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PubMed1992

Actin-dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization.

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from dark-adapted green sunfish and cultured in the light, they elongate 15 microns at 0.3-0.6 microns/min. Elongation was inhibited 65% by 0.1 microM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.

Actin Cytoskeleton↗
PubMed1991

Mechanism of the insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin.

Insertin, a protein purified from chicken gizzard smooth muscle, has been shown to retard but not to inhibit actin polymerization at the barbed ends of actin filaments. This effect has been explained by a model in which insertin remains bound to the barbed ends of actin filaments and new actin molecules are inserted into filaments between the barbed ends and barbed end-bound insertin molecules. In this paper we discuss the mechanism of the insertion reaction on a molecular level. A number of simple models were devised and were judged by their agreement with available experimental data. In one class of models insertin was assumed to dissociate from filament ends and to re-associate with the ends. Actin monomers would then bind to a filament end between a dissociation and an association reaction of insertin. In one of the two proposed models in this class insertin binds to an ATP-containing terminal subunit with higher affinity than to an ADP-containing terminal subunit. Dissociation of insertin is brought about by ATP hydrolysis at the terminal filament subunit. Insertion was then thought to re-associate with a filament end following binding of an ATP-containing actin monomer to the filament end. In the other of the two models' insertin was assumed to occur in two conformations which bind to filament ends with different affinities. Association and dissociation of insertin is caused by interconversion between the two forms of insertin. Both models turned out to be incompatible with experimental data. All types of models in which retardation of actin polymerization is brought about by dissociation and re-association of insertin with filament ends can be excluded by a common argument. As 10 nM insertin retards polymerization of 2 microns monomeric actin with maximal efficiency, the rate constant of binding of insertin to a filament end must be considerably higher (greater than 2 microM/10 nM = 200-fold). As the rate of association of actin with a barbed end is almost diffusion-controlled, assembly of insertin with a filament end would have to exceed the rate of a diffusion-controlled reaction. In the other class of models it was assumed that insertin remains permanently bound to filament ends during association or dissociation of an actin molecule and to move towards the terminal subunit of filaments. These models are compatible with experimental data. Thus, models are favoured where insertin remains bound to filament ends during polymerization and depolymerization of actin.

Actin Cytoskeleton↗
PubMed1998

Analysis of actin and actin-related protein 3 (ARP3) gene expression following induction of hyphal tip formation and apolar growth in Neurospora.

The genes encoding actin and ARP3 in the filamentous fungus Neurospora crassa were cloned and sequenced. The actin structural gene is interrupted by four introns and encodes a polypeptide of 375 amino acids, which shows very high degree of identity with actin from other sources. N. crassa ARP3 is 439 amino acids in length and is 71% to 80% identical to ARP3s from five other organisms, while actin is 40% to 50% identical to these same ARP3s. Transcript levels for actin and ARP3 decrease upon induction of asexual development (i.e. conidiation) and subsequently increase slightly when conidia are being formed. A concentration of filamentous actin is typically seen at sites of growth in eukaryotic organisms and, using indirect immunofluorescence, we showed that filamentous actin is localized primarily to hyphal tips in N. crassa. To determine if the level of actin increases in response to an increase in the number of growth sites and in the area of the growing surface, we used the temperature-sensitive mutants cot-1 and mcb. Growth of the cot-1 and mcb mutants at restrictive temperature induces hyphal tip formation and a loss of growth polarity, respectively. Unexpectedly, almost no increase in actin levels is observed following a > 20-fold increase in the number of hyphal tips or an increase in the area of the growing surface resulting from a loss of growth polarity. The results suggest that the level of actin monomers within N. crassa hyphae is sufficient to accommodate the need for additional actin patches and filaments that arises when the number of hyphal tips and the area of growing surface per unit length of hypha greatly exceeds that in wild-type.

Actin-Related Protein 3↗
PubMed1999

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.

Actin-Related Protein 2↗
PubMed2000

Regulation of the actin cycle in vivo by actin filament severing.

Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.

Actin Depolymerizing Factors↗
PubMed1992

Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes.

Actin Cytoskeleton↗
PubMed2003

Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor.

Human actin-depolymerizing factor (ADF) and cofilin are pH-sensitive, actin-depolymerizing proteins. Although 72% identical in sequence, ADF has a much higher depolymerizing activity than cofilin at pH 8. To understand this, we solved the structure of human cofilin using nuclear magnetic resonance and compared it with human ADF. Important sequence differences between vertebrate ADF/cofilins were correlated with unique structural determinants in the F-actin-binding site to account for differences in biochemical activities of the two proteins. Cofilin has a short beta-strand at the C terminus, not found in ADF, which packs against strands beta3/beta4, changing the environment around Lys96, a residue essential for F-actin binding. A salt bridge involving His133 and Asp98 (Glu98 in ADF) may explain the pH sensitivity of human cofilin and ADF; these two residues are fully conserved in vertebrate ADF/cofilins. Chemical shift perturbations identified residues that (i) differ in their chemical environments between wild type cofilin and mutants S3D, which has greatly reduced G-actin binding, and K96Q, which does not bind F-actin; (ii) are affected when G-actin binds cofilin; and (iii) are affected by pH change from 6 to 8. Many residues affected by G-actin binding also show perturbation in the mutants or in response to pH. Our evidence suggests the involvement of residues 133-138 of strand beta5 in all of the activities examined. Because residues in beta5 are perturbed by mutations that affect both G-actin and F-actin binding, this strand forms a "boundary" or "bridge" between the proposed F- and G-actin-binding sites.

Actin Depolymerizing Factors↗
PubMed1994

Interactions of tensin with actin and identification of its three distinct actin-binding domains.

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

Actin Cytoskeleton↗
PubMed1992

Actin-binding proteins regulate the work performed by myosin II motors on single actin filaments.

Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, "The Enzymes," Vol. 18, Orlando, FL: Academic Pres, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or alpha-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level.

Actins↗
PubMed1986

Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum.

We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.

Actins↗
PubMed1990

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos.

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.

Actins↗
PubMed1994

H2O2-induced increases in cellular F-actin occur without increases in actin nucleation activity.

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) approximately 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.

Actins↗
PubMed2000

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function.

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73. Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.

Acetylation↗
PubMed2001

Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics.

Cross-links between protomers in F-actin can be used as a very sensitive probe of both the dynamics and structure of F-actin. We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction. The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments. This finding is in conflict with refined models but in agreement with the original Holmes et al. model for F-actin. Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin.

Actins↗
PubMedDate not supplied

Small angle X-ray scattering and electron cryomicroscopy study of actin filaments: role of the bound nucleotide in the structure of F-actin.

Actin filaments (F-actin) complexed to various nucleotides (ADP (adenosine diphosphate) ADP-P(i) (P(i), inorganic phosphate), and ADP-BeF3- (BeF3-, beryllium fluoride)) have been studied by small angle X-ray scattering and electron cryomicroscopy. The small angle X-ray scattering data show that the value of the cross-radius of gyration (2.55 +/- 0.15 nm) and the mean helical symmetry of the filaments are independent of the nature of the bound nucleotide. The scattering profiles of all the F-actin suspensions exhibit secondary maximal located at similar positions. However, the intensities of the secondary maxima depend upon both the protein concentration and the type of nucleotide bound to the F-actin. These variations indicate that F-actin has different properties (such as its tendency to aggregate and the structure of the filament), which depend upon the type of nucleotide bound. Electron cryomicroscopy of vitrified suspensions shows that F-actin may have different conformations. The nonequatorial layer lines, which constitute the Fourier transforms of the filament images, have different signal to noise ratios depending on the type of nucleotide bound to the actin. The 3-D reconstructions of the actin filaments show an inner and outer domain. In the maps, the visibility of the outer domain depends upon the type of nucleotide bound to the actin. It respectively increases for ADP-P(i), ADP, and ADP-BeF3-. We attribute these differences to the disorder of the filaments, which in turn depends upon the nucleotide bound. These variations in disorder imply that there are structural changes (contacts, structure, or orientation) in the actin subunits forming the filaments. These changes may have an important role in the motility processes in which actin is involved.

Actins↗
PubMed1994

Evidence for an F-actin like conformation in the actin:DNase I complex.

We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucleotide in F-actin, but fails to bind to G-actin. TNP-ATP is also able to bind to actin in the actin:DNase I complex, suggesting that the nucleotide binding site in the actin:DNase I complex adopts a conformation similar to that found in F-actin. This result is consistent with the hypothesis that the two major domains of actin on either side of the cleft are able to "flex" or move relative to each other in G-actin, but that this flexing motion is limited as a consequence of either polymerisation or DNase I binding. F-actin, in which approximately 80% of the bound nucleotide is TNP-ADP, appears to be functionally similar to native ADP-F-actin. It can superprecipitate with myosin and, following regulation with troponin-tropomyosin, exhibits a Ca(2+)-sensitivity during superprecipitation. Sonication induced nucleotide exchange in regulated F-actin was not sensitive to the presence of Ca2+ which argues against a significant conformational change in the vicinity of the nucleotide binding site during Ca(2+)-sensitive thin filament regulation.

Actins↗
PubMed1986

The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments.

A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchin Tripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentration is increased, the initial rate of actin assembly is accelerated. Moreover, the final polymer concentration is reduced indicating that SU45 caps the preferred end of actin filaments shifting the critical concentration (Cc) to that of the nonpreferred end. Determination of the Cc as a function of the concentration of SU45 gave an apparent KD of 1 nM. Dilution of F-actin to below its Cc, into buffers containing SU45 and Ca2+ resulted in a sharp increase in the rate of depolymerization; reducing the Ca2+ concentration attenuated this effect. Incubation of SU45 with rabbit skeletal muscle G-actin yielded a 1:1 complex which held 45Ca2+ tightly with a dissociation half-time of 10.8 days. By kinetic analyses of assembly in the presence of the SU45-actin complex and dilution-induced disassembly of filaments precapped with complex, we have estimated both the association rate constant (4.0 X 10(4)M-1s-1) and the dissociation rate constant (0.05s-1) for the nonpreferred ends of actin filaments. Finally, dilution of F-actin to below its Cc, into complex in either Ca2+ or EGTA resulted in a much slower depolymerization consistent with a rapid capping of the preferred end by the SU45-actin complex.

Actins↗
PubMed1986

Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates.

We have determined the sequences of three recombinant cDNAs complementary to different mouse actin mRNAs that contain more than 90% of the coding sequences and complete or partial 3' untranslated regions (3'UTRs): pAM 91, complementary to the actin mRNA expressed in adult skeletal muscle (alpha sk actin); pAF 81, complementary to an actin mRNA that is accumulated in fetal skeletal muscle and is the major transcript in adult cardiac muscle (alpha c actin); and pAL 41, identified as complementary to a beta nonmuscle actin mRNA on the basis of its 3'UTR sequence. As in other species, the protein sequences of these isoforms are highly (greater than 93%) conserved, but the three mRNAs show significant divergence (13.8-16.5%) at silent nucleotide positions in their coding regions. A nucleotide region located toward the 5' end shows significantly less divergence (5.6-8.7%) among the three mouse actin mRNAs; a second region, near the 3' end, also shows less divergence (6.9%), in this case between the mouse beta and alpha sk actin mRNAs. We propose that recombinational events between actin sequences may have homogenized these regions. Such events distort the calculated evolutionary distances between sequences within a species. Codon usage in the three actin mRNAs is clearly different, and indicates that there is no strict relation between the tissue type, and hence the tRNA precursor pool, and codon usage in these and other muscle mRNAs examined. Analysis of codon usage in these coding sequences in different vertebrate species indicates two tendencies: increases in bias toward the use of G and C in the third codon position in paralogous comparisons (in the order alpha c less than beta less than alpha sk), and in orthologous comparisons (in the order chicken less than rodent less than man). Comparison of actin-coding sequences between species was carried out using the Perler method of analysis. As one moves backward in time, changes at silent sites first accumulate rapidly, then begin to saturate after -(30-40) million years (MY), and actually decrease between -400 and -500 MY. Replacements or silent substitutions therefore cannot be used as evolutionary clocks for these sequences over long periods. Other phenomena, such as gene conversion or isochore compartmentalization, probably distort the estimated divergence time.

Actins↗