Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate.     

Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ

Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.     

Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels.

Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.     

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.     

Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d- cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co- localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.     

Mutual effects of alpha-actinin, calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

Simultaneous interaction of actin with alpha-actinin and calponin

Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.     

Alpha-actinin and actin in the outer retina: a double immunoelectron microscopic study

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.     

Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with alpha-actinin.

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.     

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.     

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes

Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the model’s ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.     

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.     

Cytochalasin B and the structure of actin gels

We analyzed the structure of gels formed when macrophage actin-binding protein crosslinks skeletal muscle actin polymers and the effect of the fungal metabolite cytochalasin B on this structure. Measurement of the actin filament length distribution permitted calculation of the critical concentration of crosslinker theoretically required for gelation of actin polymer networks. The experimentally determined critical concentration of actin-binding protein agreed sufficiently with the theoretical to conclude that F-actin-actin-binding protein gels are networks composed of isotropically oriented filaments crosslinked at intervals. The effects of cytochalasin B on these actin networks fits this model. Cytochalasin B (1) bound to F-actin (but not to actin-binding protein), (2) decreased the length of actin filaments without increasing the quantity of monomeric actin, (3) decreased the rigidity of actin networks both in the presence and absence of crosslinking proteins and (4) increased the critical concentration of actin-binding protein required for incipient gelation by a magnitude predicted from network theory if filaments were divided and shortened by the extents observed. The effects of cytochalasin B on gelation were highly dependent on actin concentration and were inhibited by the actin-stabilizing agent phalloidin. Therefore, cytochalasin B diminishes actin gel structure by severing actin filaments at limited sites. The demonstration of gel-sol transformations in actin networks caused by limited actin filament cleavage suggests a new mechanism for the control of cytoplasmic structure.     

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.     

Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure

LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate.