Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Abstract

Abstract. 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.

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

“…␤-actin does not substitute for muscle actin in either the formation of stress fibers (17) or myofibrils in cardiomyocytes (18). In addition, it seems to interact more tightly with certain actin binding proteins that may function at the leading edge of crawling cells.…”

“…In this hypothesis, local accumulation of a nonfilamentous form of actin that could be released suddenly upon stimulation of motility would determine the location of actin polymerization. Potential storage particles containing nonfilamentous actin have been identified by comparing the localization patterns of vitamin D-binding protein, which binds to G-actin with 5 nM k d , and phalloidin, which binds to actin (17). These stores of nonfilamentous actin are found at the leading edge and are located adjacent to sites of actin polymerization and in the region of the cell where the ␤-actin mRNA is also present.…”

Features of senescence and cell death induced by doxorubicin in A549 cells: organization and level of selected cytoskeletal proteins

“…The second, indirect procedure for staining of nonfilamentous actin was performed as previously described (Cao et al 1993;Meijerman et al 1997;Akisaka et al 2001). Cells grown on coverslips were washed with PBS and fixed with 3.7% (w/v) paraformaldehyde (Serva) in PBS for 20 min at room temperature.…”

Regulation of Phospholipase D Activity by Actin

“…PLD1 Binds to Membrane-associated G-actin-Both monomeric G-actin and actin filaments are associated with eukaryotic membranes (59,60). Therefore, we sought to determine whether the membrane-localized interaction between actin and PLD1, as evidenced by the co-immunoprecipitation results, was due to G-actin, F-actin, or both.…”