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PubMed1983

The quantitation of G- and F-actin in cultured cells.

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.

Actins↗
PubMed1991

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.

Actins↗
PubMed1984

The influence of contact-inhibited growth and of agents which alter cell morphology on the levels of G- and F-actin in cultured cells.

The amounts of G-actin and F-actin were measured in cultured cells grown under various conditions. The percent of total actin as F-actin in monolayer cultures of asynchronous cells was 72.4% in Chinese hamster ovary (CHO) cells, 57.7% in HeLa cells, 69.8% in V79 cells, and 79.5% in 1080 cells. Actin comprises 2.4-3.1% of the total protein in these cell lines. Treatment of cells with 20 microM cytochalasin B (CB) caused different cytological effects from treatment with 10 microM colchicine, but the effects characteristic of each drug were observed throughout the range of cell lines used. Of the five cell lines treated with CB only the V79 and CHO cells showed a decrease (5-8%) in the level of F-actin. Colchicine treatment of HeLa cells resulted in a 13% increase in the percent F-actin, but similar treatment of CHO cells caused no significant change in F-actin. Therefore, a change in the steady state level of F-actin is not necessary for the observed cell shape change. The F-actin levels in CHO cells treated with 7 mM procaine decreased from 72 to 65% over the first 15 min of exposure, a time during which the cells rounded. After continuous exposure of the cells to procaine for 1 h, the F-actin percentage returned to control levels and the cells, though abnormal in appearance, flattened on the culture dish. The relationship between the level of F-actin and cell density was studied on the culture dish. The relationship between the level of F-actin and cell density was studied in the 10T 1/2 cells, a cell line which demonstrates density-dependent growth regulation. While contact inhibition was accompanied by a decrease in the F-actin percentage (from greater than 95% to about 60%) in one strain of 10T1/2 cells, two other strains of the same cells progressed from log phase growth (highly motile) to late plateau phase (non-motile, contact-inhibited) with a constant level of F-actin (about 60%). A spontaneous transformant of this cell line, which no longer demonstrated contact-inhibited cell growth, also maintained the same constant F-actin (60%). Thus, the maintenance of contact-inhibited growth control does not appear to depend upon the net distribution of actin between the globular and filamentous forms.

Actins↗
PubMed1991

The isolated 21 kDa N-terminal fragment of myosin binds to actin in an ATP and ionic strength-dependent manner.

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the 'consensus' ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the 'consensus' ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the 'consensus' site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83.10(7) M-1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the 'consensus' ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143-147 stretch, which may participate in forming the actin binding site.

Actins↗
PubMed1995

Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.

We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.

Actins↗
PubMed1987

Functional, chymotryptically split actin and its interaction with myosin subfragment 1.

We have prepared chymotryptically split actin that retains the characteristic properties of intact actin. Chymotryptic digestion of G-actin produces an intermediate 35-kilodalton (kDa) fragment and from this a final product of 33 kDa known as the C-terminal "core". These fragments remain attached to an N-terminal 10-kDa fragment. The 35-kDa-10-kDa complex is able to polymerize upon addition of KCl and MgCl2, like intact actin, whereas the 33-kDa-10-kDa complex is not. The 35-kDa-10-kDa complex is here termed "split actin". In the rigor state, split actin binds to myosin subfragment 1 (S-1) strongly, with the same stoichiometry as intact actin. In the rigor state, split actin forms a carbodiimide-induced cross-linked product with S-1; the cross-linking sites on the split actin and on S-1 were proved to be the N-terminal 10-kDa fragment of split actin and the 20-kDa domain of S-1. There was no cross-linking between the 50-kDa domain of S-1 and the 10 kDa of actin. Therefore, the structure of the split actin-S-1 complex differs somewhat from that of the complex with intact actin. The cross-linking of split actin to S-1 causes superactivation of S-1 ATPase to approximately the same extent as does cross-linking of intact actin, whereas non-cross-linked split actin activates S-1 ATPase to a lesser extent. The N-terminus of the 35-kDa fragment was found to be residue 45 (Val-45) by amino acid sequence analysis; so there is no residue missing in split actin.

Actins↗
PubMed1988

Rate of binding of tropomyosin to actin filaments.

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.

Actins↗
PubMed2004

Expression of a nonpolymerizable actin mutant in Sf9 cells.

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.

Actins↗
PubMed2000

Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.

Actins↗
PubMed2000

Remodeling of the actin cytoskeleton in the contracting A7r5 smooth muscle cell.

It has been proposed that the reorganization of components of the actin cytomatrix could contribute to force development and the low energy cost of sustained contraction in contractile cells which lack a structured sarcomere (A.S. Battistella-Patterson, S. Wang and G.L. Wright (1997) Can J Physiol Pharmacol 75: 1287-1299). However, there has been no direct evidence of an apropos actin reorganization specifically linked to the contractile response in cells of this type. Remodeling of the alpha- and beta-actin domains was studied in A7r5 smooth muscle cells during phorbol 12,13 dibutyrate (PDB)-induced contraction using immunohistologic staining and beta-actin-green fluorescent protein (beta-actin-GFP) fusion protein expression. Cell stained with phalloidin as well as cells expressing beta-actin-GFP showed densely packed actin stress cables, arranged in parallel and extending across the cell body. PDB caused approximately 85% of cells to contract with evidence of forcible detachment from peripheral adhesion sites seen in many cells. The contraction of the cell body was not uniform but occurred along a principal axis parallel to the system of densely packed beta-actin cables. During the interval of contraction, the beta-actin cables shortened without evidence of disassembly or new cable formation. The use of cytochalasin to inhibit actin polymerization resulted in the dissolution of the actin cables at the central region of the cell and caused the elongation of precontracted cells. In unstimulated cells, alpha-actin formed cables similar in arrangement to the cell spanning beta-actin cables. Within a short interval after PDB addition; however, the majority of alpha-actin cables disassembled and reformed into intensely fluorescing column-like structures extending vertically from the cell base at the center of clusters of alpha-actin filaments. The alpha-actin columns of contracting cells showed strong colocalization of alpha-actinin suggesting they could be structurally analogous to the dense bodies of highly differentiated smooth muscle cells. The results indicate that the alpha- and beta-actin domains of A7r5 cells undergo a highly structured reorganization during PDB-induced contraction. The extent and nature of this restructuring suggest that remodeling could play a role in contractile function.

Actins↗
PubMed1992

Chemical evidence for the existence of activated G-actin.

Globular actin (G-actin) will polymerize to form filamentous actin (F-actin) under physiological ionic conditions, and is known to be regulated by univalent and bivalent cations, such as K+ and Mg2+. The current concept of this process involves four steps: activation, nucleation, elongation and annealing. Evidence for the existence of activated G-protein has been suggested by changes in the resistance to proteolysis [Rich & Estes (1976) J. Mol. Biol. 104, 777-792] and u.v.-light absorption [Rouayrenc & Travers (1981) Eur. J. Biochem. 116, 73-77]. More recently we [Liu et al. (1990) Biochem. J. 266, 453-459] have provided direct chemical evidence for extensive conformational changes during the transformation of G-actin into F-actin. In this study we now present direct chemical evidence for the existence of a short-lived species, an activated form of G-actin, which can be detected by changes in the accessibility of the free thiol groups on the G-actin molecule when modified by a specific thiol-group-targeted reagent, 7-dimethylamino-4-methyl-3-N-maleimidylcoumarin (DACM). The presence of K+ and/or Mg2+ ions caused a large increase in the accessibility of the thiol groups of Cys-217 and Cys-374, but not those of Cys-10 and Cys-257. Mg2+ effected relatively faster changes than did K+ ions. The results suggest that the function of these ions is to convert G-actin into an activated form, and further suggest that the change in conformation is mainly confined to the large domain. Such changes at least involve certain portions of the G-actin molecule that contain Cys-217 and Cys-374. On the other hand, little or no significant change could be observed in the small domain of G-actin as reflected by the accessibility of Cys-10. The bound nucleotide remained as ATP during the activation of G-actin and was hydrolysed to ADP on polymerization. The activated G-actin had a life-time of about 8 min or less depending on the concentration of G-actin. At higher protein concentration, its life-time was much shorter, probably owing to the earlier onset of polymerization, which apparently is governed by the concentration of the activated form. The life-time of this new species can be extended by lowering the temperature and is less affected by actin concentration. This new species is considered to be an activated form of G-actin, since polymerization renders all the thiol groups on actin inaccessible to the reagent DACM.

Actins↗
PubMed1998

Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin.

Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (Kd = 0.1 microM) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments.

Acanthamoeba↗
PubMed2003

Tropomodulin contains two actin filament pointed end-capping domains.

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.

Actins↗
PubMed1982

Actin filaments undergo limited subunit exchange in physiological salt conditions.

The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent-labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half-time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.

Actins↗
PubMed2000

Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse.

The smooth muscle (SM) alpha-actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal alpha-actins. SM alpha-actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue-specific expression of SM alpha-actin is thought to be required for the principal force-generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM alpha-actin gene expression actually relates to an actin isoform's function. Analysis of SM alpha-actin null mice indicated that SM alpha-actin is not required for the formation of the cardiovascular system. Also, SM alpha-actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM alpha-actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal alpha-actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM alpha-actin null mice. However, even with a modest increase in skeletal alpha-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM alpha-actin-defective mice. This study supports the concept that SM alpha-actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.

Actins↗
PubMed1989

Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells.

During pressure-load hypertrophy of the adult heart in vivo, there is up-regulation of the mRNA encoding skeletal alpha-actin, the sarcomeric actin iso-mRNA characteristic of mature skeletal muscle and the fetal/neonatal heart. We have shown previously that during alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes, the induction of skeletal alpha-actin mRNA is greater than that of the mRNA encoding cardiac alpha-actin, the sarcomeric actin iso-mRNA characteristic of the adult heart. To determine if this actin iso-mRNA switch during cardiac hypertrophy reflects changes in the transcriptional status of the myocyte nucleus, we quantified the rate of transcription of actin mRNAs and total RNA, using an in vitro run-on transcription assay with nuclei isolated from the cultured myocytes after stimulation with norepinephrine (NE). Transcription of skeletal alpha-actin was increased at 3 h after NE, reached a maximum 6.1-fold increase at 12 h, and returned to the control level at 24 h. The EC50 for NE was 200 nM, and pharmacologic studies indicated alpha 1-receptor specificity. Transcription of cardiac alpha-actin was also increased rapidly by NE (maximum 4.6-fold vs. control at 3 h). However, cardiac alpha-actin transcription had returned to the control level at 6 h, when NE-stimulated skeletal alpha-actin transcription was still increasing. Transcription of the cytoskeletal (beta) actin gene was not changed significantly by NE treatment. Total RNA transcription was not increased until 6 h after NE (1.5-fold vs. control) and remained elevated through 24 h. Inhibition of protein synthesis did not attenuate NE-stimulated actin gene transcription. Thus the alpha 1-adrenoceptor mediates a rapid, transient, and selective increase in transcription of the sarcomeric actin isogenes during cardiac myocyte hypertrophy. Skeletal alpha-actin, the fetal/neonatal isogene, is induced preferentially to cardiac alpha-actin, the adult isogene. The different kinetics of actin isogene and total RNA transcription and the independence of transcription from protein synthesis suggest that transcriptional induction via the alpha 1 receptor is complex and may involve preexisting regulatory factors. These results are the first to demonstrate that the alpha 1-adrenergic receptor is a molecular mediator of transcriptional changes underlying an isogene switch that is known to be associated with cardiac myocyte hypertrophy.

Actins↗
PubMed1992

Dissociation of actin polymerization and enzyme inactivation in the hormonal regulation of type II iodothyronine 5'-deiodinase activity in astrocytes.

T4 dynamically regulates the levels of type II iodothyronine 5'-deiodinase in the brain. Using an astrocyte cell culture model, we have shown that thyroxine increases inactivation of this enzyme through a mechanism using the actin cytoskeleton. In the absence of T4, the filamentous actin (F-actin) stress fibers are absent, and deiodinase inactivation is relatively slow. T4 increases inactivation of type II 5'-deiodinase by 1) restoring the F-actin stress fibers, 2) promoting the binding of the enzyme to F-actin, and 3) stimulating enzyme internalization. To determine whether inactivation of the deiodinase is due solely to the restoration of stress fibers by T4 or also involves direct thyroxine-mediated enzyme-F-actin interactions, we examined the effects of retinoids on both actin polymerization and type II 5'-deiodinase activity in cultured astrocytes, as these hormones have been shown to alter cytoskeletal organization in other tissues. In thyroid hormone-deficient astrocytes, retinoic acid increased F-actin levels, with no change in total cell actin. The F-actin content increased approximately 40% within 30 min after the addition of retinoic acid. After a plateau of 6-8 h, the F-actin content increased further to approximately 90% of the total cell actin and was associated with the reappearance of stress fibers. Only this latter retinoid-stimulated increase in F-actin content was blocked by actinomycin-D. Restoration of the F-actin stress fibers by retinoids did not increase the turnover of the type II 5'-deiodinase (t1/2, 1.99 h-1) or promote binding of the enzyme to F-actin in the absence of T4. Similarly, retinoids did not affect the rapid T4-mediated turnover (t1/2, 0.18 h-1) of type II 5'-deiodinase. These data show that an intact F-actin cytoskeleton in the absence of T4 is inadequate to alter the inactivation of type II 5'-deiodinase and that specific T4-enzyme-F-actin interactions are necessary to initiate the rapid inactivation/internalization of this enzyme.

Actins↗
PubMed1993

Intracellular relationship between actin and alpha-actinin in a whole corneal epithelial tissue.

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of alpha-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that alpha-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the alpha-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and alpha-actinin. In epithelial cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and alpha-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin networks from both cell layers were eliminated and replaced by aggregates scattered throughout the cytoplasm. The alpha-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The alpha-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the alpha-actinin. The alpha-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actinin; however, there was some overlap with G-actin.

Actinin↗