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Membrane Fusion Mediated by Protein Fusogens

Leonid V. Chernomordik, PhD
  • Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology
  • Eugenia Leikina, DVM, Senior Research Assistant
  • Kamram Melikov, PhD, Staff Scientist
  • Margarita S. Popova, Summer Student
  • Sergei A. Pourmal, Summer Student
  • Elvira Rafikova, PhD, Visiting Fellow
  • Jean-Philippe Richard, PhD, Visiting Fellow
  • Sung-Tae Yang, PhD, Visiting Fellow
  • Elena Zaitseva, PhD, Research Fellow

Disparate membrane remodeling reactions are tightly controlled by protein machinery but are also dependent on the lipid composition of the membranes. Whereas each kind of protein has its own individual personality, membrane lipid bilayers have rather general properties manifested by their resistance to disruption and bending. Our long-term goal is to understand how proteins remodel membrane lipid bilayers in important cell biology processes. The starting point in our analysis is a consideration of the physical factors that determine the tendency of the membrane bilayers to change their topology. We expect that the analysis of the molecular mechanisms of important and diverse membrane rearrangements will bring about new ways of controlling them and clarify the generality of emerging mechanistic insights. This year, we focused on the late stages of cell-to-cell fusion, essential in normal development and in pathophysiology, and on membrane fusion in post-mitotic re-assembly of the nuclear envelope and endoplasmic reticulum.

Fusion pore expansion in cell-to-cell fusion initiated by influenza virus hemagglutinin

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 work on various cell-cell fusion reactions indicated that the cytoskeleton plays an important role in syncytium formation. However, due to the complexity of these reactions and the multifaceted contributions of the cytoskeleton in cell physiology, it remained unclear whether the 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) argued against the former hypothesis. In our most recent study, we explored cellular reorganization associated with fusion pore expansion in fusion between the murine embryonic fibroblasts of the NIH3T3 cell line, expressing the well-characterized fusogen influenza virus hemagglutinin. We uncoupled early fusion stages dependent on protein fusogens from the subsequent fusion pore expansion stage. While the opening of fusion pores requires the presence of only 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.

Transmembrane protein-free membranes fuse into Xenopus nuclear envelope and promote assembly of functional pores

The membrane fusion stage of post-mitotic re-assembly of the nuclear envelope differs markedly from cell-cell fusion. The nuclear envelope (NE), which encloses the genetic material in higher eukaryotic cells, 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. NPCs consist of multiple copies of about 30 cytosolic and transmembrane proteins and facilitate and regulate the exchange of materials (proteins such as transcription factors, and RNA) between the nucleus and the cytoplasm. Incorrect assembly of NPCs and resulting changes in their functionality, lateral distribution over the NE, and nuclear morphology underlie many hereditary diseases (including X-linked Emery-Dreifuss muscular dystrophy, Hutchinson-Gilford progeria syndrome, Allgrove syndrome, and myeloid leukemia). NPCs also play key roles in several viral infections. NE and NPC 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 a membrane-free cytosolic extract along with membrane vesicles. 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, such as transmembrane proteins. To identify the functions that do not require transmembrane proteins to be present on each of the membranes involved, we replaced some of the membrane vesicles with vesicles functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free phosphatidylcholine liposomes. We found that functionally impaired membrane vesicles and liposomes in the presence of interphase, but not mitotic, cytosol undergo fusion with each other and native vesicles and participate in the formation of the tubular ER network. Like fusion between native membrane vesicles, this cytosol-dependent fusion involving membranes without functional transmembrane proteins was inhibited by non-hydrolysable forms of GTP, by N-ethylmaleimide pre-treatment of cytosol, and by addition of the soluble N-ethylmaleimide-sensitive fusion protein (NSF)–attachment protein (a-SNAP), an inhibitor of SNARE machinery. These findings substantiate the hypothesis that membrane fusion in ER and NE assembly is mediated by NSF/a-SNAP/SNARE machinery and raise an interesting possibility that this machinery involves transmembrane domain–lacking SNAREs that may be present both on the membranes and in the interphase cytosol.

In NE assembly, vesicle-liposome fusion allowed us to add membrane material without adding transmembrane proteins. Co-incubation of interphase cytosol and chromatin with membrane vesicles in concentrations lower than the standard concentrations of membrane vesicles in the NE assembly reconstitution system resulted in the formation of nuclei that were smaller than the control ones, had fewer NPCs, and failed to actively replicate DNA and import substrates. Addition of functionally impaired membrane vesicles or liposomes to the reaction mixture and their cytosol-dependent fusion to native membrane vesicles compensated for the shortage of membrane material and increased the sizes of the nuclei. Furthermore, the liposome-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 proteins residing exclusively at membrane vesicles were required for generating the characteristic morphology of the branched ER network and of fully extended and functional NE, cytosolic proteins conferred on liposomes the ability to fuse and, in the presence of native vesicles, to participate in formation of ER and NE. In contrast to nuclei formed at a lowered concentration of membrane vesicles, liposome-rescued nuclei with most of the membrane material provided by liposomes had normal function (nuclear import and DNA replication) and spatial distribution of NPC. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of the envelope growth. Recovery of functionality of NPCs by adding protein-free membrane material suggests that pore assembly involves the transition from immature (=dysfunctional) to mature (=functional) pores upon an increase in the nuclear envelope area that provides certain minimal distances between the pores. These data identify a novel and intriguing feedback mechanism that connects the assembly of functional NPCs with an expansion of the nuclear envelope and connects the function of the pores with their lateral distribution.

Additional Funding

  • NIAID’s 2009-2011 Trans-NIH Biodefense Program

Publications

  • Chen A, Leikina E, Melikov K, Podbilewicz B, Kozlov MM, Chernomordik LV. Fusion-pore expansion during syncytium formation is restricted by an actin network. J Cell Sci 2008 21:3619-3628.
  • Chernomordik LV, Kozlov MM. Mechanics of membrane fusion. Nat Struct Mol Biol 2008 15:675-683.
  • Richard J-Ph, Leikina E, Chernomordik LV. Cytoskeleton reorganization in influenza hemagglutinin-initiated syncytium formation. Biochim Biophys Acta Biomembranes 2009 1788:450-457.
  • Rafikova ER, Melikov K, Ramos C, Dye L, Chernomordik LV. Transmembrane protein-free membranes fuse into Xenopus nuclear envelope and promote assembly of functional pores. J Biol Chem 2009 284:29847-29859.

Collaborators

  • Ori Avi-Noam, MS, Technion-Israel Institute of Technology, Haifa, Israel
  • Louis Dye, BS, Microscopy and Imaging Core, NICHD, Bethesda, MD
  • Michael M. Kozlov, PhD, DHabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
  • Benjamin Podbilewicz, PhD, Technion-Israel Institute of Technology, Haifa, Israel
  • Corinne Ramos, PhD, Division of Biological Sciences, University of California, San Diego, La Jolla, CA

Contact

For more information, email chernoml@mail.nih.gov.

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