An Optical Flow Based Methodology for Visualizing Dynamic Sucellular [sic] Organiztion [sic] Demonstrated Through Profilin and Rho GTPase Microdomains

Live cell imaging has enabled the collection of movies of subcellular protein dynamics at a submicron resolution. Statistical time series analysis can greatly expand our understanding of subcellular interactions in minimally perturbed systems. This was previously achieved for the leading edge of migrating cells in select cases. Importantly no strategy existed to simultaneously analyze every subcellular location. Building on existing optical flow based non-linear image registration we developed an approach to remap a migrating cell to a common cell footprint while preserving the characteristics of our signal of interest at a spatial granularity necessary for understanding micron scale biological interactions. This tool enabled us to discover that Profilin fluctuations are organized in living cells. This organization was found to be dependent on cell polarization and actin binding capability. Expanding on this ability to query all subcellular locations, we developed a feature set and feature projection strategy to map molecular biosensor movies of Rho GTPase signaling into micron scale regions of internally consistent signaling dynamics or "microdomains". Microdomains of GTPases match literature descriptions of signaling organization and in an optogenetic study were found to almost precisely match the perturbation footprint.