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PubMed1990

Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.

Actins↗
PubMed1996

Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity.

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.

Actins↗
PubMed1981

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

The distribution and polymerization state of actin in metaphase rat kangaroo cells was studied by fluorescence microscopy. Formaldehyde-fixed, acetone-extracted cells were labeled with either of two types of actin probes. The first, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin, has high affinity for F actin and does not bind monomeric G actin. The second was a conjugate of DNase I labeled with either tetramethylrhodamine or fluorescein. DNase binds with high affinity to G actin and with lesser affinity to F actin. The polymerization state of actin was deduced by comparing the fluorescence distribution of the phallacidin derivative with that of the fluorescent DNase. The results indicate that the pole-to-chromosome region of the metaphase spindle contains G actin but little if any conventional F actin. F actin is found concentrated in a diffuse distribution outside the spindle region in metaphase cells and returns to the interzone area between the chromosomes by early telophase. These results exclude spindle models for chromosomal movement that require more than about five F actin filaments per chromosome, support the hypothesis that F actin is involved in force generation for cell cleavage, and are not inconsistent with the possibility that actin outside the spindle may be involved in chromosomal movement.

Actins↗
PubMed1988

Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.

The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.

Actins↗
PubMed1991

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution.

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.

Actins↗
PubMed1983

Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin.

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.

Actins↗
PubMed2004

TetraThymosinbeta is required for actin dynamics in Caenorhabditis elegans and acts via functionally different actin-binding repeats.

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.

Actins↗
PubMed1990

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function.

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.

Actins↗
PubMed1979

Interaction of myosin subfragment-1 with actin. III. Effect of cleavage of the subfragment-1 heavy chain on its interaction with actin.

To determine the reason why the Mg2+-ATPase activity of subfragment-1 prepared with chymotrypsin was activated more by actin than that of subfragment-1 prepared with trypsin was and the reason why the former could enhance the polymerization of actin and the latter could not, we digested subfragment-1, prepared with chymotrypsin, with trypsin and examined the actin activated Mg2+-ATPase activity and the ability to polymerize actin. It was found that cleavage of the heavy chain decreased the actin activated Mg2+-ATPase activity of subfragment-1 prepared with chymotrypsin but did not affect its ability to polymerize actin. Trypsin attacked the subfragment-1 heavy chain at two sites and produced 26 K, 50 K, and 21 K fragments. From the comparison of the time course of tryptic digestion with that of the decrease in actin activation, it was deduced that cleavage of the 50 K-21 K junction was mainly responsible for the decrease in actin activation. We also measured the length and the amount of F-actin polymerized by the addition of different amounts of subfragment-1. It was found that the amount of F-actin increased with the increase in the amount of subfragment-1 added and that the length of F-actin also increased though slightly. We concluded from the results that subfragment-1 enhanced the polymerization not only by facilitating the nucleus formation but also by strengthening the bond between actin monomers in forming F-actin.

Actins↗
PubMed1982

Actin modulating proteins in the sea urchin egg. I. Analysis of G-actin-binding proteins by DNase I-affinity chromatography and purification of a 17,000 molecular weight component.

Two groups of protein species which interact with G-actin were detected in unfertilized sea urchin eggs by DNase-I affinity chromatography in the presence of Ca2+. One of the protein groups, which comprised of six major proteins, was eluted by EGTA. One of these proteins was tentatively identified as calmodulin. The other protein group, comprising of four major proteins, could be dissociated from the immobilized DNase I at a higher ionic strength. One of these proteins, showing a molecular weight of 17,000 (17 K protein), was purified to homogeneity. In its action on actin, 17 K protein revealed properties quite similar to those of a protein called depactin isolated from unfertilized starfish oocytes, but different from those of profilins isolated from mammalian tissues or Acanthamoeba. 17 K protein co-migrated with depactin on an SDS-gel. It inhibited actin polymerization and quickly depolymerized F-actin. When added to G-actin before polymerization, 17 K protein suppressed the final extent of actin polymerization. This inhibition was not released by the addition of sonicated F-actin nuclei. When added to F-actin, 17 K protein rapidly reduced the viscosity and increased the G-actin concentration of the actin solution. In both cases, the final extent of actin polymerization strictly depended on the molar ratio of 17 K protein to actin, indicating a stoichiometric association between 17 K protein and actin.

Actins↗
PubMed1989

The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin.

The stoichiometric actin--DNase-I complex was used to study the actin--nucleotide and actin--divalent-cation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin--DNase-I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size-exclusion chromatography. This effect is due to a loss of actin-bound nucleotide but is prevented by the presence of 0.1-0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ ('physiological conditions') the association rate constants for ATP, ADP and epsilon ATP (1,N6-ethenoadenosine 5'-triphosphate) and the dissociation rate constant for epsilon ATP were determined. Both the on and off rates were found to be reduced by a factor of about 10 when compared to uncomplexed actin. Thus the binding constant of epsilon ATP to actin is almost unaltered after complexing to DNase I (2.16 x 10(8) M-1). Titrating the increase in DNase I activity of the actin--DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation-free form of actin--DNase I complex was found to be 5 x 10(3) M-1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high-affinity metal-binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide-free and metal-ion-free actin--DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.

Actins↗
PubMed1991

Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

Actins↗
PubMed1997

Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function.

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter 'actin crossovers' ( approximately 75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.

Actin Cytoskeleton↗
PubMed2000

The carboxy terminus of AFAP-110 modulates direct interactions with actin filaments and regulates its ability to alter actin filament integrity and induce lamellipodia formation.

The actin filament-associated protein AFAP-110 is an SH2/SH3 binding partner for Src. AFAP-110 contains several protein-binding motifs in its amino terminus and has been hypothesized to function as an adaptor molecule that could link signaling proteins to actin filaments. Recent studies using deletional mutagenesis demonstrated that AFAP-110 can alter actin filament integrity in SV40 transformed Cos-1 cells. Thus, AFAP-110 may be positioned to modulate the effects of Src upon actin filaments. In this report, we sought to determine whether (a) AFAP-110 could interact with actin filaments directly and (b) deletion mutants could affect actin filament integrity and cell shape in untransformed fibroblast cells. The data demonstrate that the carboxy terminus of AFAP-110 is both necessary and sufficient for actin filament association, in vivo and in vitro. Analysis of the carboxy terminus revealed a mean 40% similarity with other known actin-binding motifs, indicating a mechanism for binding to actin filaments. AFAP-110 can also induce lamellipodia formation. Contiguous with the alpha-helical, actin-binding motif is an alpha-helical, leucine zipper motif. Deletion of the leucine zipper motif (AFAP(Deltalzip)) followed by cellular expression enabled AFAP(Deltalzip) to alter actin filament integrity and cell shape in untransformed cells as evidenced by the induction of lamellipodia formation. We hypothesize that AFAP-110 may be an important signaling protein that can directly modulate changes in actin filament integrity and induce lamellipodia formation.

3T3 Cells↗
PubMed2000

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.

Actin Cytoskeleton↗
PubMed2006

Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface.

ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR-actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments.

Actin Depolymerizing Factors↗
PubMed2006

Dynamic polymorphism of single actin molecules in the actin filament.

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.

Actin Cytoskeleton↗
PubMed2004

Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL.

Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry.

Actin Depolymerizing Factors↗