Secretory vesicle elasticity and mobility

Child Health And Human Development

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Abstract

While primarily focusing on a putative proteinaceous calcium sensor in studies on biological membrane fusion, a calcium-induced modulation of the mechanical properties of the membrane and their role in fusion are often overlooked. As a first step in studying the effects of calcium binding to the lipid bilayer on the mechanical properties of the membrane, the elastic moduli of the membranes are being measured using both biological and artificial membranous vesicles exposed to calcium. The elastic modulus of the membrane can be derived from measuring the osmotic swelling of the vesicles. We have investigated osmotic swelling of isolated sea urchin yolk granules which are fusion competent and are abundant in sea urchin eggs. The osmotic gradient was produced by varying concentration of glycine (within the range of zero to 0.5 M) in the medium. The size of those vesicles, measured by dynamic light scattering (DLS) was found to increase with decreasing medium osmolarity. However, the increase was too steep to be accounted for by swelling alone. Differential interference contrast and phase contrast microscopy showed that in fact the increase in mean size of the vesicles observed in DLS experiments resulted entirely from vesicle aggregation since the decrease in osmolarity did not affect vesicle mean size measured microscopically. The aggregation was presumably speeded up by a release of vesicle internal content of the vesicles due to their rupture progressing with decreasing medium osmolarity. Whether the elastic modulus may be derived for the membrane of cortical granules by their osmotic swelling is to be tested. It is also possible that the rupture of yolk granules masks their swelling assessed by averaging microscopically measured sizes of the vesicles due to a changing vesicle population. Sizing microscopically the same vesicles immobilized on a cover glass in a perfusion chamber will be used to clarify that point. When the mesh size of polymer network is close to the particle size, the particle may penetrate into the network by two mechanisms: either squeezing through the mesh and occupying the available space or by disrupting polymer entanglements and locally deforming the polymer network. The latter mechanism dominates the penetration of rigid particles while the former mechanism may dominate that of the elastic ones (Radko and Chrambach 2002 Electrophoresis 23, 1957). Different dependencies of particle retardation on particle size, rate of the penetration, and polymer concentration are expected for those mechanisms. For membranous vesicles, the switch between the mechanisms of penetration may depend on the elastic modulus of the membrane. To test that hypothesis, we have investigated the dependencies of retardation of extruded liposomes (80 to 200 nm in mean diameter, PC:PS:cholesterol ratio of 3:1:1), subjected to capillary zone electrophoresis in PEG solutions, on their size, applied voltage, and PEG concentration. Retardation of the liposomes was found to increase with vesicle size within the size range studied, thus differing from that of rigid polystyrene latexes. The dependence of retardation on membrane elasticity modulated by cholesterol content or by lipid composition (producing lipid bilayers in either a gel or liquid state at the experimental temperature of 25oC) is under investigation.