Membrane Fusion Mediated by Viral and Developmental Protein Fusogens

Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology
Eugenia 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

While our earlier work on mechanisms of protein-mediated membrane fusion focused on membrane rearrangements yielding a fusion pore (FP), we have recently studied the mechanisms of FP expansion. Transition from nanometer-sized nascent FPs to an open lumen of cell-size diameter is an important and unexplored stage of cell-to-cell fusion in both development and pathophysiology. Using confocal microscopy, we studied expansion of the micron-scale FPs in syncytium formation initiated by baculovirus gp64. In contrast to the opening of a pore, FP growth requires cell metabolism. While the actin cortex has been proposed to drive FP expansion, our model demonstrates that actin structures slow expansion and disassemble under the pore. We plan to test the applicability of the mechanisms of syncytium formation to biologically relevant cell fusion processes. We also explored intracellular delivery of the chemical conjugates of RNase-resistant phosphorodiamidate morpholino oligomers (PMO), which are promising reagents for redirecting the splicing machinery. We varied the charge and hydrophobicity of these conjugates and monitored their cellular uptake and splicing correction efficiency. Our work identifies the stage of endosomal escape of PMO as a limiting stage in PMO delivery.

Fusion pore expansion during syncytium formation is restricted by an actin network

Chen, Leikina, Melikov, Chernomordik; in collaboration with Kozlov, Podbilewicz

Cell-cell fusion in animal development involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Earlier work on fusion mechanisms focused on the initial stages of the fusion pathways, which yield fusion pores of a few nanometers in diameter. In syncytium formation, these pores expand to ones readily detectable by fluorescence microscopy (diameter greater than about 0.2 µm) and finally yield an open lumen with an approximate cell-size diameter of 10–15 µm. Little is known about the properties of these larger pores and the mechanisms that underlie the enlargement of cytoplasmic bridges from early fusion pores to syncytia. For instance, we still do not know whether the enlargement is driven by the fusogens, proceeds spontaneously, or is driven by the cytoskeleton, membrane tension, or other as yet unidentified cell machinery.

In our most recent work, we explored the enlargement of micron-scale pores in syncytium formation initiated by baculovirus gp64, a well-characterized fusogen. Low pH–triggered conformational change in gp64 results in a fast opening of fusion pores and inactivation of the fusogen. Thus, these properties of gp64-mediated cell fusion allowed effective uncoupling of fusogen-specific early fusion stages from the pore expansion stages. To visualize the three-dimensional morphological changes in the contact zone during the opening and expansion of the fusion pore(s), we labeled Sf9^Op1D cells expressing gp64 with fluorescent lipids and imaged the cells with three-dimensional time-lapse confocal microscopy as they underwent fusion. In both live-cell experiments and after fixing cells, we analyzed the development of fusion pores large enough to be detected by light.

The pores were roughly circular and expanded radially until they came close to one another, whereupon their shapes begin to distort. Fusion pore expansion within the zone of tight contact was accompanied by an increase in the cell contact area, suggesting that fusion pores grow by displacement of membrane material toward the contact zone periphery. Pore growth is driven neither by membrane tension nor by microtubule cytoskeleton but rather depends on cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Polymerization and depolymerization of actin filaments inhibited and promoted pore expansion and syncytium formation, respectively, indicating that the actin cytoskeleton restricts rather than drives the expansion of fusion pores. We propose that the growth of the strongly bent fusion pore rim is restricted by dynamic resistance of the actin network and driven by membrane-bending proteins involved in the generation of highly curved intracellular membrane compartments.

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;121:3619-3628.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure activity studies

Yang, Chernomordik; in collaboration with Abes R, Moulton, Clair, Abes S, Kamran, Melikov, Prevot, Youngblood, Iversen, Lebleu

Several arginine-rich peptides have been shown to traverse the cell membrane and reach the cytosol and nucleus. These peptides, referred to as cell-penetrating peptides (CP), have been shown to facilitate intracellular delivery of conjugated (or fused) macromolecules while retaining their biological activity. In our earlier work, we established that cationic CPP enter the cell by endocytosis, although the mechanisms by which CP cross the endosomal membrane to be delivered into the cytosol and nucleus remain unknown. Recent studies have indicated that cationic peptides such as oligoarginines and Tat 48-60 are relatively inefficient in transporting uncharged oligonucleotide (ON) analogues such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) largely because CP-conjugated material remains trapped in endocytic vesicles. Given that redirecting the splicing machinery through the hybridization of high-affinity, RNase-incompetent PNA and PMO might lead to important clinical applications, our collaborators recently explored the efficiency of splicing correction by a number of PMO conjugates. Among these conjugates, (R-Ahx-R)₄ (Ahx stands for 6-aminohexanoic acid) was the most efficient in the tested group. Importantly, (R-Ahx-R)₄-PMO conjugates are effective in mouse models of various viral infections and Duchenne’s muscular dystrophy.

To identify structural features facilitating efficient nuclear delivery and to uncover limiting steps in the internalization pathway of conjugated PMO, we carried out structure-activity characterization. We observed a significant correlation among splicing correction efficiency, affinity for heparin, and the ability to destabilize model synthetic vesicles but found no correlation with efficiency of cellular uptake. To evaluate the ability of CP-PMO conjugates to escape from endosomes, we employed a liposome leakage assay. It is known that late endosomes are characterized by an unusual lipid composition—they are enriched in bis(monooleoylglycero) phosphate—and that their lumen is acidic (pH 5.5). We therefore prepared liposomes from the lipid mixture mimicking the lipid composition of late endosomes and explored the effect of low pH on the CP-PMO–induced dye escape. In correlation with the data on splicing activity, the (R-Ahx-R)₄-PMO conjugate induced significantly more low pH-dependent leakage than other tested conjugates. Interestingly, the more hydrophobic (R-AbuL-R)₄-PMO (Abu stands for 4-aminobutyric acid) induced considerably less leakage than (R-Ahx-R)₄-PMO. Our findings indicate that the efficiency of delivery of the splice-redirecting ON analogues correlates with their ability to destabilize membranes with a lipid composition mimicking that of the late endosomal membranes.

Our work substantiates the hypothesis that entry of CP proceeds through binding to cell-surface heparin sulfates, endocytosis, and escape from endosomal compartments, with the escape stage dramatically limiting the efficiency of entry. We expect future work on the mechanisms of low pH– and lipid composition–dependent endosomal escape to bring about better cell-penetrating reagents required for drug delivery and to provide new insights into intracellular trafficking of internalized material.

Abes R, Moulton HM , Clair P, Yang S-T, Abes S, Melikov K, Prevot P, Youngblood DS, Iversen PL, Chernomordik LV, Lebleu B. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies. Nucleic Acids Res 2008;36:6343-6354.
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;121:3619-3628.
Chernomordik LV, Kozlov MM. Mechanics of membrane fusion. Nat Struct Mol Biol 2008;15:675-683.
Sapir A, Avinoam O, Podbilewicz B, Chernomordik LV. Conservation and divergence of viral and developmental cell fusion mechanisms. Dev Cell 2008;14:11-21.

Collaborators

Rachida Abes, PhD, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France
Said Abes, PhD, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France
Ori Avi-Noam, MS, Technion-Israel Institute of Technology, Haifa, Israel
Philippe Clair, PhD, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France
Patrick L. Iversen, PhD, AVI BioPharma, Corvallis, OR
Michael M. Kozlov, PhD, DHabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Bernard Lebleu, PhD, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France
Hong M. Moulton, PhD, AVI BioPharma, Corvallis, OR
Benjamin Podbilewicz, PhD, Technion-Israel Institute of Technology, Haifa, Israel
Paul Prevot, PhD, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France
Corinne Ramos, PhD, Division of Biological Sciences, University of California, San Diego, La Jolla, CA
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
Derek S. Youngblood, PhD, AVI BioPharma, Corvallis, OR

For further information, contact chernoml@mail.nih.gov.