Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.

ABSTRACT

Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.