|
Binding/unbinding kinetic rates of cytoskeletal proteins at the cytofacial surface of the plasma membrane of smooth muscle cells in culture are measured and imaged by total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) microscopy. Cells are first injected with either rhodamine monomeric actin or rhodamine phalloidin, which binds to polymeric actin. A TIR beam, which illuminates approximately 80 nm deep into the cell, is then used to preferentially observe the labeled actin in the vicinity of the membrane. Fluorophores at cell-substrate contact regions within the evanescent field illumination are then photobleached by a prolonged flash. The subsequent recovery, excited by a series of briefer flashes at 1 or 6 frames per minute, is recorded by a cooled CCD. The resulting stack of images can be curve-fit pixel-by-pixel to produce a spatially resolved image of the unbinding rate (the reciprocal of residency time) of protein reversibly adsorbed at the submembrane surface. For rhodamine actin and for rhodamine phalloidin, the two ways of marking actin in the cell, the average characteristic unbinding times are somewhat different: 308 plus or minus 142 sec and 833 plus or minus 140 sec, respectively. The spatially resolved images of rhodamine phalloidin, pseudo-colorized to show average unbinding rates at each pixel, reveal a considerable variation over the cell, ranging over an order of magnitude.
|
