Mechanism of Actin Polymerization in Cellular ATP Depletion

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 β4 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-PK1. 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 F-actin 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 β4 from 4 to 11 μm. 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. 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 β4 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-PK1. 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 F-actin 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 β4 from 4 to 11 μm. 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 (1Nelson W.J. Nature. 2003; 422: 766-774Crossref PubMed Scopus (578) Google Scholar, 2Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). However, our understanding of actin dynamics and the roles of actin-binding proteins under conditions of cellular stress pertinent to pathophysiology is currently limited (3Atkinson S.J. Molitoris B.A. Molitoris B.A. Finn W.F. Acute Renal Failure: A Companion to Brenner and Rector's The Kidney. 1st Ed. W. B. Saunders, Philadelphia2001Google Scholar). 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 (4Gerlach E. Deuticke B. Dreisbach R. Rosarious C. Pflügers Arch. 1963; 278: 296-315Crossref Scopus (79) Google Scholar, 5Weinberg J.M. Kidney Int. 1991; 39: 476-500Abstract Full Text PDF PubMed Scopus (307) Google Scholar). 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 (6Sutton T.A. 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This is of considerable functional importance for the cell, because the rate of actin polymerization is directly proportional to the local free monomer concentration (17Cooper J.A. Annu. Rev. Physiol. 1991; 53: 585-605Crossref PubMed Scopus (243) Google Scholar). However, this high concentration requires the deployment of actin-binding proteins to maintain the monomer pool because, in the absence of other factors, all actin in excess of the dissociation equilibrium constant for subunit binding (commonly termed the critical concentration) polymerizes. The high concentration of actin monomer is maintained by the activity of actin monomer-binding proteins (18Sun H.Q. Kwiatkowska K. Yin H.L. Curr. Opin. Cell Biol. 1995; 7: 102-110Crossref PubMed Scopus (172) Google Scholar). In most metazoan cells a large fraction of the monomer pool is probably associated with thymosins (T) 1The abbreviations used are: TthymosinTRITCtetramethylrhodamine isothiocyanate. (19Nachmias V.T. Curr. Opin. Cell Biol. 1993; 5: 56-62Crossref PubMed Scopus (97) Google Scholar). These low molecular weight (∼5,000) proteins act as monomer-sequestering factors in which thymosin-bound ATP-G-actin is unable to associate with filament ends or other monomers. The predominant forms of thymosin are thymosin β4 and, to a much lesser extent, thymosin β10. In contrast, profilin-bound monomers are able to associate with actin filament barbed ends (fast growing ends; the predominant site for actin polymerization in the cell), but association with filament pointed ends and spontaneous nucleation, or de novo polymerization, is blocked (20Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (239) Google Scholar, 21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (131) Google Scholar). In most vertebrate cell types the concentration of the various forms of thymosin far exceeds that of profilin so that under normal physiological conditions the bulk of the monomer pool is associated with thymosin (22Cassimeris L. Safer D. Nachmias V.T. Zigmond S.H. J. Cell Biol. 1992; 119: 1261-1270Crossref PubMed Scopus (138) Google Scholar). The association and dissociation rates for the interaction between thymosin and actin are rapid and result in a very small pool of free G-actin (concentration <1 μm) and rapid flux of monomers between the thymosin-bound pool and F-actin (23Carlier M.F. Jean C. Rieger K.J. Lenfant M. Pantaloni D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5034-5038Crossref PubMed Scopus (135) Google Scholar). thymosin tetramethylrhodamine isothiocyanate. Actin monomers bind either ATP or ADP, and both forms of G-actin are competent for polymerization. However, the nature of the bound nucleotide modulates the kinetics of association and dissociation, with distinct effects at the barbed and pointed ends of the filament (24Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (604) Google Scholar). Moreover, the affinities of ATP- and ADP-G-actin monomers for thymosin and profilin differ considerably. Both profilin and thymosin β4 bind ATP monomers with higher affinity than ADP monomers (21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (131) Google Scholar, 23Carlier M.F. Jean C. Rieger K.J. Lenfant M. Pantaloni D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5034-5038Crossref PubMed Scopus (135) Google Scholar), but the difference in affinity is greater by far for thymosin (KD(ADP) 100 μm, KD(ATP) 0.6 μm) (23Carlier M.F. Jean C. Rieger K.J. Lenfant M. Pantaloni D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5034-5038Crossref PubMed Scopus (135) Google Scholar). In normal cells this preference for ATP monomers ensures that the pool of unpolymerized actin consists almost entirely of ATP-G-actin with no significant amount of ADP-G-actin in the thymosin-sequestered pool (25Safer D. Nachmias V.T. BioEssays. 1994; 16: 473-479Crossref PubMed Scopus (91) Google Scholar, 26De La Cruz E.M. Ostap E.M. Brundage R.A. Reddy K.S. Sweeney H.L. Safer D. Biophys. J. 2000; 78: 2516-2527Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Conceivably, conditions such as ischemia that result in the depletion of ATP-G-actin and accumulation of ADP-G-actin would result in decreased levels of thymosin-bound actin as a direct result of the differential affinity of thymosin for ATP- and ADP-actin (Fig. 1B). The actin monomers thus released would increase the free monomer concentration to levels that exceed the critical concentration for polymerization, resulting in rapid polymerization at available nuclei or even, perhaps, initiation of new filament growth. To directly test this hypothesis, which predicts that ATP depletion will result in decreased concentrations of ATP-actin monomers and increased free thymosin β4, we measured the concentrations of ATP, ADP, ATP-monomer, ADP-monomer, and thymosin-monomer using the mitochondrial poison antimycin A and substrate depletion to induce ATP depletion and mimic ischemia in LLC-PK1 cells. Cell Culture—A clonal line of LLC-PK1 cells was grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal bovine serum containing penicillin/streptomycin, passaged once per week with 0.5% trypsin/EDTA, and seeded at a 1:8 dilution. Cultures typically reached confluence 2 or 3 days after seeding, and experiments after confluence in or and from ATP by in modified Eagle's medium or containing antimycin A for to 30 min as B.A. R. Hosford M. Am. J. Physiol. Google Scholar). For experiments the effect of cellular levels of ATP on actin polymerization, cells in depletion containing In cells in D for 30 min depletion and during cellular ATP depletion. Cellular G-actin in Cells with of in and 2 with and and with A and per of to free at for min at 4 To actin with bound we used J. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar). content was to the in Cellular ATP and ADP by high as Am. J. Physiol. 2000; PubMed Google Scholar). Cellular F-actin content was measured by an of the of Zigmond S.H. J. Cell Biol. 1991; PubMed Scopus Google for cells grown in a cells with in for 30 min at and for with a containing 0.5% in was measured on a at for actin and for and Cellular β4 with as and by a molecular for at thymosin was in the and thymosin bound to G-actin in the was on was measured by of and by or in in min for or min for to to in by of or profilin of and La was used for of and by to to the of Safer D. PubMed Scopus Google Scholar). complexes by the in of actin on with the complexes by for actin and profilin under of and by Renal using an of the of Weinberg J.M. J. Clin. Invest. PubMed Scopus Google Scholar). Kidney was in in a with at for 30 and a The from by was by the ratio of from after a with and concentration of of intracellular was by the per by the intracellular per ATP in of to used ATP depletion in a clonal line of LLC-PK1 cells as a of ischemic injury Geerdes Molitoris B.A. Am. J. Physiol. 1991; Google Scholar). Cells with the mitochondrial antimycin A μm) and with medium to both and as of The intracellular ATP concentration rapidly in to this treatment (Fig. from levels of to after min of depletion. The concentration of ADP to a of at min of depletion, to at 30 The ratio of from to by min (Fig. and decreased to by 30 min of ATP depletion. To the effect of intracellular ATP on the actin we measured the fraction of and monomeric actin as a function of the duration of ATP depletion (Fig. cellular F-actin was by the binding of that but not monomeric to cells, and G-actin was on to the activity of ATP depletion of to 30 min there was a increase in the fraction of to a higher than that observed under physiological conditions (Fig. a decrease in the monomeric fraction of to the under physiological conditions, with the increase in The total intracellular actin concentration measured by was μm, the G-actin concentration was the observed in and F-actin levels a decrease in monomeric actin from to μm, with a corresponding increase in F-actin from to that are the in the ATP depletion 30 min did not result in significant in the F-actin which at line after min of ATP depletion. We measured the of F-actin in a fraction using high to large or the bulk (Fig. The F-actin after 30 and min of depletion was the that was in the high significant small was observed not Actin polymerization by ATP depletion was not the result of of actin as treatment no effect on F-actin levels during depletion or not in our and by other B.A. Geerdes A. McIntosh J.R. J. Clin. Invest. 1991; 88: 462-469Crossref PubMed Scopus (134) Google Scholar), cells actin and stress and, aggregates throughout the in the perinuclear intracellular concentrations of actin and actin-binding in a new To the nature of the to F-actin formation in cells, we cells with the D μm) to actin filament by monomer filament barbed ends (Fig. J.A. J. Cell Biol. 1987; PubMed Scopus Google Scholar). D effect on F-actin accumulation during ATP depletion and, in in increased F-actin accumulation after min of ATP depletion We to the energy which actin polymerization was To this we a of concentrations to the medium during 30 min of antimycin A treatment (Fig. In the of antimycin A the intracellular ratio as a function of the concentration a of concentrations from to concentrations greater than in which the ratio was greater than there was no effect on F-actin levels, but at in which the ADP ratio ATP and ADP concentrations of and μm, a increase in F-actin content was The F-actin content was a function of the decrease in the ratio as the concentration was decreased from to concentrations are μm, with ADP concentrations constant in the of of ATP on the to the the effect of ATP depletion on the nucleotide state of we unpolymerized G-actin monomers by binding to on and measured the fraction of ATP and ADP associated with the monomers (Fig. We and in the to nucleotide exchange J. M. J. Cell Biol. PubMed Scopus Google and polymerization or during the the cellular concentration of G-actin with ATP depletion, from to in cells after min of ATP depletion (Fig. was a corresponding decrease in the concentration of ATP However, the concentration of ADP G-actin constant at throughout the time of the potential importance of of actin monomer-binding we measured their concentrations in the LLC-PK1 cells used for experiments β4 was at high concentrations the concentration of G-actin in the cells under normal conditions The concentration of profilin was much a we measured the levels of proteins as a fraction of the total in LLC-PK1 cells and in tissue epithelial from in very to in the LLC-PK1 cells the physiological of our results for ischemic injury in the To the effect of ATP depletion on G-actin association with we used with a molecular weight of to the free fraction of thymosin β4 from the fraction associated with G-actin (Fig. In cells under normal conditions the concentration of free thymosin β4 was μm, to 11 after min of ATP depletion (Fig. This to a of all thymosin-bound actin monomers in the min of ATP depletion. We the of complexes by using with to the of the complexes observed (Fig. complexes of free actin probably because thymosin binding the of available to actin (Fig. La Cruz E.M. Ostap E.M. Brundage R.A. Reddy K.S. Sweeney H.L. Safer D. Biophys. J. 2000; 78: 2516-2527Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, D. R. Nachmias V.T. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar). of cell G-actin predominantly in the thymosin-bound fraction (Fig. in from cells to min of ATP depletion G-actin was and the was of free actin monomers (Fig. We used D. PubMed Scopus Google to the of actin monomer association with profilin (Fig. increased levels of profilin with actin monomer in from cells for min 4 and that was by the of an ATP to the In this we for a direct mechanism for actin polymerization on the differential affinity of ATP- and ADP-actin monomers for the monomer thymosin We that ATP depletion induced by substrate depletion and antimycin A in net conversion of G-actin to The increase in the fraction of cellular actin by or as by binding was in the min of depletion. in which we the ratio by the cells in concentrations of to a mechanism that directly the ATP concentration or the ratio because a critical ADP ratio which there was an between ratio and net F-actin of a direct biochemical treatment with antimycin A and substrate depletion in the of as in of both in vivo and in vivo Am. J. Physiol. 2000; PubMed Google Scholar, Weinberg J.M. J. Clin. Invest. 1988; PubMed Scopus Google Scholar), because ADP to and is to or which is from cells by and J.M. Kidney Int. 1991; 39: 476-500Abstract Full Text PDF PubMed Scopus (307) Google Scholar). F-actin formation during ATP depletion was not by treatment with and in we observed that cells F-actin than in the absence of the the concentration used μm) D actin monomers and the of monomers the barbed ends of actin J.A. J. Cell Biol. 1987; PubMed Scopus Google Scholar), with of the of ATP-actin than of results using that much of the F-actin during ATP depletion not by barbed end polymerization mechanism that for the bulk of actin polymerization induced by normal but by pointed end which is to a much lesser degree by pointed by and Pollard Pollard T.D. Biochemistry. 1991; PubMed Scopus Google Scholar), the conversion of ATP- to ADP-actin may be by which could for the higher levels of F-actin in cells with with in the absence of the The of this effect at time a of this These results that the mechanism of F-actin formation in cells is than that which ATP ADP as it in normal cells, in which pointed end polymerization is for the critical concentration for polymerization of ATP monomers is at the barbed end than at the pointed monomers bound to profilin are able to filament barbed ends but not pointed ends or to participate in de novo filament and the pointed ends of most actin in the of normal cells are for monomer because most are T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). However, in the conditions that during ischemia or in ATP depletion, that most actin monomers are in the the critical concentrations not differ between the pointed and barbed ends (24Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (604) Google Scholar), and monomers will from profilin because of the affinity of profilin for ADP monomers (21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (131) Google and thus be available for pointed end addition. (Fig. the The concentration of thymosin β4 is high in cells to a fraction of the unpolymerized We measured a concentration of thymosin β4 in LLC-PK1 cells of 11 μm, a that the for the measured concentration of G-actin in cells at ATP depletion results in a decrease in the concentration of ATP monomers but not ADP monomers. We measured a in ATP-G-actin concentration but in ADP-G-actin concentration during depletion. the ratio of monomers after 30 min of depletion (Fig. is much higher than would be from the ratio of in the at this time with the higher affinity of monomer for ATP, would a ratio of with flux of ADP monomers from the unpolymerized to the polymerized The concentration of free thymosin in to the decrease in the concentration of the G-actin Increased levels of profilin-actin complexes could increase the rate of exchange of ADP for ATP on actin monomers. The all of this proteins bind actin monomers to their effects on actin and are and during ATP depletion. The concentration of in LLC-PK1 cells is low S.L. Sandoval R.M. J.R. Molitoris B.A. Am. J. Physiol. 2003; PubMed Scopus Google but there are higher levels of that we to be to the levels of to ADP profilin for binding (21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. 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The low affinity of the interaction between ADP monomers and thymosin is to monomers to be on so that of nucleotide exchange by profilin ATP monomers for rapid filament barbed all cell types high concentrations of thymosin β4 or In for example, the concentration of profilin is on affinity for to for the monomer pool, and factors not to a significant role (21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (131) Google Scholar, V.K. D.B. Pollard T.D. J. Cell Sci. 112: Google Scholar). would be to the effect of ATP depletion in cells such as that in the absence of is to that in cell types that significant of thymosin is associated with including formation of actin that are with the of ischemia or ATP depletion K. K. M. M. R. PubMed Scopus Google Scholar). is to the we mechanism that has to to an energy in the it has been that of actin dynamics in to ATP depletion result in levels of cell K. K. M. M. R. PubMed Scopus Google Scholar). However, because our mechanism to result directly from the of the of actin and monomer-binding proteins this may not be the is that cells such as the free and that are to direct in of of energy depletion are the very cell types that not thymosin as of their actin T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar, 21Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (131) Google Scholar, V.K. D.B. Pollard T.D. J. Cell Sci. 112: Google Scholar). is by the monomer by thymosin may be by the effects of the of a large fraction of the pool the cell is will be to polymerization of monomers by the mechanism is directly to cells. We and for We are to Nachmias for and with for of thymosin and to Cooper for important and