Measurements of plasma membrane architecture during hypoxia using multiple fluorescent spectroscopic techniques

Alterations in plasma membrane structure and function seem to be of primary importance in the pathogenesis of cell injury, calling for more understanding of the changes in plasma membrane lipid structure (e.g., lipid order, lateral diffusion, dependence of phase states, and viscoelasticity) during the evolution of hypoxic injury in hepatocytes using multiple fluorescent spectroscopic techniques. Following hypoxic injury, fluorescence recovery after photobleaching was used to monitor plasma membrane lipid diffusion, resonance energy transfer microscopy was used to detect the lipid topography (domain formation), and the laser trapping technique was used to measure the plasma membrane viscoelasticity. The use of these different kinds of fluorescent spectroscopic techniques coupled with the authors' previous studies using digitized fluorescence polarization microscopy which was used to measure lipid order (fluidity) allowed the delineation of alterations in membrane structure during hypoxic injury and a model of membrane architecture during hypoxic injury, which could not be obtained from the use of any of these techniques alone. A model is proposed in which gel- and fluid-phase lipid islands form during hypoxic cell injury. Formation of these lipid domains promotes cell surface bleb formation, with eventual weakening of plasma membrane integrity, bleb rupture, and cell death. 11