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PubMed1994

The kinetics of the interaction between the actin-binding domain of alpha-actinin and F-actin.

Measurement of the binding equilibrium for the interaction of alpha-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle alpha-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 x 10(5), 1.8 x 10(6) and 4 x 10(6) M-1.s-1 at 5 degrees C, 15 degrees C and 25 degrees C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6s-1, giving equilibrium constants of 0.83 and 2.4 microM, respectively. Optical changes on mixing intact alpha-actinin with F-actin were dominated by secondary bundling events.

Actinin↗
PubMed1989

Botulinum C2 toxin treatment increases the G-actin pool in intact chicken cells: a model for the cytopathic action of actin-ADP-ribosylating toxins.

Botulinum C2 toxin ADP-ribosylates actin in intact chicken embryo cells in a concentration-dependent manner. This effect correlates with an enhancement in the inhibitory potency of the respective cell lysates on DNAse I activity, indicating an increase in the cellular G-actin content of toxin-treated cells. The data support our view, that ADP-ribosylation of cellular actin with subsequent depolymerization of cytoskeleton-associated F-actin to monomeric G-actin is involved in the cytotoxic effects of botulinum C2 toxin. A model of the cytopathic action of actin-ADP-ribosylating toxins is presented.

Actins↗
PubMed1991

Mechanism of the active movement of actin: is actin sliding directly or indirectly related to binding with myosin and activation of myosin ATPase?

In the present work we examined the effect of crosslinking of polymerized and monomeric actin with glutaraldehyde, EDC and DSS on: 1) binding of actin to HMM in solution; 2) activation of HMM ATPase; 3) sliding movement of actin on glass-attached myosin; 4) properties of actin itself, like polymerizability and exchangeability of tightly bound nucleotide. The obtained data show that inhibition of sliding cannot be explained only by changes in the extent of activation of HMM ATPase and binding of actin to HMM; this result emphasizes the role of structural properties of actin in the mechanism of movement generation.

Actins↗
PubMed1986

Purification of rat adrenocortical actin and its use in an immunoprecipitation assay to quantitate cellular actin.

In an earlier study on the long-term effects of the pituitary hormone corticotropin (adrenocorticotropin, ACTH), we reported that ACTH promotes the loss of 20-25% of the total actin in isolated rat adrenocortical cells. In a continuation of this work, we report here the purification to near homogeneity of rat adrenal actin. We demonstrate that the protein purified is adrenal actin, and use this actin in the DNAase immunoprecipitation assay of Snabes et al. to confirm our earlier observation of the effect of ACTH on adrenocortical actin. Finally, we describe several changes made in the original protocol of this immunoassay which we believe significantly improve the accuracy of the assay in estimating actin contents in tissue and cell samples.

Actins↗
PubMed2003

Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium.

The model of myosin regulation by a continuous tropomyosin chain is generalized to a chain of tropomyosin-troponin units. Myosin binding to regulated actin is cooperative and initially inhibited by the chain as before. In the absence of calcium, myosin is further inhibited by the binding of troponin-I to actin, which through the whole of troponin pins the tropomyosin chain in a blocking position; myosin and TnI compete for actin and induce oppositely-directed chain kinks. The model predicts equilibrium binding curves for myosin-S1 and TnI as a function of their first-order affinities K(S1) and L(TI). Myosin is detached by the actin binding of TnI, but TnI is more efficiently detached by myosin when the kink size (typically nine to ten actin sites) spans the seven-site spacing between adjacent TnI molecules. An allosteric mechanism is used for coupling the detachment of TnI to calcium binding by TnC. With thermally activated TnI kinks (kink energy B approximately k(B)T), TnI also binds cooperatively to actin, producing cooperative detachment of myosin and biphasic myosin-calcium Hill plots, with Hill coefficients of 2 at high calcium and 4-6 at low calcium as observed in striated muscle. The theory also predicts the cooperative effects observed in the calcium loading of TnC.

Actins↗
PubMed1997

The 3'-end of the human beta-actin gene enhances activity of the beta-actin expression vector system: construction of improved vectors.

The human beta-actin promoter has been widely used to drive expression of genes of interest in mammalian cell lines and transgenic mice. The original form of the human beta-actin expression vector contains upstream sequences, 5'UTR (untranslated region) and intron 1 from the beta-actin gene linked to a three restriction site polylinker and SV40 (Simian Virus 40) 3'UTR. We have modified this vector now to contain the highly conserved beta-actin 3'UTR plus flanking region which replaces the SV40 sequences. An additional modification has removed the mRNA peripheral localization sequences present in the beta-actin 3'UTR. The new vectors also contain an improved polylinker. The activity of these two new vectors has been compared with that of the original vector and that of a vector using the popular cytomegalovirus (CMV) promoter. Mouse C2 myoblasts were transfected with each vector driving expression of enhanced green fluorescent protein (EGFP) and analyzed for EGFP mRNA levels. We find that both new vectors drive twice the level of mRNA accumulation of the original vector and over 30-times that of the CMV promoter. This suggests that these new vectors will provide a substantial elevation in levels of expression by virtue of inclusion of the beta-actin 3'UTR plus flanking region.

Actins↗
PubMed2002

Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates.

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.

Actins↗
PubMed1995

Coordinated regulation of synapsin I interaction with F-actin by Ca2+/calmodulin and phosphorylation: inhibition of actin binding and bundling.

The synapsins are a family of synaptic vesicle phosphoproteins whose role seems to be to limit the availability of small synaptic vesicles for exocytosis by linking them to the cytoskeleton. One member of the family, synapsin I, has been shown to bind calmodulin in a Ca(2+)-dependent manner. In this study, we have examined whether or not calmodulin can regulate one of the activities of synapsin I, namely, its interaction with F-actin. Synapsin I is an actin bundling protein: this activity is controlled by phosphorylation. Here we show that calmodulin in the presence of Ca2+ is a competitive inhibitor of both actin binding and bundling by synapsin I. Under the conditions of our assay (0.45 microM synapsin I, 4 microM F-actin), half-maximal inhibition of actin binding and bundling by unphosphorylated synapsin I was found with 4.3 and 3.7 microM calmodulin, respectively. The actin binding activity of synapsin I phosphorylated by cAMP-dependent protein kinase or by calmodulin-dependent protein kinase II showed similar sensitivity to calmodulin inhibition to unphosphorylated synapsin I. However, inhibition of bundling was potentiated. Half-maximal inhibition of bundling by synapsin I phosphorylated by cAMP-dependent kinase was achieved at approximately 0.5 microM calmodulin. Half-maximal inhibition of bundling by synapsin I phosphorylated by calmodulin-dependent protein kinase II was achieved at less than 0.2 microM calmodulin, although the maximum binding under the conditions of the assay was lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1982

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads.

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.

Actins↗
PubMed1980

Acanthamoeba profilin interacts with G-actin to increase the rate of exchange of actin-bound adenosine 5'-triphosphate.

A sevenfold molar excess of Acanthamoeba profilin, a 12 000-dalton protein that inhibits actin polymerization, increases the rate of exchange of ATP bound to G-actin with ATP in solution about 17-fold, i.e., from 7.7 x 10(-4) to 1.3 x 10(-2) S-1, at 25 degrees C, 0.033 mM Ca2+, and 0.1 mM ATP, pH 7.5. Detailed analysis of the equilibrium isotope-exchange data shows that profilin and actin form a 1:1 complex with KD = 4.7 x 10(-5) M and that the binding of profilin to actin is rapid and reversible. The actin-profilin complex binds 1 mol of ATP/mol, as does G-actin. Profilin does not interact with ATP or Ca2+.

Actins↗
PubMed2002

Preparation of irreversibly sickled cell beta-actin from normal red blood cell beta-actin.

We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein beta-actin is primarily responsible for locking the irreversibly sickled red blood cells (ISCs) of sickle cell anemic patients into the sickle shape. To support studies on biological and chemical characterization of the oxidized beta-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized beta-actin from normal red blood cell (RBC) beta-actin by a chemical reaction, expecting a product equivalent to that found in ISCs. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) was used for the oxidation. We proved the absence of accessible sulfhydryl groups in the oxidized product using liquid chromatography (LC) with both UV and fluorescence detection. Polymerization assays indicated that the chemically produced ISC actin demonstrated the same kinetics as ISC actin obtained from patients with sickle cell disease. The effect of the oxidation could be reversed by the use of the reducing agent tris(carboxyethyl)phosphine (TCEP).

Actins↗
PubMed1996

Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer".

Actin cross-linked between cys 374 and cys 10 via a disulfide-containing bridge, c-A, is completely unpolymerizable even in the presence of phalloidin. Upon the addition of dithiothreitol, c-A polymerizes with high yield, indicating that denaturation due to the modification was almost absent. In the present study we show that cross-linked actin is a useful model for studying the properties of monomeric actin under polymerization conditions. Addition of salt, for example, produced fluorescence changes possibly reflecting conformational transitions but did not lead to the development of phalloidin binding capacity. Cross-linking of the two cysteine residues also caused a decrease in the nucleotide exchange rate by a factor of ca. 3, an effect that was fully reversed by the addition of KCl. Cross-linked actin inhibits DNase I to the same extent as G-actin and binds thymosin beta 4 and profilin as shown by cross-linking studies. Capping capacity for the barbed end of the filament was not observed, although it might have been expected from the fact that both ends of the cross-link are anchored to subdomain 1. Using the 61-FITC derivative of c-A we showed that c-A is able to bind to myosin S1 with a KD in the microM range. In agreement with this, c-A shows actomyosin ATPase activity with a Kapp comparable to that of F-actin, but a Vmax decreased by a factor of ca. 11. The c-A myosin S1 complex provides the hitherto smallest model of actomyosin, which appears promising for crystallization and X-ray analysis.

Actins↗
PubMed1999

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.

Actins↗
PubMed2001

Clathrin hub expression dissociates the actin-binding protein Hip1R from coated pits and disrupts their alignment with the actin cytoskeleton.

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin-Hip1R interaction is involved in the cytoskeletal organization of coated pits.

3T3 Cells↗
PubMed1999

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

Actins↗
PubMed1990

Peptide mimetics of an actin-binding site on myosin span two functional domains on actin.

The sites on the myosin heavy chain that interact with actin and are responsible for force generation are ill-defined: crosslinking and experiments with isolated domains of the myosin head implicate regions in both the 50K and 20K (molecular weights in thousands) domains of the myosin head (subfragment 1, S1) in this process. We have synthesized peptides from the sequence around the fast-reacting SH1 thiol residue in the 20K domain of S1 in order to delineate precisely an actin-binding site. We used a combination of 1H-NMR and enzyme inhibition assay and also assessed the effects of peptides on skinned rabbit psoas muscle fibres to show that the region of amino acids 690-725 contains an actin-binding site. Peptides from this region bind to actin, act as mixed inhibitors of the actin-stimulated S1 Mg2(+)-ATPase, and influence the contractile force developed in skinned fibres, whereas peptides flanking this sequence are without effect in our test systems. Remarkably, peptides from the N-terminal half of this segment 690-725 increase force development in skinned fibres at submaximal activating concentrations of Ca2+, that is, they behave as calcium-sensitizers; C-terminal peptides, however, inhibit force development without effecting sensitivity to calcium. These different responses indicate that this region is probably binding at two functionally distinct sites on actin.

Actins↗
PubMed2004

Actions by actin: reciprocal regulation of cortactin activity by tyrosine kinases and F-actin.

The polymerization of actin is catalysed by the Arp (actin-related protein) 2/3 complex, which acts downstream of a variety of receptors and signalling cascades. Intermediary molecules such as cortactin bind to the Arp2/3 complex and stimulate its activity, thus promoting actin polymerization and actin filament stabilization. New data in this issue of the Biochemical Journal by the Kapus group suggest that cortactin is reciprocally regulated by filamentous (F) actin and tyrosine kinases. This suggests a new paradigm for considering the cellular processes that regulate the dynamic organization of the actin cytoskeleton.

Actins↗
PubMed1995

Histamine-evoked chromaffin cell scinderin redistribution, F-actin disassembly, and secretion: in the absence of cortical F-actin disassembly, an increase in intracellular Ca2+ fails to trigger exocytosis.

Histamine is a known chromaffin cell secretagogue that induces Ca(2+) -dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca2+ utilized in histamine-evoked secretion. Here we report that histamine-H1 receptor activation induces redistribution of scinderin, a Ca(2+)-dependent F-actin severing protein, cortical F-actin disassembly, and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca2+ and were triggered by release of Ca2+ from intracellular stores. The trigger for the release of Ca2+ was inositol 1,4,5-trisphosphate because U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP3 and intracellular Ca2+ levels, scinderin redistribution, cortical F-actin disassembly, and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca2+, blocked the rise in intracellular Ca2+ concentration, scinderin redistribution, F-actin disassembly, and catecholamine secretion in response to histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca2+ from intracellular stores. This suggests that protein kinase C is involved in histamine-induced responses. The results also show that in the absence of F-actin disassembly, rises in intracellular Ca2+ concentration are not by themselves capable of triggering catecholamine release.

Actins↗