Non-Invasive Imaging of F-Actin Dynamics in Living Cells by Atomic Force Microscopy

Abstract

Active and dynamic regulation of actin filaments (F-actin) is a key process in both constitutive and adaptive maintenance of cellular activities. We utilized a high-speed atomic force microscope (AFM) combined with an optical microscopy system, to visualize the dynamics of sub-membrane actin networks with sub-second time scale. Direct AFM imaging of a living COS-7 cell surface in growth medium revealed a cell-surface topography and membrane dynamics including endo/exocytosis. Increasing the scanning force enabled visualization of individual F-actin fibers constituting cortical actin networks. These filaments formed a three-dimensional meshwork with sizes of 1.7-14.0×104 nm2, which is in good agreement with the previous observations by super-resolution microscopy. Sub-second imaging of these actin networks over time revealed various dynamic rearrangements of the actin fibers and movements of actin-bound complexes. Fast polymerization of actin fibers was observed at a rate of >0.25 µm/sec, which is significantly faster than in vitro analyses (< 0.10 µm/sec). We occasionally observed actin-bound particles with various shapes (globule and rod) and sizes (100-500 nm along the long axis) moving along the fiber. Detailed analysis of the movement revealed that they moved in one direction along the fiber intermittently with an average velocity ranging 16-30 nm/sec. This is the first study which visualizes dynamics of single F-actin fibers with their surrounding environment in a live cell with high temporal and spatial resolution, and provides a novel non-invasive technique to investigate F-actin dynamics in a live cell.