Patterned Two-Photon Photoactivation Illuminates Spatial Reorganization in Live Cells

Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value de composition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1-2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.