Membrane Rearrangements And Protein Refolding

Child Health And Human Development

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Abstract

Membrane remodeling is an important stage of diverse cell biological processes, including exo- and endo-cytosis, fertilization, membrane trafficking and different stages of enveloped virus infection. We hypothesize that specialized proteins mediate the remodeling by promoting the changes in the membrane shape that both prime the membranes for the bilayer rearrangement and drive it to the completion. More specifically, we hypothesize that membrane rearrangements from local hemifusion, merger of contacting membrane monolayers, to an opening of an expanding fusion pore are driven by lateral interactions between fusion proteins. In our recent work on the pathway of lipid bilayer fusion we have theoretically analyzed the possible mechanisms of the transition between stalk, a local hemifusion intermediate, and pore. We have also focused on the properties of the hemifusion stage in the prototype fusion reaction driven by influenza virus hemagglutinin (HA) and found that hemifusion is generated by fusion machinery that is distinct and independent from the machinery that form expanding fusion pores. Finally, we have studied the mechanisms by which short cationic peptides remodel cell membranes to enter into cytoplasm without degradation. Our data indicate that these cell-penetrating peptides are internalized by endocytic pathway. The work can be divided into 3 projects. 1. Theoretical analysis of formation, structure, and decay of hemifusion diaphragm. While widely considered to be a key stage in membrane fusion, hemifusion is poorly understood both on the structural level and in terms of the physical forces involved. In our recent work we analyzed theoretically the pathways of the intermediate membrane structures emerging in the course of hemifusion and the forces driving evolution of these intermediates into a fusion pore. This evolution can proceed by one of the following scenarios. The first stalk-pore pathway suggests that the stalk expands radially and brings the distal monolayers of the two membranes together into a single bilayer. Opening of a fusion pore within this hemifusion diaphragm (HD) completes the fusion reaction. To allow formation of the pore rim, the HD radius has to exceed a certain value approximately equal to the lipid monolayer thickness. In the second model, the fusion pore forms directly from the stalk and constitutes from the very beginning a bilayer connection between the membranes. We show that a fusion stalk spontaneously expands into an HD only if the contacting monolayers of the fusing membranes consist of lipids such as dioleoyl phosphatidylethanolamine or its mixtures with other lipids. For "bilayer" lipid, such as dioleoyl phosphatidylcholine, stalk expansion requires an external force. In case of biological fusion, this force might be generated by the fusion proteins that pull apart the diaphragm rim. To address the mechanism of fusion pore formation, we analyze the distribution of the lateral tension emerging in the HD due to the establishment of lateral equilibrium between the deformed and relaxed portions of lipid monolayers. We show that this tension concentrates along the HD rim and reaches high values sufficient to rupture the bilayer and form the fusion pore. The prediction of a crack-like pore propagating along the HD rim readily explains an earlier observation of the lack of movement of aqueous dyes while total fusion pore conductance increases that has been observed for HA-mediated fusion. In general, our analysis supports the hypothesis that transition from a hemifusion to a fusion pore involves radial expansion of the stalk. 2. Kinetically differentiating influenza hemagglutinin fusion and hemifusion machines. HA-mediated membrane fusion yields different phenotypes depending on the surface density of activated HAs. A key question is whether different phenotypes arise from different fusion machines or whether different numbers of identical fusion machines yield different probabilistic outcomes. If fusion were simply a less probable event than hemifusion, requiring a larger number of identical fusion machines to occur first, then two predictions can be made. First, fusion should have a shorter average delay time than hemifusion, since there are more machines. Second, fusion should have a longer execution time of lipid mixing after it begins than hemifusion, since the full event cannot be faster than the partial event. To compare fusion machines that yield different fusion phenotypes we have developed a novel, simple and rigorous computer driven kinetic analysis of video fluorescence microscopy of fusion between HA expressing cells and fluorescently labeled erythrocytes. This assay provides the quantitative information about the waiting time for initiation of dye spread and the time required for dye spread to be complete for a large number of individually fusing pairs. Using this technique, we screened many individual HA-expressing cells fusing with erythrocytes and identified cell pairs with either full or only partial redistribution of fluorescent lipids. Only the full lipid mixing phenotype represented fusion with contents mixing. Partial lipid mixing is found to correspond to unrestricted hemifusion phenotype (lipid mixing without content mixing) where lipidic connection between the bilayers dissociated prior to full redistribution of lipid dye. The new technique allowed us to test the above predictions. Since i) there was no correlation between the waiting times for the onset of lipid mixing for fusion and the unrestricted hemifusion phenotypes; and ii) the execution time for fusion was faster than that for hemifusion, we conclude that distinct and independent machines generate these two fusion phenotypes. 3. Cell-penetrating peptides: a re-evaluation of the mechanism of cellular uptake. During the last decade, several proteins, such as HIV-1 Tat and Drosophila Antennapedia homeoprotein, have been shown to traverse cell membrane by a process called protein transduction, and to reach the nucleus while retaining their biological activity. Short cationic peptides derived from protein-transduction domains that are responsible for the cellular uptake of these proteins (cell penetrating peptides or CPP) can be internalized in most cell types and, more importantly, allow the cellular delivery of conjugated (or fused) biomolecules. Internalization of CPP has been ascribed in literature to a mechanism, which do not involve endocytosis. This interpretation has been based mainly on CPP translocation studies that relied on the fluorescence microscopy on fixed cells and fluorescence-activated cell sorter analysis. In our recent work, we have demonstrated that cell fixation, even in mild conditions, leads to the artefactual uptake of CPP peptides. Moreover, these peptides bind strongly to the cell plasma membrane and remain associated with cells even after repeated washings. As a result, flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake is similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat conjugated peptide nucleic acids. These data indicate that the cellular internalization of CPP proceeds by endocytic pathway. Future work will clarify the specific mechanisms by which CPP promote remodeling of membranes to facilitate either endocytosis of the peptide-conjugated cargo and/or its fast escape from endosome into cytoplasm and nucleus of living cells to avoid degradation.