Mechanism of Actin Polymerization in Cellular ATP Depletion

Abstract

Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin ␤ 4 for ATP-and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK 1 . ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce Factin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin ␤ 4 from 4 to 11 M. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.Recent progress in understanding the function of actin-binding proteins has clarified their role in a number of processes in normal cells, including motility and the establishment of cell polarity (1, 2). However, our understanding of actin dynamics and the roles of actin-binding proteins under conditions of cellular stress pertinent to pathophysiology is currently limited (3). For example, in tissue ischemia the lack of oxygen and nutrients is known to rapidly result in decreased intracellular ATP levels and increased ADP levels, varying in degree with the severity and duration of ischemic time (4, 5). These results have been associated with a concomitant decrease in cellular G-actin and a corresponding increase in the fraction of polymerized actin observed both in vivo and in vitro (6 -11), with the resultant F-actin accumulating as dispersed aggregates throughout the cytoplasm (12, 13), notably in the perinuclear region (14 -16).Cells maintain a high potential energy for actin polymerization by maintaining a high total actin concentration and reserving a large fraction of this actin in a pool of monomers available for polymerization. This is of considerable functional importance for the cell, because the rate of actin polymerization is directly proportional to the local free monomer concentration (17). However, this high concentration requires the deployment of actin-binding proteins to maintain the monomer poo...

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