Imaging of Dynamic Changes of the Actin Cytoskeleton in Microextensions of Live NIH3T3 Cells with a GFP Fusion of the F-Actin Binding Domain of Moesin

C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent.Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.Lamellipodia, filopodia, retraction fibers and microspikes are dynamic and often transient membraneous structures on the surface of most cells. They can readily be observed in spreading, moving and dividing cultured cells, but also in migrating cells during development and inflammation, or in invading cancer cells in vivo. Recent evidence suggests that small GTPases of the rho family regulate this protrusive cell surface activity [1,2]. Using a permeabilized Swiss3T3 cell system, Mackay et al. [3] have shown recently that moesin, and possibly its relatives ezrin or radixin, are necessary for cellular responses induced by rho, namely the formation of lamellipodia, focal adhesion complexes and stress fibers in serum-starved fibroblasts. One or more members of this protein family is also required for the formation of filopodia in growth cones of neuronal cells [4], but how moesin interacts with the actin cytoskeleton