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 beta4 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 beta4 from 4 to 11 microm. 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.     

Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.     

Beta thymosins as actin binding peptides

The beta thymosins are a highly conserved family of strongly polar 5 kDa polypeptides that are widely distributed among vertebrate classes; most are now known to bind to monomeric actin and inhibit its polymerization. One beta thymosin, beta four, (Tβ⁴) is the predominant form in mammalian cells, present at up to 0.5 mM. Many species are known to produce at least two beta thymosin isoforms, in some cases in the same cell. Their expression can be separately regulated. When present outside the cell, the N-terminal tetrapeptide of beta four appears to affect cell cycle regulation; beta thymosins or smaller fragments derived from them may have additional regulatory functions. We suggest that many developmental changes in beta thymosin levels within cells and tissues may be related to changes in G-actin pool size.     

Localization of thymosin β10 in breast cancer cells: relationship to actin cytoskeletal remodeling and cell motility

Beta-thymosins are polypeptides involved in the regulation of actin polymerization and thymosin β10 and β4 have been implicated in sequestration of monomeric (G-) actin. Additionally, experimental overexpression of thymosin β10 has been found to result in increases in F-actin bundles as well as in cell motility and spreading. We have studied the distribution of endogenously expressed thymosin β10 in cultured human breast cancer cell lines. Both unperturbed monolayer cultures and wound-healing models were examined using double-staining for thymosin β10 and polymerized (F-) actin. Our findings show that thymosin β10 is expressed in all three-cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-231) studied. No or little staining was detected in confluent cells, whereas strong staining occurred in semiconfluent cells and in cells populating monolayer wounds. Importantly, the distribution of staining for thymosin β10 was inverse of staining for F-actin. These data support a physiological role for thymosin β10 in sequestration of G-actin as well as in cancer cell motility.     

beta-Thymosins, small acidic peptides with multiple functions

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.     

Identification of a cofilin-like actin-binding site on translationally controlled tumor protein (TCTP)

Translationally controlled tumor protein (TCTP) expression is suppressed during cancer cell reversion to a non-malignant phenotype. We identified a primary sequence of TCTP with homology to ADF/cofilin. We confirm that a synthetic peptide corresponding to this sequence binds specifically to actin and is displaced from actin by cofilin. TCTP peptide has higher affinity for G-actin than F-actin and does not block actin-filament depolymerization by cofilin. These results suggest that TCTP may channel active cofilin to F-actin, enhancing the cofilin-activity cycle in invasive tumor cells. Loss of TCTP may result in sequestration of active cofilin by a monomeric pool of actin.     

Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin

Thymosin beta(4) possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin beta(4) serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin beta(4) may serve as a useful tool for further investigations in cell biology. Thymosin beta(4) could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin beta(4) concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin beta(4) may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Chemotactic peptide modulation of actin assembly and locomotion in neutrophils

To determine the relationship between the state of actin polymerization in neutrophils and the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced changes in the locomotive behavior of neutrophils, the mean rate of locomotion (mROL), the percent G-actin, and the relative F-actin content of neutrophils were determined. The mROL was quantified by analysis of the locomotion of individual cells; the percentage of total actin as G-actin was measured by DNase I inhibition; and the F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained neutrophils. Neutrophils stimulated with fMLP exhibit a change in their mROL that is biphasic and dose dependent. The mROL of neutrophils exposed to 10(-8) M fMLP, the KD, is 11.9 +/- 2.0 micron/min (baseline control 6.2 +/- 1.0 micron/min). At 10(-6) M fMLP, the mROL returns to baseline levels. Stimulation of neutrophils with fMLP also induces action polymerization. Evidence for actin polymerization includes a 26.5% reduction in G-actin and a twofold increase in the amount of NBD-phallacidin staining of cells as determined by FACS analysis. The NBD-phallacidin staining is not due to phagocytosis, is inhibited by phalloidin, requires cell permeabilization, and is saturable at NBD-phallacidin concentrations greater than 10(-7)M. The fMLP-induced increase in NBD-phallacidin staining occurs rapidly (less than 2 min), is temperature dependent, and is not due to cell aggregation. Since NBD-phallacidin binds specifically to F-actin, the increase in fluorescent staining of cells likely reflects an increase in the F-actin content of fMLP-stimulated cells. FACS analysis of NBD-phallacidin-stained cells shows that the relative F-actin content of neutrophils stimulated with 10(-11)-10(-8)M fMLP increases twofold and remains increased at concentrations greater than 10(-8)M fMLP. Therefore, the fMLP-induced increase in F-actin content of neutrophils as determined by FACS analysis of NBD-phallacidin-stained cells coincides with a decrease in G-actin and correlates with increased mROL of neutrophils under some (10(-11)-10(-8)M fMLP) but not all (greater than 10(-8)M fMLP) conditions of stimulation. Quantification of the F-actin content of nonmuscle cells by FACS analysis of NBD-phallacidin-stained cells may allow rapid assessment of the state of actin polymerization and correlation of that state with the motile behavior of nonmuscle cells.     

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.     

Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.     

Latrunculin a can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.     

Plasma levels of F-actin and F:G-actin ratio as potential new biomarkers in patients with septic shock

Objective: To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls.

Results: F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay’s lowest detection range (78?ng/ml).

Conclusions: F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.     

Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin beta 4 in migrating IEC-6 cells

The cause of reduced migration ability in polyamine-deficientcells is not known, but their actin cytoskeleton is clearly abnormal.We depleted polyamines with -difluoromethylornithine (DFMO) inmigrating cells with or without stimulation by epidermal growth factor(EGF) and investigated filamentous (F-) actin, monomeric (G-) actin,and thymosin 4 (T4), using immunofluorescent confocal microscopy,DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cellinterior, increased it in the cell cortex, redistributed G-actin, andincreased nuclear staining of T4. However, DFMO did not affect theamount of T4 mRNA. EGF caused a rapid increase in the staining ofF-actin in control cells, but DFMO prevented this response to EGF.Despite the visible changes shown by immunocytochemistry, statisticallysignificant changes in the amount of either actin isoform or of totalactin did not occur. We propose that DFMO reduces migration byinterfering with the sequestration of G-actin by T4 and theassociation of F-actin with activated EGF receptors.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor

Human actin-depolymerizing factor (ADF) and cofilin are pH-sensitive, actin-depolymerizing proteins. Although 72% identical in sequence, ADF has a much higher depolymerizing activity than cofilin at pH 8. To understand this, we solved the structure of human cofilin using nuclear magnetic resonance and compared it with human ADF. Important sequence differences between vertebrate ADF/cofilins were correlated with unique structural determinants in the F-actin-binding site to account for differences in biochemical activities of the two proteins. Cofilin has a short beta-strand at the C terminus, not found in ADF, which packs against strands beta3/beta4, changing the environment around Lys96, a residue essential for F-actin binding. A salt bridge involving His133 and Asp98 (Glu98 in ADF) may explain the pH sensitivity of human cofilin and ADF; these two residues are fully conserved in vertebrate ADF/cofilins. Chemical shift perturbations identified residues that (i) differ in their chemical environments between wild type cofilin and mutants S3D, which has greatly reduced G-actin binding, and K96Q, which does not bind F-actin; (ii) are affected when G-actin binds cofilin; and (iii) are affected by pH change from 6 to 8. Many residues affected by G-actin binding also show perturbation in the mutants or in response to pH. Our evidence suggests the involvement of residues 133-138 of strand beta5 in all of the activities examined. Because residues in beta5 are perturbed by mutations that affect both G-actin and F-actin binding, this strand forms a "boundary" or "bridge" between the proposed F- and G-actin-binding sites.