Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization

Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolymerization from the pointed end were measured in fluorescence assays using pyrenylactin filaments capped at the barbed end by villin. The initial pointed end depolymerization rate in the presence of tropomyosin averaged 56% of the control rate. Elongation from the pointed filament end in the presence of tropomyosin occurred at a lower free G-actin concentration, although the on rate constant, kappa p+, was not greatly affected. Furthermore, in the presence of tropomyosin, the free G-actin concentration was lower at steady state. Therefore, nonmuscle tropomyosin stabilizes the pointed filament end by lowering the off rate constant, kappa p-.     

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.     

Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomer. Formed filaments keep a constant length of about 1 μm. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomers and can stabilize the pointed ends of actin filaments. We observed that tropornyosintroponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.     

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491–1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin. Received: 1 October 1999 / Accepted: 29 November 1999     

Changes in Xenopus oocyte nucleus and cytoplasm organization after actin filaments depolymerization by latrunculin

Actin-containing filaments have been visualized inside the Xenopus oocyte nuclei due to combination of fluorescence and transmission electron microscopy. It has been shown that these filaments contact with nucleoli, spherical bodies and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing latrunculin causes membrane vesiculation in the cytoplasm, and disruption of the nucleoplasm and nuclear envelope integrity. We suppose that actin-containing filaments belong to crucial cell components which are involved in coordination of nuclear-cytoplasmic interactions as well as distribution and transport of intranuclear components in growing Xenopus oocytes.     

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.     

Kinetic analysis of actin assembly suggests that tropomyosin inhibits spontaneous fragmentation of actin filaments

The time-course of actin assembly was measured in the absence and in the presence of tropomyosin. The polymerization was followed by the fluorescence enhancement of a 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole label attached to actin molecules or by light-scattering. The kinetic curves measured in the absence and in the presence of tropomyosin revealed characteristic differences. Tropomyosin was found to retard actin polymerization and to cause the final constant actin monomer concentration to be reached slowly. In the absence of tropomyosin, the final constant actin monomer concentration was approached considerably faster. The time-course of polymerization was interpreted quantitatively in terms of inhibition of actin filament fragmentation by tropomyosin molecules bound along the filaments. Within the limits of this model, actin monomers are consumed slowly in the presence of tropomyosin because the creation of new filament ends by spontaneous fragmentation is inhibited by tropomyosin.     

Lysophosphatidic acid inhibits anti-Fas-mediated apoptosis enhanced by actin depolymerization in epithelial ovarian cancer

Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.     

Purification and characterization of a new mammalian serum protein with the ability to inhibit actin polymerization and promote depolymerization of actin filaments

A protein with capacity to bind G-actin and the ability to inhibit polymerization and promote depolymerization of actin filaments has been isolated from the serum of rabbit. The protein, SAIP (for serum actin inhibitory protein), has been purified by affinity chromatography of serum over actin-Sepharose followed by protein fractionation with ammonium sulfate and chromatography over DEAE-cellulose. Five milligrams of purified SAIP is obtained from 100 mL of serum. Rabbit SAIP is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two closely related polypeptides of 60000 and 56000 daltons, respectively (ratio 5.1:1). Each of these polypeptides consists of two isoelectric variants. SAIP binds to monomeric actin with a stoichiometry of 1:1 and a Kd of 0.12 microM. The SAIP-actin complex binds to DNase I. Actin polymerization is completely inhibited by incubation of actin with an equal concentration of SAIP. At equimolar concentrations to F-actin, SAIP induces complete depolymerization of the actin filaments. SAIP is also present in calf serum.     

Calcium dependence of villin-induced actin depolymerization

"Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium (in addition to potassium and magnesium). It was observed that at high calcium concentrations (200 microM) the time course of depolymerization could not be described by the single exponential that defines it at low calcium and low villin levels. Instead, at high calcium, the exponent increased with time and the rate of depolymerization became greater than that of controls in the absence of villin. This contrasts with the inhibition of depolymerization by villin at low calcium. The latter inhibition is a consequence of the capping of the barbed filament end by villin as are the inhibition of filament elongation and the elevation of the critical concentration. Evidence is presented that the effects of villin at high calcium are the result of cutting of the actin filaments by villin. It thus appears that different calcium binding sites control capping and cutting and that the calcium binding sites regulating cutting have a much lower affinity for calcium than the sites regulating capping of the barbed filament ends.     

A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments

The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca(2+)-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.     

Formin-induced nucleation of actin filaments

Formins are proteins best defined by the presence of the unique, highly conserved formin homology domain 2 (FH2). FH2 is necessary and sufficient to nucleate an actin filament in vitro. The FH2 domain also binds to the filament's barbed end, modulating its elongation and protecting it from capping proteins. FH2 itself appears to be a processive cap that walks with the barbed end as it elongates.     

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo.     

INF2 Is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization

Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed "diaphanous formins" are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory domain (DID) and a C-terminal diaphanous autoregulatory domain (DAD). Here, we found several striking features for the mouse formin, INF2. First, INF2 interacted directly with actin through a region C-terminal to the FH2. This second interacting region sequesters actin monomers, an activity that is dependent on a WASP homology 2 (WH2) motif. Second, the combination of the FH2 and C-terminal regions of INF2 resulted in its curious ability to accelerate both polymerization and depolymerization of actin filaments. The mechanism of the depolymerization activity, which is novel for formin proteins, involves both the monomer binding ability of the WH2 and a potent severing activity that is dependent on covalent attachment of the FH2 to the C terminus. Phosphate inhibits both the depolymerization and severing activities of INF2, suggesting that phosphate release from actin subunits in the filament is a trigger for depolymerization. Third, INF2 contains an N-terminal DID, and the WH2 motif likely doubles as a DAD in an autoinhibitory interaction.     

The hyaluronate receptor is associated with actin filaments

The cell-surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., A. L. Thurn, and B. E. Lacy, 1985, J. Biol. Chem., 260:8128-8133) that is thought to mediate many of the effects that hyaluronate has on cell behavior, such as migration, angiogenesis, and phagocytosis. To determine if the receptor is associated with the underlying cytoskeleton, Swiss 3T3 cells were extracted with a solution of Triton X-100, which solubilized most of the cellular components, but which left behind an insoluble residue containing the cytoskeleton. This detergent-insoluble residue was found to contain the bulk of the hyaluronate-binding activity, suggesting that the receptor might indeed be associated with the cytoskeleton. To further define the cytoskeletal element with which the receptor interacts, 3T3 cells were extracted with Triton X-100 under a variety of different ionic conditions. In each case, the amount of hyaluronate-binding activity in the detergent-insoluble residue was related to the amount of actin present, but not to either tubulin or vimentin. In addition, the recovery of hyaluronate-binding activity was dramatically enhanced (to 100% in most cases) if the cells were extracted in the presence of phalloidin, a drug that stabilizes actin filaments. However, the recovery of binding activity was dramatically decreased when whole cells were treated with cytochalasin B before extraction, and when extracted cells were treated with DNase I, which promotes the depolymerization of actin filaments. In addition, preincubating an extract of SV-40-transformed Swiss 3T3 cell membranes with DNase I caused a change in the elution profile of the receptor as judged by molecular-sieve chromatography. Presumably this decrease in the size of the receptor is due to the loss of associated actin filaments. The results of these experiments strongly suggest that the receptor for hyaluronate is associated either directly or indirectly with cytosolic actin filaments.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

The Yersinia effector protein YpkA induces apoptosis independently of actin depolymerization

The pathogenicity of the plague agent Yersinia pestis is largely due to the injection of effector proteins that potently block immune responses into host cells through a type III secretion apparatus. One Yersinia effector protein, YpkA, a putative serine/threonine kinase, has been reported to act by depolymerizing actin and disrupting actin microfilament organization. Using YpkA-GFP fusion proteins to directly visualize cells expressing YpkA, we found instead that YpkA triggered rapid cell death that can be blocked by caspase inhibitors and Bcl-xL, but was not dependent on caspase-8. The actin depolymerization promoted by YpkA was only seen in cells with other features of apoptosis, and was blocked by inhibiting apoptosis, indicating that actin filament disruption is likely to be a result, rather than a cause of YpkA-induced apoptosis. A region including aa 133-262 in YpkA was sufficient for inducing apoptosis independent of localization to the plasma membrane. These data suggest that YpkA can act as a direct inducer of cell death.     

Slow down of actin depolymerization by cross-linking molecules

The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.