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Vesicle and Membrane Biophysical Chemistry Department of Chemistry, GWU Research Instrumentation Publications The cell membrane is a complex assortment of actors. Also, as the gateway and guardian to the interior cell, it accommodates ever changing physical and chemical environments. The lipids and cholesterol components form a fluid matrix for the various transmembrane proteins and receptors to inhabit. The relationship between the lipid bilayer and membrane proteins is not well-understood even ~35 years after Singer and Nicolson first described the structure. (1) Lipid organization in rafts, clusters or nano-domains, or lack there of, is the subject of constant debate with many competing theories.(2-8) In cellular shape transformation, transmembrane pores mediate salt concentrations, cell volume, and solute movement across the cell membrane. Enzymes (flippases) aid in re-orienting lipids to accommodate the curvature demands.(9) Vesicles have demonstrated shape transformations, which require lipid re-arrangement between the leaflets and volume modulations without these facilitators.(10-13) For a mobile cell, conformation to new surfaces and volume regulation are key responses to an ever changing landscape. The ability of cells to condense and flatten, due to volume and to actin de-polymerization, and to change their dimensions allows them to crawl across varied topographies quickly. As well, extracellular scaffolds offer embedded clues to cells in their three-dimensional pockets and individual grooves. For cells to interact and to respond to these nuanced features, the membrane must conform to the surface irregularities. The response of vesicles to surface demands is vital to our understanding of the cell membrane lipid role, specifically, the lipid raft and the lipid-phase contributions.
Figure: (a) An example of a vesicle imaged using a confocal microscope. A stack of XY slices renders a three dimensional image, as shown in the schematic. When vesicles (GUV, giant unilamellar vesicles, 5-50 m) are cooled without perturbations, they form round, spherical constructs. (b) A vesicle is enmeshed in a three dimensional flexible peptide matrix, similar to a collegen extracellular matrix. (a) The three dimensional reconstruction of the vesicles reveals a highly deformed vesicle with significant undulations to accommodate the surrounding matrix, indicating the conformation of the vesicle to the available space in the peptide scaffold. When the surface of the vesicle is examined, many small scale undulations are evident where it accommodates the individual tendrils of the matrix. When viewed in profile, there are several deviations form the normal spherical shape of the vesicle. In the YZ reconstruction, the vesicle has a curved, inverted bowl profile, instead of a full spherical shape, with a large protrusion to form an incomplete circle. In the XZ reconstruction, the surface undulations render the view blurry in contrast to the sharp edges of Fig. 1. (b) In the cut-away views, the edges are blurred due to the undulations on the surface of the vesicle, shown in (a). The XZ cut away reveals indentations along the profile not evident in the three dimensional reconstruction. In (c), the PuraMatrix is visible after a vesicle has ruptured and labeled the tendrils with the lipid dye. As evident in the image, the scaffold formed by PuraMatrix is complex with small rope-like tendrils, forcing the vesicles to conform to the rich topography with feature sizes larger and smaller than a micrometer. As my research progresses, I expect to develop biomimetic surfaces on a platform that incorporates surface plasmon resonance analysis. On it, I expect to probe the influences of surface chemistry on vesicle adhesion and to investigate adhesion events of cells at specifically tailored distances. The vesicle behavior will allow us to test the various theoretical models of lipid organization in the cell membrane. From the SPR instrumentation, we expect to record the timeline of the adhesion complex assembly. 1. S. J. Singer, G. L. Nicolson, Science 175, 720 (1972). |
