Paxillin focal adhesions, localization and implication: insight from Photo-Activated Localization Microscopy

Abstract

Photo-Activated Localization Microscopy (P.A.L.M.) as described by E. Betzig (2006) optically resolves selected subsets of photo-activatable fluorescent probes within cells at mean separations of less than 25 nanometers through serial photo-activation and subsequent photobleaching of numerous sparse subsets of photo-activated fluorescent protein molecules.The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this work COS-7 and ST14A tdEos-Paxillin transfected cells were used. We observed some features that limit the versatility of PALM, both in this setup and in its present version. It takes actually hours to go through the cycles of photo-activation and image acquisition, to collect all of data needed and to generate a single high-resolution image limiting the use to fixed specimens which precludes PALM.'s use for imaging of live cells. More important is the loss of data. Depending on the spatial concentration of the PA-FPs, most of the information about the position of molecules is lost during the photo-activation photobleaching phase, especially during the first cycles of data collection. From the biological point of view, we observe small paxillin clusters along the focal adhesions. Supported by U54 GM064346 CMC (MD, EG), NIH-P41-RRO3155 (EG, FC), P50-GM076516 (EG).Sponsored by Enrico Gratton