preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Nanyang Technological University

Dysfunction in the endolysosome, a late endosomal to lysosomal degradative intracellular compartment, is an early hallmark of some neurodegenerative diseases, in particular Alzheimer's disease. However, the subtle morphological changes in compartments of affected neurons are difficult to quantify quickly and reliably, making this phenotype inaccessible as either an early diagnostic marker, or as a read-out for drug screening.

Multivariate profiling of neurodegeneration-associated changes in a subcellular compartment of neurons via image processing.

We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin and cholesterol over a range of compositions - both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradients, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling events occur. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental giant unilamellar vesicles through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms to compensate for osmotic stress during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery.

Osmotic gradients induce bio-reminiscent morphological transformations in giant unilamellar vesicles.

Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR-FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR-FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200,000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR-FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross-correlation functions (ΔCCF). With the determination of the PSF by ITIR-FCS and the optimization of measurement conditions ITIR-FCS becomes a calibration-free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.

Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.

We report the experimental observation of osmotically induced transient pearling instabilities in vesicular membranes. Giant phospholipid vesicles subjected to negative osmotic gradient, which drives the influx of water in to the vesicular interior, produces transient cylindrical protrusions. These protrusions exhibit a remarkable pearling intermediate, which facilitates their subsequent retraction. The pearling front propagates from the distal free end of the protrusion toward the vesicular source and accompanies gradual shortening of the protrusion via pearl-pearl coalescence. Real-time introduction of a positive osmotic gradient, on the other hand, drives vigorous shape fluctuations, which in turn produce cylindrical, prolate- and pear-shaped intermediates presumably due to an increased vesicular area relative to the encapsulated volume. These intermediates transiently produce a pearled state prior to their fission. In both cases, the transient pearling state gives rise to an array of stable spherical daughter vesicles, which may be connected to one another by fine tethers not resolved in our experiments. These results may have implications for self-reproduction in primitive, protein-free, cells.

Article	Year
Multivariate profiling of neurodegeneration-associated changes in a subcellular compartment of neurons via image processing. BioData mining\| PubMed ID: 19014586	2008
Osmotic gradients induce bio-reminiscent morphological transformations in giant unilamellar vesicles. Frontiers in physiology\| PubMed ID: 22586404	2012
Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 22615144	2012
Transient pearling and vesiculation of membrane tubes under osmotic gradients. Faraday discussions\| PubMed ID: 23805742	2013

Rachel S. Kraut

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