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PubMed1997

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.

Actins↗
PubMed1997

Actin assembly by cadmium ions.

Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca2+ concentrations or compete with Ca2+ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8-1.0 mM) concentrations, CdCl2 causes actin denaturation. At such Cd2+ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd2+ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl2 is more effective as an actin polymerizing agent than both MgCl2 and CaCl2. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C(c) ranges from 0.25 to 0.5 microM), while Ca-polymerized actin shows a polymerization extent markedly lower (C(c) = 4.0 microM). By both the fluorescent Ca2+ chelator Quin-2 assay and limited proteolysis of actin by trypsin and alpha-chymotrypsin, the real substitution of G-actin-bound Ca2+ by Cd2+ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl2 indicates that, in our experimental conditions, Ca2+ tightly-bound to actin is partially (60-70%) replaced by Cd2+, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61-69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd2(+)-treated cells; however, considering the positive effect of Cd2+ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd2+ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable.

Actins↗
PubMed2006

Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells.

BACKGROUND INFORMATION: The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. RESULTS: In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. CONCLUSIONS: Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.

Actins↗
PubMed1980

Immunological differences between actins from cardiac muscle, skeletal muscle, and brain.

The antigenic similarities and differences between various actins were explored by use of antisera against purified bovine cardiac actin and chicken embryo brain actin. In double-antibody coprecipitation tests, purified iodinated actins from bovine cardiac muscle, rabbit skeletal muscle, chicken embryo brain, and bovine brain all bound to antiserum against chicken embryo brain actin. This result demonstrates the presence of shared antigenic determinants among these actins. Cardiac actin antiserum, on the other hand, bound cardiac and skeletal actin, but failed to bind significantly either brain actin. In radioimmunoassay, all four unlabeled actins were capable of some degree of inhibition of binding of (125)I-labeled chicken embryo brain actin to homologous antiserum. The results confirm the existence of shared or similar antigenic determinants, but also show that the molecules are not antigenically identical. In the cardiac actin radioimmunoassay, unlabeled cardiac and skeletal muscle actins inhibited the binding of (125)I-labeled cardiac actin to homologous antiserum, but neither brain actin inhibited the binding. Thus, the muscle actins possess at least one antigenic determinant not expressed by the brain actins, in addition to the shared determinants. Furthermore, cardiac actin and skeletal actin generated different inhibition curves in the cardiac actin radioimmunoassay, demonstrating that, although antigenically related, they are not identical. Correlations with existing sequence data imply that substitutions in only a few residues alter the antigenic properties of actin.

Actins↗
PubMed1997

Dystrophin-glycoprotein complex is monomeric and stabilizes actin filaments in vitro through a lateral association.

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (Mr 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind G-actin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophin-glycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes F-actin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.

Actins↗
PubMed1995

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.

Actins↗
PubMed1998

State of actin in gastric parietal cells.

Remodeling of the apical membrane-cytoskeleton has been suggested to occur when gastric parietal cells are stimulated to secrete HCl. The present experiments assayed the relative amounts of F-actin and G-actin in gastric glands and parietal cells, as well as the changes in the state of actin on stimulation. Glands and cells were treated with a Nonidet P-40 extraction buffer for separation into detergent-soluble (supernatant) and detergent-insoluble (pellet) pools. Two actin assays were used to quantitate actin the deoxyribonuclease I binding assay to measure G-actin and F-actin content in the two pools and a simple Western blot assay to quantitate the relative amount of actin in the pools. Functional secretory responsiveness was assayed by aminopyrine accumulation. About 5% of the total parietal cell protein is actin, with about 90% of the actin present as F-actin. Stimulation of acid secretion resulted in no measurable change in the relative amounts of G-actin and cytoskeletal F-actin. Treatment of gastric glands with cytochalasin D inhibited acid secretion and resulted in a decrease in F-actin and an increase in G-actin. No inhibition of parietal cell secretion was observed when phalloidin was used to stabilize actin filaments. These data are consistent with the hypothesis that microfilamentous actin is essential for membrane recruitment underlying parietal cell secretion. Although the experiments do not eliminate the importance of rapid exchange between G- and F-actin for the secretory process, the parietal cell maintains actin in a highly polymerized state, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

Actins↗
PubMed1991

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells.

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F-actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F-actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5-isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F-actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F-actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1980

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin.

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.

Actins↗
PubMedDate not supplied

Actinic keratosis. Current treatment options.

Actinic keratoses are hyperkeratotic skin lesions that represent focal abnormal proliferation of epidermal keratinocytes. Some actinic keratoses evolve into squamous cell carcinoma of the skin, while others resolve spontaneously. The conversion rate of actinic keratosis to squamous cell carcinoma is not accurately known, but appears to be in the range of 0.25 to 1% per year. Although there is a low rate of conversion of actinic keratoses to squamous cell carcinoma, 60% of squamous cell carcinomas of the skin probably arise from actinic keratoses. The main cause of actinic keratoses in otherwise healthy Caucasians appears to be the sun. Therapy for actinic keratoses begins with prevention which starts with sun avoidance and physical protection. Sunprotection with sunscreens actually slows the return of actinic keratoses in patients already getting actinic keratoses. Interestingly, a few studies are available that demonstrate that a high fat diet is associated with the production of more actinic keratoses than is a low fat diet. One of the mainstays of therapy has been local destruction of the actinic keratoses with cryotherapy, and curettage and electrodesiccation. A new addition to this group of therapies to treat individual actinic keratoses is photodynamic therapy with topical aminolevulinic acid and light. In patients who have numerous actinic keratoses in an area of severely sun damaged skin, therapies which are applied to the whole actinic keratosis area are used. The goal of treating such an area of skin is to treat all of the early as well as the numerous clinically evident actinic keratoses at the same time. The classical approaches for treating areas of photodamaged skin without treating actinic keratoses individually include: the use of topically applied fluorouracil cream, dermabrasion, and cutaneous peels with various agents like trichloroacetic acid. Both topically as well as orally administered retinoids have been used to treat actinic keratoses but retinoids alone are probably not an optimal monotherapy. Photodynamic therapy with topical aminolevulinic acid and light is a new therapy for actinic keratoses. Aminolevulinic acid is a precursor of protoporphyrin IX (PpIX) which is synthesized in the actinic keratosis when it is treated with aminolevulinic acid, and the PpIX photosensitizes the actinic keratosis so that light exposure can lead to its destruction. Photodynamic therapy with topical aminolevulinic acid is approved in the US to treat multiple individual actinic keratoses on the face and scalp and has similar cure rates to those reported for cryotherapy and fluorouracil therapy.

Humans↗
PubMed2000

Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments.

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.

Actin Cytoskeleton↗
PubMed1996

Beta cap73: a novel beta actin-specific binding protein.

Whereas actin-binding proteins (ABPs) regulate network formation during the cell cycle, it is not known whether ABPs also function to sequester or target isoactins to specific subcellular compartments. Recently, we have shown that ezrin indirectly associates with beta, but not alpha actin filaments in a calcium- and cytochalasin-sensitive manner [Shuster and Herman, 1995: J. Cell Biol. 128:837-848]. To identify the beta actin-specific binding protein that fosters ezrin-beta actin interactions, we developed an isoactin affinity fractionation and F-isoactin overlay/Western blotting technique. Results reveal that a 73 kd polypeptide that co-precipitates with ezrin and beta actin [Shuster and Herman, 1995: J. Cell Biol. 128:837-848] can also binds directly to filaments of beta, but not alpha actin by isoactin overlay. In an effort to establish whether p73 plays a role in regulating beta actin dynamics in cells, we produced monoclonal antibodies by immunizing BALB/c mice with p73-containing lamellar lysates or high salt elutions from beta actin affinity columns. Two monoclonal antibodies were cloned that react with p73 present in fractions released from beta actin Sepharose-4B or purified to homogeneity by DEAE chromatography. Anti-p73 Western blots reveal that there is a 16-fold difference in p73 binding to beta actin vs. alpha actin affinity columns when experiments are performed in physiological salts. To characterize p73-beta actin binding in vitro and establish whether p73 binds along the lengths or at the barbed end of the beta actin filament, we asked whether cytochalasin D (CD) could displace p73 pre-bound to beta actin-Sepharose 4B. Anti-p73 Western blotting reveals that nanomolar concentrations of CD are capable of selectively eluting p73 and ezrin from beta actin Sepharose 4B, indicating that p73 binds beta actin via the barbed end. Simultaneous double antibody localization studies using anti-beta actin IgG and anti-p73 IgM reveal that p73 and beta actin are co-localized in the forward aspects of motile cytoplasmic domains, in close proximity to the plasma membrane. Because of its isoform-specific interactions with the barbed end of beta actin filaments, we have named this molecule beta cap73. These results indicate that isoform-specific actin-binding proteins can be identified from cortical cytoplasm, and suggest that beta cap73 may not only act to spatially regulate the intracellular distribution of isoactins, but may also facilitate forward protrusion formation through the regulated release of free filament ends during cell motility.

Actins↗
PubMed1994

Caldesmon, N-terminal yeast actin mutants, and the regulation of actomyosin interactions.

N-Terminal yeast actin mutants were used to assess the role of N-terminal acidic residues in the interactions of caldesmon with actin. The yeast actins differed only in their N-terminal charge: wild type, two negative charges; 4Ac, four negative charges; DNEQ, neutral charge; delta DSE, one positive charge. Caldesmon inhibition of actomyosin subfragment 1 ATPase was affected by alterations in the N-terminus of actin. This inhibition was similar for skeletal muscle alpha-actin and the yeast 4Ac and wild-type actins (80%), but much smaller for the neutral and deletion mutants (15%). However, cosedimentation experiments revealed similar binding of caldesmon to polymerized rabbit skeletal muscle alpha-actin and each yeast actin. This result shows that the N-terminal acidic residues of actin are not required for the binding of caldesmon to F-actin. Caldesmon-actin interactions were also examined by monitoring the polymerization of G-actin induced by caldesmon. Although the final extent of polymerization was similar for all actins tested, the rates of polymerization differed. Skeletal muscle and 4Ac actins had similar rates of polymerization, and the wild-type actin polymerized at a slower rate. The neutral and deletion mutants had even slower rates of polymerization by caldesmon. The slow polymerization of DNEQ G-actin was traced to a greatly reduced binding of caldesmon to this mutant G-actin when compared to wild-type and alpha-actin. MgCl2-induced actin polymerization proceeded at identical rates for all actins.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1981

Gene switching in myogenesis: differential expression of the chicken actin multigene family.

We described the construction of an alpha-actin complementary deoxyribonucleic acid (cDNA) clone, pAC269 [Schwartz, R. J., Haron, J. A., Rothblum, K. N., & Dugaiczyk, A. (1980) Biochemistry 19, 5883], that was used as a hybridization probe in the current investigation to examine the induction of actin messenger ribonucleic acid (mRNA) during myogenesis. A Tm difference of 10-13 degrees C between skeletal muscle alpha-actin and nonmuscle beta- and gamma-actin mRNAs and pAC269 allowed us to establish the highly stringent hybridization conditions necessary to measure separately the content of alpha-actin mRNA and beta- and gamma-actin mRNA during muscle development in culture. We observed low levels of alpha-actin mRNA (approximately 130 molecules/cell) in replicating prefusion myoblasts. The vast majority of actin mRNA (2000 molecules/cell) present at this stage was accounted for by beta- and gamma-actin mRNA. Beginning at myoblast fusion, alpha-actin mRNA accumulated and within 30 h reached a level 270-fold greater than that observed in the undifferentiated state. At 95 h in culture when myotube formation was completed, alpha-actin content was at its peak (36 000 molecules/nucleus). Conversely, beta- and gamma-actin mRNA content began to decline at the beginning of fusion, and by the end of myotube formation beta- and gamma-actin mRNAs were undetectable by our techniques. A rapid depression of alpha-actin mRNA levels was observed after 95 h in the absence of cell death. At 6 days after the initiation of myotube formation, the content of alpha-actin mRNA was reduced by 80% in comparison of peak values and remained at that level. The switching of actin mRNA species was inhibited in myoblasts treated with bdU. The accumulation of alpha-actin mRNA and the disappearance of beta- and gamma-actin mRNA were observed following the reversal of the bdU block and coincident with the onset of myoblast fusion. We found that the expression of actin genes within the actin multigene family is switched in myogenesis through a strict developmental pattern.

Actins↗
PubMed1998

Interaction of caldesmon with actin subdomain-2.

The polymerization-resistant maleimidobenzoyl-G-actin (MBS-G-actin), which behaves as a functional analogue of native G-actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028-6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085-9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross-linking reactions. MBS-G-actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 degrees C, with a high affinity (Kd approximately 0.04 microM) and with a 1:1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS-G-actin cleaved at the subdomain-2 loop with subtilisin decreased fivefold (Kd approximately 0.20 microM). Conversely, caldesmon strongly protected subdomain-2 of MBS-G-actin from tryptic digestion at the susceptible peptide bond at positions 68-69. Furthermore, caldesmon induced the dissociation of native G-actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS-G-actin-caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS-G-actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain-2, to caldesmon, producing in high yield a 205-kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS-F-actin. MBS-G-actin could be also cross-linked to caldesmon by 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, producing a three-band pattern identical to that of F-actin and caldesmon and previously shown to reflect the covalent union between the NH2-terminal segment of actin and the COOH-terminal actin-binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain-2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain-1 as well as the top portion of its subdomain-2.

Actins↗
PubMed1997

Evidence for a conformational change in actin induced by fimbrin (N375) binding.

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

Actins↗
PubMed1983

Structural and dynamic states of actin in the erythrocyte.

Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 mug/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing ("pointed") ends of free actin filaments. This suggests that these ends of the actin "protofilaments" in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 mug/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 mug/ml of cells, compared with 270 mug/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.

Actins↗
PubMed1988

Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line.

HUT-14 cells, tumorigenic human fibroblasts, express a mutant beta-actin which has a single amino acid substitution at position 244 (glycine to aspartic acid), in addition to normal beta- and gamma-actin. In order to characterize the biochemical function of the mutant beta-actin, actins were extracted and purified from HUT-14 cells. The partially purified actin fraction contained beta-, gamma-, and mutant beta-actins in the ratio of 1:1:1, the same ratio as in the cells. When the actin of this fraction was purified through a polymerization step, mutant beta-actin was always less incorporated into actin filaments than beta- and gamma-actin. When the polymerization ability of purified HUT-14 actins was examined by sedimentation technique, it was lower than those of muscle and of cytoplasmic actins from another human cell line (HUT-11) which expresses only normal beta- and gamma-actin, in the ratio of 2:1. The deficient polymerization of mutant beta-actin was also observed by examining the ratio of beta-, gamma-, and mutant beta-actins incorporated into actin filaments. The ratio of mutant beta-actin in polymerized actins under all conditions examined was always less than that before polymerization. These results indicate that the single amino acid substitution at position 244 caused the reduction of incorporation of the mutant beta-actin into actin filaments in vitro.

Actins↗