Actin Dynamics at the Living Cell Submembrane Imaged by Total Internal Reflection Fluorescence Photobleaching

Abstract

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics.

Spatial and temporal traction response in human airway smooth muscle cells

“…Measurements of the actin dynamics in cultured smooth muscle cells (30) showed that actin kinetic parameters display a cell-wide gradient. The unbinding rate of actin near the plasma membrane at the cell-substrate contact regions, as measured using total internal reflection microscopy and fluorescence recovery after bleaching, was found to be spatially nonuniform throughout the cell (30). In some cell regions actin kinetics was fast, whereas in other regions it was slower.…”

mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration

“…To visualize F-actin, cells were microinjected with X-Rhodamine-labeled actin either alone or together with the inhibitory mDia2 antibody. To assay F-actin dynamics in the ventral cortex of cells, we used total internal reflection fluorescence recovery after photobleaching (TIR-FRAP) (Sund and Axelrod, 2000). FRAP of fluorescent actin in the ventral cell cortex using TIRF microscopy allows quantification of the rate and completion of fluorescence recovery, indicating how quickly fluorescent actin in the cortex cycles between the cytoskeleton-bound and cytoplasmic pools.…”

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

“…However, it should be taken into account that in comparison with conventional fluorescence microscopy, TIRFM signals are by about a factor 100 lower and need very sensitive optical detection systems in order to obtain good signal-to-noise ratios. TIRFM has been first described in 1981 (14), and since that time has been used increasingly for measuring cell-substrate contacts (15)(16)(17), membrane or protein dynamics (18), membrane proximal ion fluxes (19) as well as endocytosis or exocytosis (20)(21)(22).…”