Real-time analysis of F-actin fluctuation in living cells with quasi super-resolution technique

Abstract

Actin filaments play significant roles in many essential cellular processes including muscle contraction, cell motility, vesicle and organelle movement, cell signaling, and mechano-sensing mechanism of cells. However, one of the important cellular processes, the mechano-sensing mechanism of cells, is still debatable. To elucidate these mechanisms of cellular processes, it is important to observe the remodeling of the F-actin structure in cells. Although there are many reports about the remodeling of the F-actin structure during cellular processes, it needs to consider that the intracellular and extracellular proteins are fluctuating with thermal energy. We assumed that these small fluctuations affect the mechano-transduction in cells. We focused on an F-actin small fluctuation and tried to observe that in a living cell. Here, we propose the analyzing method of F-actin fluctuation in a living cell using the quasi super-resolution technique. To visualize F-actin filaments in living cells, Lifeact-GFP plasmid vector was transfected to NIH3T3 cells. Ten images of F-actin in living cells were taken every second under static culture conditions. First, we analyzed the small fluctuation of F-actin in living cells with Gaussian smoothed images. In this analysis, the amplitude of F-actin fluctuation was approximately 0.2 to 0.3 μm, which is almost the same as the optical resolution. Therefore, we have reconstructed the F-actin image in cell from a cross-sectional fluorescent profile with the quasi super-resolution technique. We analyzed the fluorescent profile at several points along one F-actin filament, and the X and Y coordinates were picked up from the peak fluorescent in each fluorescent profile. From these X and Y coordinates in images, F-actin filament was approximated as a quadratic curve. We reconstructed the F-actin filament each time. The obtained spatial resolution was 0.08 μm which was 2.5 times higher than the optical resolution. In quasi super-resolution images, the fluctuation amplitude of F-actin was 0.42 to 0.53 μm in the peripheral region and 0.27 μm in the center region. The frequency of F-actin fluctuation in both center and peripheral regions was approximately 3 Hz. The F-actin fluctuation was irregular, and several fluctuation patterns were observed. These phenomena might be caused due to the influence of restriction of the cytoskeletal network.