies that one general-purpose fusogen such as EFF-1 is not sufficient to account for all somatic cell fusions. AFF-1 is indeed a specialized fusogen required for particular fusion events that involve small membrane domains and limited timing of action; the events are tightly controlled by transcriptional and post-translational mechanisms. The regulated expression of distinct fusogens might establish the formation of developmental barriers between adjacent syncytia that may represent a general characteristic of developmental fusion.
Gattegno T, Mittal A, Valansi C, Nguyen KC, Hall DH, Chernomordik LV, Podbilewicz B. Genetic control of fusion pore expansion in the epidermis of Caenorhabditis elegans. Mol Biol Cell 2007;18:1153-66.
Podbilewicz B, Leikina E, Sapir A, Valansi C, Suissa M, Shemer G, Chernomordik LV. The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo. Dev Cell 2006;11:471-81.
Sapir A, Choi J, Leikina E, Avinoam O, Valansi C, Chernomordik LV, Newman AP, Podbilewicz B. AFF-1, a FOS-1-regulated fusogen, mediates fusion of the anchor cell in C. elegans. Dev Cell 2007;12:683-98.
Transmembrane proteins are not required for early stages of nuclear envelope assembly
The nuclear envelope (NE) of metazoa consists of an outer membrane, which is continuous with the endoplasmic reticulum, and an inner membrane, which is supported by the lamina, a network of intermediate filaments. Reformation of the NE around the segregated chromosomes at the completion of mitosis involves many steps, including binding of membrane vesicles (MVs) to decondensed chromatin and GTP-dependent fusion of the vesicles at the surface of chromatin. These steps have all been studied in the Xenopus cell-free system, whereas protein fusogens that initiate membrane fusion at the early stage of tightly controlled NE assembly have yet to be identified. As all well-studied protein fusogens are anchored in the membranes by one or more transmembrane domains (TMDs), we questioned whether transmembrane proteins are required for fusion at the early stages of NE assembly.
More specifically, to explore whether transmembrane proteins are required for MV binding to the surface of the chromatin and for fusion itself, we replaced MV in the Xenopus cell-free system with fluorescently labeled dioleoylphosphatidylcholine liposomes (LSs). We incubated LSs with cytosol and then isolated LSs with bound cytosolic proteins (Cyt-LSs) by flotation through a sucrose density gradient. Analysis of the binding between Cyt-LSs and chromatin revealed that cytosolic proteins on LSs mediated the interactions between Cyt-LSs and chromatin. These interactions involve lamins, as evidenced by the finding that cytosol immunodepleted from lamin does not support Cyt-LS binding to decondensed chromatin. To study the fusion stage of NE assembly, we incubated decondensed chromatin with a mixture of Cyt-LSs labeled with fluorescent lipids and unlabeled Cyt-LSs. Using fluorescence resonance energy transfer and dequenching assays, we detected lipid mixing by fluorescence microscopy and spectrofluorometry. As in nuclear envelope assembly, the lipid mixing was dependent on the presence of decondensed chromatin and GTP and inhibited by 1 mM GTP-gamma-S. We observed no lipid mixing when Cyt-LSs pretreated with interphase cytosol were replaced with Cyt-LSs pretreated with heat-inactivated cytosol or with interphase cytosol converted to a mitotic state by adding cyclin B. We then tested whether Cyt-LS fusion involved a small GTPase Ran, which plays an important role in NE assembly. Indeed, we found Ran on the Cyt-LSs by immunoblotting. Immunodepletion of Ran from cytosol strongly inhibited lipid mixing, indicating the direct involvement of Ran or a Ran-binding protein in Cyt-LS fusion on decondensed chromatin. In brief, lipid mixing between the Cyt-LSs bound to decondensed chromatin reconstitutes many properties of the biologically relevant fusion of MV in NE assembly, including dependencies on the cell cycle, the presence of decondensed chromatin, and Ran-dependent GTP-hydrolysis.
The early stages of nucleus assembly, including docking and fusion of MV at the chromatin surface, are followed by the assembly of nuclear pore complexes, additional fusion events, and further chromatin swelling. While neither Cyt-LSs nor LSs in the presence of cytosol formed nuclei on their own, they were able to join MV in the assembly of a functional nucleus. We first incubated rhodamine-tagged LS and cytosol with chromatin and then added MV, producing fluorescent nuclei morphologically indistinguishable from those observed in the absence of LSs. Very rapid and complete recovery of fluorescence at the fluorescent rim of the nuclei after photobleaching indicated that lipids freely diffuse over distances much exceeding the approximate 100 nm diameter of our LSs. Thus, lipids were distributed in the unified bilayers at the chromatin surface rather than limited in their diffusion, as expected for the bound LSs or in the case of LS hemifusion, in which only the outer monolayers of the adjacent LS would merge and the inner monolayers would remain distinct.
In conclusion, we reconstituted early stages of post-mitotic reassembly of NE with liposomal membranes carrying tightly associated cytosolic factors but lacking transmembrane proteins. Our data emphasize the importance of cytosolic regulation of membrane targeting to the chromatin and suggest that, in contrast to transmembrane proteins involved in viral and intracellular SNARE-dependent fusion, proteins that mediate membrane fusion in NE assembly lack transmembrane domains. While unexpected, the finding that membrane anchoring via transmembrane domains is not a prerequisite for fusion proteins is in line with the known ability of peripheral membrane proteins to drive membrane remodeling in budding and fission.
Ramos C, Rafikova ER, Melikov K, Chernomordik LV. Transmembrane proteins are not required for early stages of nuclear envelope assembly. Biochem J 2006;400:393-400.
Point-like protrusion as a prestalk intermediate in membrane fusion pathway
The widely accepted pathway of membrane fusion begins with the fusion stalk representing the initial intermediate of hemifusion. Yet, important questions remain unanswered about the structures and energies of lipid intermediates preceding fusion stalk formation and the possibility that the intermediates limit the rate of the entire fusion reaction. Indeed, in order to form a stalk, the membranes have to establish, at least locally, a dehydrated contact allowing them both to perturb the continuity of their surfaces and to merge, without exposure of the hydrophobic moieties of lipids to the aqueous surrounding. Such membrane contact requires the membranes to overcome the resistance of powerful short-range repulsion forces. Owing to these hydration forces, the conventional wave-like membrane bulges approaching each other would have energies of hundreds of kT, thereby making the bulges highly unlikely. To overcome the energy problem, we suggest an energetically feasible structure of prestalk intermediate that has a sharp tip allowing for establishment of a point-like rather than extended dehydrated contact between membranes. Such a point-like protrusion (PLP) has significantly lowered energy of the hydration repulsion between the membranes. The critical issue is whether the PLP will have very high elastic energy resulting from a sharp bend of the membrane monolayers. We analyzed the overall energy of the PLP by using the elastic tilt-splay model for the membrane deformations and the hydration force model for the intermembrane repulsion and found that the interplay between the splay of the lipid hydrocarbon chains and their tilt with respect to the monolayer surface decreases elastic energy costs of the PLP to modest values. Further, we found that, given the shape of the PLP along with the discreteness of the hydration centers, the hydration repulsion energy of PLP formation remains in the range of few tens of kT. For the relevant lipid compositions of membrane monolayers, the overall energy of the PLP is lower than that of the fusion stalk. Consequently, the PLP is not expected to limit the rate of hemifusion, and the lipid dependency of hemifusion is determined by the energetics of the stalk and the hemifusion diaphragm. Moreover, a point-like dehydrated contact between the PLP and the target membrane facilitates stalk formation. We consider the new prestalk intermediate an important addition to the fusion-through-hemifusion pathway.
Efrat A, Chernomordik LV, Kozlov MM. Point-like protrusion as a pre-stalk intermediate in membrane fusion pathway. Biophys J 2007;92:L61-3.
1 Corinne Ramos, PhD, former Postdoctoral Fellow, now at the University of California San Diego, La Jolla, CA
COLLABORATORS
Ori Avi-Noam, MS, Technion-Israel Institute of Technology, Haifa, Israel
Jaebok Choi, PhD,Baylor College of Medicine, Houston, TX, and Washington University Medical Center, St. Louis, MO
Avishay Efrat, PhD, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Faculty of Sciences, Holon Institute of Technology, Holon, Israel
Michael M. Kozlov, PhD, Dhabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Faculty of Sciences, Holon Institute of Technology, Holon, Israel
Anna P. Newman, PhD, Baylor College of Medicine, Houston, TX
Benjamin Podbilewicz, PhD, Technion-Israel Institute of Technology, Haifa, Israel
Amir Sapir, PhD, Technion-Israel Institute of Technology, Haifa, Israel
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 chernoml@mail.nih.gov.
