This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation, are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with either human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the """"""""flickering"""""""" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore. Shape dynamics and permeability of a membrane neck connecting a vesicle and plasma membrane were also considered. The neck is modeled by a lipid membrane tubule extended between two parallel axisymmetric rings. Within a range of lengths, defined by system geometry and mechanical properties of the membrane, the tubule has two stable shapes, catenoidal microtubule and cylindrical nanotubule. Their permeabilities, measured as ionic conductivity of the tubule interior, differ by up to four orders of magnitude. Near the critical length the transitions between the shapes occur within less than a millisecond. Theoretical estimates show that the shape switching is controlled by a single parameter, the tubule length. Thus the tubule connection can operate as a conductivity microswitch toggling the release of vesicle content in such cellular processes as kiss-and-run exocytosis. In support of this notion, bistable behavior of membrane connections between vesicles and the cell plasma membrane in macrophages is demonstrated. In a third study, we introduced a new method for human gene therapy. Unlike oncoretroviruses, lentiviral vectors can insert large genes and can target both dividing and nondividing cells; thus they hold unique promise as gene transfer agents. To enhance target range, the native lentiviral envelope glycoprotein is replaced (pseudotyped) with vesicular stomatitis virus G (VSVG), and the genes of interest are packaged in nonreplicating vectors by transient transfection with three plasmids. However, because of cytotoxic effects of VSVG expression in producer cells (293T cells) it has been difficult to generate a packaging cell line, required for even modest scale-up of vector production. Here we introduce a pseudotyped lentivirus vector using the baculovirus GP64 envelope glycoprotein. Compared with VSVG, GP64 vectors exhibited a similar broad tropism and similar native titers. GP64-pseudotyped vectors were usually highly concentrated without much loss of titer. Because, unlike VSVG, GP64 expression does not kill cells, we generated 293T-based cell lines constitutively expressing GP64. Our results demonstrate that the baculovirus GP64 protein is an attractive alternative to VSVG for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications.
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