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PubMed2002

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro.

We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament "seeds" and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.

Actin Cytoskeleton↗
PubMed1999

Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.

Actin Depolymerizing Factors↗
PubMed1989

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.

Actins↗
PubMed1991

Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].

Actins↗
PubMed2007

Expression patterns of ubiquitin, heat shock protein 70, alpha-actin and beta-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei.

Crustacean muscle growth is discontinuous due to molt cycle. To characterize molt-related gene expression patterns, we studied the mRNA levels of molecular chaperone-ubiquitin and heat shock protein 70 (Hsp 70) in comparison with muscle protein alpha-actin and beta-actin in marine shrimp Litopenaeus vannamei. Total RNA from abdominal muscle was isolated from 3-month-old animals in six different molt stages. The mRNA levels of target genes were detected by reverse-transcriptase-multiplex PCR and expressed as the ratio to elongation factor-1alpha. Ubiquitin mRNA levels were relatively steady over all stages of the molt cycle. Hsp70 levels were not detectable in early postmolt and late premolt stages, but showed a progressive increase from late postmolt to intermolt stages. Expression levels of alpha-actin gene were lower during postmolt, reached a plateau in intermolt and remained relatively high in premolt stage. Levels of beta-actin increased progressively from postmolt to intermolt, reaching a maximum value in premolt. Therefore, the mRNAs encoding for ubiquitin and Hsp 70 in abdominal muscle did not increase significantly in premolt stages, which is typically associated with claw muscle degradation. Muscle structural alpha-actin and cytoskeletal beta-actin were increased during intermolt and premolt stages, suggesting high muscle growth during these stages in the abdominal muscle of the L. vannamei.

Abdominal Muscles↗
PubMed1996

Cytoskeletal F-actin polymerization from cytosolic G-actin occurs in the phagocytosing immunocytes of arthropods (Limulus polyphemus and Gromphadorhina portentosa): does [cAMP]i play any role?

Phagocytosis is a major defense reaction in arthropods and is accomplished by two blood cells (hemocytes), the granulocyte (GRs) and plasmatocytes (PLs), collectively called immunocytes. Immunocytes (principally the GRs) from two arthropods, Limulus polyphemus (horseshoe crab) and Gromphadorhina portentosa (Madagascar hissing cockroach) effectively phagocytose fluorescein isothiocyanate (FITC)-conjugated fluoresbrite microspheres (FITC-FM) and chicken (Gallus domesticus) erythrocytes within 1 hr of incubation. Although actin polymerization and changes in intracellular cAMP ([cAMP]i) levels occur during the early stages of phagocytosis in vertebrates, these two phenomena have not been studied in arthropod immunocytes. Using the DNase I inhibition assay, we found a decrease in cytosolic G-actin and an increase in the cytoskeletal F-actin in the phagocytosing immunocytes; the total actin in both resting and phagocytosing immunocytes remained constant. These results showed an 86% increase in F-actin in G. portentosa immunocytes and a 29% increase in those of L. polyphemus after 1 hr of initial incubation with FITC-FM. As in some vertebrates, the role of [cAMP]i in the early stages of phagocytosis in these two animals- and perhaps in arthropods in general-is variable; although we detected some negligible amounts of [cAMP]i (0.10-0.80 pmol/cell at different time intervals) in L. polyphemus immunocytes, it was inconclusive whether those in G. portentosa also contained [cAMP]i. Even in L. polyphemus, the difference in the amounts of [cAMP]i in resting and phagocytosing cells was insignificant (P > 0.05). It was also inconclusive whether [Ca2+]i and/or [Mg2+]i play any roles in the early stages of phagocytosis in the two arthropods in this study. These results suggest that the two phenomena (F-actin polymerization and levels of [cAMP]i in arthropods) are basically similar to those in vertebrate neutrophils and macrophages, which suggests that certain immunological mechanisms are conserved in nature.

Actins↗
PubMed1998

Fluorescent phalloidin enables visualization of actin without effects on myosin's actin filament sliding velocity and hydrolytic properties in vitro.

Recent reports have demonstrated an activating effect of phalloidin in striated muscle. Furthermore, modeling of X-ray diffraction and crystallographic data suggests that phalloidin binding may induce conformational changes in actin. To determine whether phalloidin affects the mechanics of the actomyosin interaction, the velocity of actin filaments variably labeled with rhodamine-phalloidin was measured. In addition, solution actin-activated myosin subfragment-1 ATPase activity with phalloidin-labeled actin was compared to unlabeled actin. Here we found that phalloidin does not significantly effect actin filament velocity or parameters of ATPase, namely Vmax and K(m). Possible differences between muscle strip data and these in vitro results are discussed.

Actins↗
PubMed2001

Two conformations of G-actin related to two conformations of F-actin.

In summary, a number of different conformational states of F-actin have been described by several different laboratories. Crystal structures have revealed that an opening of the nucleotide-binding cleft, produced by a large rotation of subdomain 2, can occur in G-actin. We have shown that two crystal states of beta-actin, in an open and closed form, can provide a very good model for the conformational difference in F-actin between yeast the wild-type and a V159N mutant. This suggests that some of the dynamics associated with G-actin may provide insights into dynamic processes within the F-actin filament.

Actins↗
PubMed1995

Actin interaction with purified dystrophin from electric organ of Torpedo marmorata: possible resemblance with filamin-actin interface.

We have purified dystrophin from Torpedo marmorata electric tissue by means of alkaline extraction in conjunction with an affinity chromatography column using anti-peptide antibodies. Using solution (cosedimentation) and solid phase experiments (sedimentation with Sepharose filamentous actin and ELISA), we have demonstrated that purified dystrophin is able to bind filamentous and monomeric actin. Using ELISA coupled with biotin labelled peptides and taking advantage of strong affinity binding of streptavidin-biotin complex, we have identified two exposed sequences of the actin molecule implicated in dystrophin binding: fragment 40-113, further restricted to peptide 75-106 and peptide 360-372. In a previous study, we have shown that fragment 40-113 displays binding site(s) for filamin but probably not for alpha-actinin. Moreover, we have recently reported that alpha-actinin and filamin display divergent behaviours towards conformational changes of actin. In this study, we have demonstrated that, similarly to filamin, dystrophin binding is insensitive to the locking of actin in a monomeric conformation. Taken together, these results lead us to favour the idea that dystrophin could share properties in common with filamin in its binding of actin.

Actinin↗
PubMed1988

Scanning microfluorometric measurement of TRITC-phalloidin labelled F-actin. Dependence of F-actin content on density of normal and transformed cells.

A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting. The relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considering a possible involvement of F-actin in growth control.

Actins↗
PubMed1986

Kinetics of actin monomer exchange at the slow growing ends of actin filaments and their relation to the elongation of filaments shortened by gelsolin.

The kinetics of actin monomer exchange with the slow growing pointed ends of actin filaments have been determined by measuring rates of monomer addition to or loss from filaments with their fast-growing barbed ends blocked by the protein gelsolin. Direct measurements of filament length by electron microscopy confirmed that each gelsolin acts as a nucleus for an actin filament. The rate constants ascertained are k- = 0.03 s-1; k+ = 0.06 microM-1 s-1 at 23 degrees C and k- = 0.11 s-1; k+ = 0.09 microM-1 s-1 at 37 degrees C. They are approximately independent of pH from 7.0 to 8.0 at both temperatures. These rates are far slower than those reported on the basis of some electron microscopic studies of filaments assembled on to actin bundles. The rate constants also predict a higher critical monomer concentration for the pointed end at 37 degrees C than at room temperature, consistent with direct measurements of this quantity. The relative slowness of the monomer exchange at the pointed end suggests that actin filaments with blocked barbed ends are relatively stable. The rate of redistribution of actin monomers from filaments stabilized at their barbed ends by gelsolin-calcium complex to longer filaments was measured following removal of Ca2+, which decreases the capacity of gelsolin to nucleate filaments. The elongation occurs at a rate consistent with the measured rates of monomer exchange and is quantitatively described by Hill's model for filament elongation by random exchange of monomers from one end.

Actins↗
PubMed1987

The sequence of a sea urchin muscle actin gene suggests a gene conversion with a cytoskeletal actin gene.

We report the nucleotide sequence of the single muscle actin gene of the sea urchin Strongylocentrotus purpuratus. Comparison of the protein-coding sequence of this muscle actin gene (pSpG28) with that of two linked sea urchin cytoskeletal actin genes (pSpG17 and CyIIa) reveals a region of exceptional sequence conservation from codon 61 through codon 120. Furthermore, when silent nucleotide changes are compared, the conservation of this region is still evident (7.9% silent site differences in the conserved region vs 43.3% silent site differences in the rest of the gene when pSpG28 and CyIIa are compared), indicating that the conservation is not due to particularly stringent selection on the portion of the protein encoded by this region of the genes. These observations suggest that a gene conversion has occurred between the muscle actin gene and a cytoskeletal actin gene recently in the evolution of the sea urchin genome. Gene conversion between nonallelic actin genes may thus play a role in maintaining the homogeneity of this highly conserved gene family.

Actins↗
PubMed1997

The actin gene of the glaucocystophyte Cyanophora paradoxa: analysis of the coding region and introns, and an actin phylogeny of eukaryotes.

We isolated the actin gene of the glaucocystophyte alga Cyanophora paradoxa and analyzed the coding region and its introns. Phylogenetic analyses of the actin coding region and the inferred protein sequence in data sets containing 47 other actin sequences show Cyanophora to be a member of the eukaryotic crown-group radiation in agreement with ribosomal DNA sequence analyses. Four of the five Cyanophora actin introns are relatively short (55-59 nt) and occupy novel positions in a catalogue of actin introns containing 56 distinct sites. The fifth intron has a length of 171 nt and occurs also in actin genes from green algae and the crustacean Artemia.

Actins↗
PubMed2004

Ha-VP39 binding to actin and the influence of F-actin on assembly of progeny virions.

We present evidence that actin is necessary for the successful assembly of HaNPV virions. Purified nucleocapsid protein Ha-VP39 of Heliothis armigera nuclear polyhedrosis virus (HaNPV) was found to be able to bind to actin in vitro without assistance, as demonstrated by Western blot and isothermal titration calorimeter. DeltaH and binding constants (K) detected by isothermal titration calorimeter strongly suggested that Ha-VP39 first binds actin to seed the formation of hexamer complex of actin, and the hexamers then link to each other to form filaments, and the filaments finally twist into cable structures. The proliferation of HaNPV was completely inhibited in Hz-AM1 cells cultivated in the medium containing 0.5 microg/ml cytochalasin D (CD) to prevent polymerization of actin, while its yield was reduced to 10(-4) in the presence of 0.1 microg/ml CD. Actin concentration and the viral DNA synthesis were not significantly affected by CD even though the progeny virions assembled in the CD treated cells were morphologically different from normal ones and resulted in fewer plaques in plaque assay.

Actins↗
PubMed1989

Substoichiometric concentrations of ATP-G-actin are required to anneal actin polymerized by calcium ions.

At 3 degrees C and pH 7.0, the addition of 40 nM ATP-G-actin to F-actin (12 microM as the monomer), polymerized in the presence of 4 mM CaCl2, determines a substantial and rapid increase of the viscosity of the solution, which is accompanied by the incorporation of the ATP-G-actin added into the polymer. The hypothesis that the presence of ATP-actin at the filament end(s) promotes the annealing reaction is substantiated by the finding that, after the addition of ATP-G-actin, the average filament length is increased. This finding is relevant, not only because it provides evidences in favour of the existence of annealing but also because it shows that the concentration of ATP-G-actin influences the filaments length distribution through a mechanism different from the elongation reaction.

Actins↗
PubMed1986

Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.

In 8-day-old embryos stereocilia can be identified on the hair cells of the chick cochlea; within each is a small population of actin filaments which extend from the tip of the stereocilium to the apical cytoplasm of the cell. These filaments are not ordered in a regular way, however, and tend to be found near the lateral margins of the stereocilia with large spaces between adjacent filaments. By 9 days the spaces between adjacent filaments are reduced and there are regions where the crossover points of adjacent actin helices are in register even though in cross section the actin filaments do not lie on a regular lattice. By 10-11 days the actin filaments become progressively more crossbridged together and we can recognize in longitudinal section horizontal stripes caused by the periodicity of the crossbridges. In transverse section the filaments begin to lie on a hexagonal lattice. Each stereocilium, however, contains less than 100 actin filaments. Evidence is presented that once crossbridging is maximal and the filaments hexagonally packed (Days 11-12), the stereocilia increase in width by the orderly addition of actin filaments to the lateral margins of the existing filament bundle so that by Day 16 we find up to 400 filaments all packed on a hexagonal lattice. Thus there are two stages in bundle formation. In the first a small number of filaments condense into a hexagonally packed, crosslinked bundle. In the second, the bundle increases in diameter by addition of filaments to the periphery of the bundle in a process akin to crystal growth. From observations on the elongation of filaments in the rootlets and stereocilia, we conclude that rootlets grow by addition of subunits at the nonpreferred end while stereocilia elongate by addition to the preferred end. What makes this interesting is that these two modes of addition occur at different developmental times.

Actins↗
PubMed1991

Peptide competition of actin activation of myosin-subfragment 1 ATPase by an amino terminal actin fragment.

The amino-terminal region of actin participates in the binding of myosin subfragment 1 (S1) during cross-bridge cycling, thereby assisting in the activation of the magnesium-dependent myosin ATPase. Effects of three actin fragments on the magnesium-dependent S1 and acto-S1 ATPase activities in solution were studied. One of the peptides, containing residues actin 1-44, mimicked the S1 ATPase-activating properties of actin and in turn inhibited acto-S1 ATPase both in a concentration-dependent manner. This suggests peptide competition for the actin binding site on myosin. The other fragments, residues actin 1-18 and 82-119, respectively, had no detectable effect on S1- and acto-S1 ATPase activity.

Actins↗
PubMed1994

Polymerization of actin from the thymosin beta 4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1.

Thymosin beta 4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin-thymosin beta 4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin beta 4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non-muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin-stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Actins↗