Mapping the Evolution of Molecular Flow Fields in Migrating Cells with Time-Resolved STICCS

Cell migration is a complex process that involves an intricate choreography between cytoskeleton and adhesion molecules with precise regulation of their transport and interactions in space and time in living cells. Although there have been many advances recently in improving spatial resolution of optical microscopy methods, temporal resolution remains a bottleneck for many methods. Previously we have reported spatio-temporal image cross-correlation spectroscopy (STICCS) in combination with TIRF microscopy to provide snap shots of flow fields of adhesion proteins and the cyctoskeleton in migrating cells. However, the temporal resolution of the STICCS transport maps was low compared to the frame time resolution of the TIRF imaging. Here we report the extension of STICCS to its absolute temporal resolution limit as set by the imaging frame rate by applying a short correlation window of 10 or fewer frames with the analysis window iterated sequentially by single frame steps. Although computationally more intensive, this enables us to capture the time evolution of the flow transport and interactions of adhesion components in living cells with a STICCS vector map for every frame of the TIRF movie. We illustrate the method with measurements of time evolving transport maps of the adhesion related macromolecules alpha5, alpha6 and alphaL integrins with paxillin, and actin within, or associated with the basal membrane in adherent U2OS and CHO.B2 cells plated on extracellular matrix components fibronectin, laminin, or ICAM-1. The time resolved cross-correlation vector maps clearly show that the dynamic interactions between alpha6 or alphaL integrins with paxillin evolve in space and time only at active adhesions in protruding and retracting regions of the cells.