Localization and dynamics of nonfilamentous actin in cultured cells

Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.