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PubMed2005

Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2.

The role of actin in endocytosis of G protein-coupled receptors is poorly defined. In the present study, we demonstrate that agents that depolymerize (latrunculin B and cytochalasin D) or stabilize (jasplakinolide) the actin cytoskeleton blocked agonist-induced endocytosis of the beta isoform of the thromboxane A(2) receptor (TPbeta) in HEK293 cells. This suggests that endocytosis of TPbeta requires active remodeling of the actin cytoskeleton. On the other hand, disruption of microtubules with colchicine did not affect endocytosis of the receptor. Expression of wild-type and mutant forms of the small GTPases RhoA and Cdc42 potently inhibited endocytosis of TPbeta, further indicating a role for the dynamic regulation of the actin cytoskeleton in this pathway. Agonist treatment of TPbeta in HEK293 cells resulted in the formation of actin stress fibers through Galpha(q/11) signaling. Because we previously showed that endocytosis of TPbeta is dependent on arrestins, we decided to explore the relation between arrestin-2 and -3 and actin in endocytosis of this receptor. Interestingly, we show that the inhibition of TPbeta endocytosis by the actin toxins in HEK293 cells was overcome by the overexpression of arrestin-3, but not of arrestin-2. These results indicate that the actin cytoskeleton is not essential in arrestin-3-mediated endocytosis of TPbeta. However, arrestin-3 could not promote endocytosis of the TPbetaY339A and TPbetaI343A carboxyl-terminal mutants when the actin cytoskeleton was disrupted. Our data provide new evidence that the actin cytoskeleton plays an essential role in TPbeta endocytosis. Furthermore, our work suggests the existence of actin-dependent and -independent arrestin-mediated pathways of endocytosis.

Actins↗
PubMed1998

The yeast V159N actin mutant reveals roles for actin dynamics in vivo.

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.

Actins↗
PubMed2002

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

Actins↗
PubMed1982

Effect of actin-binding protein on the sedimentation properties of actin.

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F-actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.

Actins↗
PubMed1992

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.

Actins↗
PubMed1987

Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells.

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.

Actins↗
PubMed2004

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells.

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

Actins↗
PubMed1979

Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules.

G actin, labelled presumably on cysteine-373 with the fluorescent chromophore N-iodoacetyl-N'-(5 sulfo-1-napthyl)-ethylenediamine and purified by Sephacryl S-200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations. It was found that these anisotropy decays could be fitted by a sum of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5 degrees C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5 degrees C) were introduced in order to characterize the F actin motions. The lower of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions. The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.

Actins↗
PubMed1994

A gelsolin-related actin-severing protein with fully reversible actin-binding properties from the tail muscle of crayfish, Astacus leptodactylus.

A Ca(2+)-dependent actin-severing protein was purified from the tail muscle of the crayfish Astacus leptodactylus. The isolation procedure involved extraction at low ionic strength in the presence of EGTA, followed by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The purified crayfish actin modulator appeared as a single band with a molecular mass of 105 kDa on SDS/PAGE. The crustacean actin modulator revealed basic functional properties in common with vertebrate gelsolin, like the Ca(2+)-activated severing of F-actin and the nucleation of actin polymerization. However, both proteins differed in major aspects: Ca2+ activation of crayfish actin modulator started at lower threshold concentrations (0.1 microM). The effect of the modulator on shortening the nucleation phase of actin polymerization was significantly weaker at lower modulator/actin ratios. The modulator formed three distinct stoichiometric complexes with G-actin, identified as binary, ternary and quaternary. Binding of G-actin occurred in a low cooperative manner and was completely reversible by EGTA. Despite some properties being similar to those of villin, crayfish actin modulator did not cross-link actin filaments. It is regarded in principle as a gelsolin-type protein, but with characteristic functional deviations from vertebrate gelsolin.

Actins↗
PubMed1991

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.

Actins↗
PubMed2002

Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells.

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-delta, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-delta were verified by Western blot analysis of immunoprecipitated proteins. PKC-delta was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-delta. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-delta. Binding of recombinant PKC-delta to NKCC1 was not detected in overlay assays. Rather, activated PKC-delta bound to actin, and this interaction was prevented by a peptide encoding deltaC2, a C2-like domain based on the amino acid sequence of PKC-delta. deltaC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-delta in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-delta indicated colocalization of PKC-delta and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-delta.

Acetophenones↗
PubMed2000

Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule.

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

Actins↗
PubMed1999

Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

Actins↗
PubMed2001

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization.

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

Actins↗
PubMed1992

Beta actin expression and organization of actin filaments in colorectal neoplasia.

Several studies have linked abnormal actin cytoskeletal organization and mutant actin genes with neoplastic transformation. Using in situ hybridization techniques we looked at expression of beta actin mRNA at a cellular level in normal, benign and neoplastic human colorectal tissues and correlated the level of expression with the extent of actin cytoskeletal organization in sequential sections. Normal tissues showed light labelling with the actin riboprobe, but non-neoplastic crypts, with evidence of regeneration and repair, showed greater levels. Higher levels of actin mRNA expression were found in adenomas and metaplastic polyps, but the highest levels were found in carcinomas, particularly those that were poorly differentiated. Cytoskeletal actin organization was, however, reduced in colorectal neoplasia. Normal mucosa showed the highest level of cytoskeletal actin organization, as assessed by phalloidin binding, and adenomas and metaplastic polyps also stained strongly. In contrast, phalloidin binding to poorly differentiated carcinomas was absent or very weak. The inverse relationship between actin mRNA expression and actin filament organization was confined to the carcinomas studied and may indicate defects in actin binding proteins or in post-transcriptional or translational events.

Actins↗
PubMed1988

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity.

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.

Acanthamoeba↗
PubMedDate not supplied

[Study of a protein complex from the brain which reduces actin viscosity. II. The complex inhibits elongation of actin filaments at the end preferable for polymerization and fragments the filaments].

Functional properties of the protein complex from bovine brain that shortens actin filaments are described. In the presence of Ca2+ complex shortens actin filaments and increases the initial rate of actin polymerization. In the absence of free calcium ions the complex loses its accelerating effect on actin polymerization, but still possesses actin filament shortening activity. Neither phalloidin nor tropomyosin prevent the shortening of actin filaments induced by the protein complex. Therefore the protein complex causes the fragmentation of actin filament. The data on actin polymerization in the presence of F-actin nuclei have indicated that the protein complex inhibits the elongation step of actin polymerization. The analysis of elongation in the presence of both the protein complex and cytochalasin D has demonstrated that the inhibition occurs on the fast-growing end of actin filaments.

Actins↗
PubMed1982

Nonlinear dependence on actin of acto-heavy meromyosin 18O exchange and ATPase rate at low actin concentrations.

Hydrolysis of highly enriched [gamma-18O]ATP in unlabeled water by acto-heavy meromyosin at low actin concentration was found to be heterogeneous in that significant amounts of both the species containing 0 or 3 18O-labeled oxygens/phosphate were formed. Detailed quantitative comparison with theoretical distributions over a wide range of actin concentrations, however, indicated that the pathway which catalyzed ATP hydrolysis with a low extent of exchange only made a significant contribution at a low actin concentration and did not represent a major fraction of the total hydrolysis seen at higher actin concentrations. This low exchange component was also detected in the dependence on actin of the steady state ATPase. At low actin the steady state ATPase rate increased more rapidly as a function of actin concentration than predicted by the Km and Vmax for actin activation observed at moderate to high actin levels. This extra ATP hydrolysis at low actin correlates with that predicted for the low exchange pathway both with respect to the fraction of the ATP hydrolyzed and to its dependence on the actin concentration.

Actins↗