We are interested in very diverse fusion reactions and this year explored both cell-to-cell fusion and intracellular fusion. Our study on cell fusion initiated by influenza virus hemagglutinin has focused on fusion stages that involve expansion of initial fusion pores. This process yields an open lumen of cell-size diameter, is essential for syncytium formation in animal development and in diseases, and is very poorly understood. Earlier works on different cell-cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it had remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Our study on baculovirus gp64 initiated fusion of insect cells (Chen et al., 2008) has argued against the former hypothesis. In most recent study we explored cellular reorganization associated with fusion pore expansion in fusion between murine embryonic fibroblasts NIH3T3-based cells expressing very well-characterized fusogen influenza virus hemagglutinin. We uncoupled early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage. While the opening of fusion pores requires only the presence of functional hemagglutinin, this transition requires cell metabolic energy and is negatively regulated by protein kinase C. Late and cell-dependent stages of fusion yielding syncytium formation are not blocked by microtubule- and actin-modifying treatments, indicating that neither the microtubule nor the actin cytoskeleton drives the enlargement of initial fusion connections. The mechanistic insights provided by our simplified experimental model based on well characterized viral fusogens will hopefully help in elucidation of complex cell-cell fusion reactions in normal development and in pathophysiology. Membrane fusion stage of post-mitotic re-assembly of nuclear envelope is very different from cell-cell fusion. In species with an open mitosis, the nuclear envelope (NE) breaks and reassembles during each cell cycle. NE reassembly starts during anaphase and involves formation of a double membrane around segregated chromosomes, insertion of multiprotein nuclear pore complexes (NPCs), and further NE expansion. NE formation has been intensively studied in vitro with demembraned chromatin and fractionated Xenopus leavis egg extract. The fractionation step results in disruption of endoplasmic reticulum (ER) and yields membrane-free cytosolic extract along with membrane vesicles (MVs) involved in ER and NE formation. In our recent study of ER and NE assembly in the Xenopus egg reconstitution system we explored the relative contributions of cytosolic proteins (defined as proteins that are present either only in cytosol or both in cytosol and, as peripheral proteins, at the membranes) and exclusively membrane-residing proteins (MR), such as transmembrane proteins. To identify the functions that do not require MR proteins to be present on each of the membranes involved, we replaced some of the MVs with MVs functionally impaired by trypsin or N-ethylmaleimide (NEM) treatments or with protein-free phosphatidylcholine liposomes. We found that functionally impaired MVs and liposomes in the presence of interphase- but not mitotic cytosol undergo fusion with each other and native MVs and participate in the formation of the tubular ER network. Like fusion between native MVs, this cytosol-dependent fusion involving membranes without functional MR proteins was inhibited by non-hydrolysable forms of GTP, by NEM pre-treatment of cytosol, and by addition of soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein (-SNAP), an inhibitor of SNARE machinery. In NE assembly, vesicle-liposome fusion allowed us to add membrane material without adding transmembrane proteins. Co-incubation of interphase cytosol and chromatin with MVs in concentrations lowered relative to the standard MV concentrations in the NE assembly reconstitution system resulted in formation of nuclei that were smaller than the control ones, had a decreased number of NPCs, and failed to actively replicate DNA and import substrates. Addition of functionally impaired MVs or liposomes to the reaction mixture and their cytosol-dependent fusion to native MVs compensated for the shortage of membrane material and increased the sizes of the nuclei. Furthermore, the liposomes-rescued nuclei appeared similar to the control ones in ultrastructure, active nuclear transport, and DNA replication. The recovery required nuclear transport and involved an increase in the number of functional NPCs. Thus, while MR proteins at MVs were required for generating the characteristic morphology of the branched ER network and of fully extended and functional NE, cytosol confered on liposomes the ability to fuse and, in the presence of native MVs, to participate in formation of ER and NE. In contrast to nuclei formed at a lowered concentration of MVs, liposomes-rescued nuclei with most of the membrane material provided by liposomes had normal function and spatial distribution of NPC. Our findings emphasize the mutual dependency of NPC assembly and NE expansion and suggest that interphase cytosol contains proteins that mediate the fusion stage of the envelope growth.
NIH ExPorter
NIH ExPorter