Role of the DNase-I-Binding Loop in Dynamic Properties of Actin Filament

Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.     

Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins

The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.     

A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers

ADF/cofilins (AC) are essential F- and G-actin binding proteins that modulate microfilament turnover. The genome of Plasmodium falciparum, the parasite causing malaria, contains two members of the AC family. Interestingly, P. falciparum ADF1 lacks the F-actin binding residues of the AC consensus. Reverse genetics in the rodent malaria model system suggest that ADF1 performs vital functions during the pathogenic red blood cell stages, whereas ADF2 is not present in these stages. We show that recombinant PfADF1 interacts with monomeric actin but does not bind to actin polymers. Although other AC proteins inhibit nucleotide exchange on monomeric actin, the Plasmodium ortholog stimulates nucleotide exchange. Thus, PfADF1 differs in its biochemical properties from previously known AC proteins and seems to promote turnover exclusively by interaction with actin monomers. These findings provide important insights into the low cytosolic abundance and unique turnover characteristics of actin polymers in parasites of the phylum Apicomplexa.     

Binding of myosin subfragment-1 to F-actin.

During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Cofilin (ADF) affects lateral contacts in F-actin

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.     

Two opposite effects of cofilin on the thermal unfolding of F-actin: a differential scanning calorimetric study

Differential scanning calorimetry was used to examine the effects of cofilin on the thermal unfolding of actin. Stoichiometric binding increases the thermal stability of both G- and F-actin but at sub-saturating concentrations cofilin destabilizes F-actin. At actin:cofilin molar ratios of 1.5-6 the peaks corresponding to stabilized (66-67 degrees C) and destabilized (56-57 degrees C) F-actin are observed simultaneously in the same thermogram. Destabilizing effects of sub-saturating cofilin are highly cooperative and are observed at actin:cofilin molar ratios as low as 100:1. These effects are abolished by the addition of phalloidin or aluminum fluoride. Conversely, at saturating concentrations, cofilin prevents the stabilizing effects of phalloidin and aluminum fluoride on the F-actin thermal unfolding. These results suggest that cofilin stabilizes those actin subunits to which it directly binds, but destabilizes F-actin with a high cooperativity in neighboring cofilin-free regions.     

Photoaffinity labeling of the nucleotide binding site of actin

Rabbit skeletal muscle actin was photoaffinity-labeled by the nucleotide analogue 8-azidoadenosine 5'-triphosphate. In both G-actin and F-actin about 25% covalent incorporation was achieved. The labeled actins were digested with cyanogen bromide, and the labeled peptides were isolated and sequenced. In F-actin the label was bound primarily to Lys-336, while in G-actin the label was bound to Lys-336 or to Trp-356. The results indicate that the nucleotide binding site is near the phalloidin binding site of actin [Vanderkerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818]. The binding of the azido group to Trp-356 in G-actin but not in F-actin may indicate that a change in the conformation of actin occurs in this region.     

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.     

Amplitude of the tension developed by glycerinated muscle fibers during rigidity depends on the nature of the structural reorganizations in F-actin induced by formation of the actomyosin complex

Dependence between the amplitude of tension, developed by glycerinated muscle fibers during rigidity, and the character of structural changes in F-actin, induced by the formation of actomyosin complex, was studied by polarized microfluorimetry and tensiometry. It is shown that during rigidity the anisotropy of intrinsic tryptophan residues as well as of rhodamine phalloidin bound to F-actin, and amplitude of tension depend on pH (6-8) and ionic strength (mu = 0.07 M-0.14 M) of solution. Greater changes in polarized fluorescence and in amplitude of tension were registered during rigidity in solutions with low ionic strength (mu = 0.07 M) and pH 8. It suggested that the amplitude of muscle fibre tension depends on the relative quantity of actin monomers, being in the "switched on" state.     

Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability

Rabbit muscle actin reacts with 2,4-dinitrophenylglutathionyldisulfide, forming a mixed disulfide in position 374. The product S-(cysteine-374)glutathionyl actin forms filaments which are easily disrupted under shearing stress. Even weak mechanical strain, as exerted, for example, during capillary viscometry or heating the solution to 37°C, leads to considerable breakage of these filaments. Because of spontaneous repair which consumes ATP, the mechanically broken filaments exhibit an approx. 6-fold enhanced steady-state ATPase activity as compared to normal F-actin. Monomers of glutathionyl actin have a reduced affinity for their bound nucleotide and a slightly increased critical concentration. Disruption of the filaments and enhanced ATPase activity are reversed by the addition of KCl or the mushroom toxin phalloidin. By the large stabilizing effects of KCl and phalloidin on glutathionyl actin filaments we propose glutathionyl actin as a tool for detecting filament-stabilizing agents and for studying the different mechanisms of filament stabilization     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.     

Phallotoxin and actin binding assay by fluorescence enhancement.

The fluorescence of five fluorophores conjugated to phallotoxins was found to be specifically enhanced upon binding to F-actin in a polymerizing buffer. Rhodamine phalloidin had the greatest fluorescence enhancement of ninefold. The fluorescence titration of rhodamine phalloidin by actin was shown to be consistent with stoichiometric binding. The fluorescence enhancement of rhodamine phalloidin at 5 microM is linearly related to F-actin concentrations up to 2 microM and therefore can be used as an easy means of F-actin quantitation. In a competition assay, other phallotoxins reduce the fluorescence enhancement that results from the binding of rhodamine phalloidin to polymerized actin. This reduction also permits a convenient measurement of the binding constants of any competing phallotoxins.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts

Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.     

Antagonistic effects of cofilin, beryllium fluoride complex, and phalloidin on subdomain 2 and nucleotide-binding cleft in F-actin

Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound epsilon-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of epsilon-ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2.     

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.     

Characterization of carbethoxylated actin

The carbethoxylation of histidine residues in G-actin impairs actin polymerization. The histidine residue essential for polymerization was identified as histidine-40 [Hegyi, G., Premecz, G., Sain, B., & Mühlrad, A. (1974) Eur. J. Biochem. 44, 7-12]. Non-polymerizable actin was separated from the polymerizable fraction after partial carbethoxylation. The non-polymerizable actin recovered the ability to polymerize following addition of phalloidin. Taking into account the evidence that phalloidin does not bind to G-actin in the absence of salt, the results indicate that the actin monomer undergoes a conformational change and subsequently binds phalloidin before polymerization. The resulting polymers activated S1 ATPase activity as effectively as control F-actin. In the presence of tropomyosin and troponin, a strong inhibition of actin-activated ATPase activity was observed in the absence of Ca2+, although no inhibition was observed in the presence of Ca2+. These results indicate that His-40 is not directly involved in a myosin binding site nor in a tropomyosin-troponin binding site.     

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.