Cytoskeletal actin filaments are typically nucleated at the plasma membrane, rather than the centrosome that acts as an organizational center for microtubules. Thus, it is the cell periphery that generally contains the highest concentration of actin filaments. When located directly underneath the plasma membrane, actin filaments comprise part of the cell cortex, which regulates the form and motion of the surface of the cell, playing a key role in whether or not a cell develops projections such as filopodia or microvilli. A number of external factors generally control actin filament nucleation and enable the cytoskeletal filaments to change their characteristics rapidly upon signaling. A group of special proteins, two of which are actin-related proteins (ARPs) that are very similar to actin, are chiefly responsible for nucleation catalysis. This ARP complex is most prominent in locations where actin filament growth needs to be a brisk process, and is associated with various signaling molecules and components of the plasma membrane.

Using double immunofluorescence the adherent culture of APM cells illustrated above was washed, fixed, permeabilized, blocked with 10-percent normal goat serum, and treated with a cocktail of rabbit anti-giantin (Golgi complex) and mouse anti-NPCP (nuclear pore complex proteins) primary antibodies. The target antigens were visualized with goat anti-rabbit and anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. The filamentous actin cytoskeletal architecture was labeled with Alexa Fluor 350 conjugated to phalloidin. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.