Responses to cholesterol depletion are often taken as evidence of a role for lipid rafts in cell function. Here, we show that depletion of cell cholesterol has global effects on cell and plasma membrane architecture and function. The lateral mobility of membrane proteins is reduced when cell cholesterol is chronically or acutely depleted. The change in mobility is a consequence of the reorganization of the cell actin. Binding of a GFP-tagged pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to the plasma membrane is reduced after cholesterol depletion. This result implies that the reorganization of cytoskeleton depends on the loss or redistribution of plasma membrane PI(4,5)P2. Consistent with this observation, agents that sequester plasma membrane PI(4,5)P2 mimic the effects of cholesterol depletion on actin organization and on lateral mobility.

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.