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1.
Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.  相似文献   

2.
The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.  相似文献   

3.
Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.  相似文献   

4.
5.
Adducin is a protein associated with spectrin and actin in membrane skeletons of erythrocytes and possibly other cells. Adducin has activities in in vitro assays of association with the sides of actin filaments, capping the fast growing ends of actin filaments, and recruiting spectrin to actin filaments. This study presents evidence that adducin exhibits a preference for the fast growing ends of actin filaments for recruiting spectrin to actin and for direct association with actin. beta-Adducin-(335-726) promoted recruitment of spectrin to gelsolin-sensitive sites at fast growing ends of actin filaments with half-maximal activity at 15 nM and to gelsolin-insensitive sites with half-maximal activity at 75 nM. beta-Adducin-(335-726) also exhibited a preference for actin filament ends in direct binding assays; the half-maximal concentration for binding of adducin to gelsolin-sensitive sites at filament ends was 60 nM, and the Kd for binding to lateral sites was 1.5 microM. The concentration of beta-adducin-(335-726) of 60 nM required for half-maximal binding to filament ends is in the same range as the concentration of 150 nM required for half-maximal actin capping activity. All interactions of adducin with actin require the myristoylated alanine-rich protein kinase C substrate-related domain as well as a newly defined oligomerization site localized in the neck domain of adducin. Surprisingly, the head domain of adducin is not required for spectrin-actin interactions, although it could play a role in forming tetramers. The relative activities of adducin imply that an important role of adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or loss of actin subunits.  相似文献   

6.
The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 microM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.  相似文献   

7.
A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.  相似文献   

8.
Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.  相似文献   

9.
We have purified a protein from rabbit serum with a molecular weight of 90,000 that inhibits the polymerization of actin measured viscometrically and that we have named "brevin" (from the Latin breviare, to shorten). From the extent of purification, we estimate that this inhibitor constitutes 0.3% of the total protein in plasma and serum. Brevin is also present in sera from humans and rats. Almost all of the activity in blood is extracellular; only 1% is present in platelets or other cellular elements. Several lines of evidence indicate that brevin is the same protein as the factor described by Fagraeus and Norberg [Fagraeus, A. & Norberg, R. (1978) Curr. Top. Microbiol. Immunol. 82, 1-13] as an actin-depolymerizing factor (ADF). If ADF and brevin are identical, then "ADF" is an inappropriate name because we find that the protein shortens actin filaments without depolymerizing them. Thus, brevin causes little change in the critical concentration of monomeric actin, even though the inhibitor binds to monomeric actin complexed to DNase I-agarose. Binding of brevin to filaments was demonstrated by sedimenting the inhibitor with F-actin. From the amounts of actin and brevin sedimented, and from the lengths of filaments measured by electron microscopy, we calculated that the stoichiometry of binding is one brevin molecule per filament over a wide range of inhibitor concentrations. This stoichiometry suggests that brevin inhibits polymerization by binding at the end of elongating actin filaments, a mechanism similar to that proposed for several intracellular actin-binding proteins and for the cytochalasins. Its abundance suggests that brevin plays an important physiological role in serum, but one not directly concerned with intracellular motility. Therefore its relationship to cytoplasmic actin-binding proteins remains to be determined.  相似文献   

10.
Actin filaments could play a role in regulation of cell swelling by two distinct mechanisms. One is by a tensile mechanism involving the coordinated interaction of actin and actin-associated proteins with all plasma membrane domains. The actin-membrane linkage would restrain cell swelling in the event of the influx of water. In shark rectal gland cells, conditions that cause massive cell swelling (i.e., high K medium, exposure to mercurials) are associated with disruption of membrane-associated actin filaments. Under conditions that result in only moderate swelling (Na-pump inhibition, Li substitution) the actin filaments remain associated with the cell membrane. Thus, in this cell type, disruption of the actin-membrane organization is correlated with increased swelling. Another mechanism by which actin could limit cell swelling is via regulation of ion transport proteins that are activated by cell swelling. This could be accomplished by a vesicle transport and insertion mechanism that delivers ion transport units to the cell membrane or by interaction with transport proteins already present in the membrane. Cell-attached patch clamp studies of RCCT-28A cells exposed to hypotonic medium demonstrated the activation of Cl-channel activity coincident with an alteration in actin. Activation of the channel was mimicked by stretching the membrane. Exposure of inside-out patches to cytochalasins also increased Cl-channel activity. Treatment of isolated patches with phalloidin inhibited stretch-induced activation. Thus, regulation of a volume-sensitive Cl-channel appears to be directly related to the state of organization of actin filaments.  相似文献   

11.
alpha-crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced damage and to share sequence similarity with small heat-shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by alpha-crystallin, alpha A-crystallin or alpha B-crystallin. However, phosphorylation of alpha-crystallin markedly decreased its protective effect. It is suggested that phosphorylation of alpha-crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain alpha-crystallin species. This communication presents biochemical evidence clearly demonstrating that alpha-crystallin is involved in actin polymerization-depolymerization dynamics. It is also shown that alpha-crystallin prevented heat-induced aggregation of actin filaments. alpha-crystallin was found to stabilize actin polymers decreasing dilution-induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 microM to 0.18 microM. Similar results were found with either alpha-crystallin or its purified subunits alpha A-crystallin and alpha B-crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between alpha-crystallin and actin monomers since the effect of alpha-crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the alpha-crystallin effect indicates that 2-3 alpha-crystallin monomers/actin monomer give maximum actin polymer stabilization.  相似文献   

12.
Actin depolymerizing factor (ADF)/cofilin is a widely distributed family of actin-binding proteins which regulate actin polymerization in a pH-dependent manner. In cultured cells, cofilin, as well as ADF, translocates from the cytoplasm into the nucleus together with actin and forms rod-like structures in response to heat shock or dimethylsulfoxide (DMSO) treatment. In order to study in vivo interaction of cofilin with actin, we examined the effects of cofilin overexpression on actin cytoskeleton in C2 myoblasts. Interestingly, no remarkable effect was observed on phalloidin-stained patterns in cells overexpressing cofilin as compared with normal cells. However, upon treatment with DMSO, cytoplasmic actin filaments were disrupted and intranuclear rod structures containing cofilin and actin were apparently larger and thicker in cells overexpressing cofilin than in normal cells. Heat shock also stimulated disruption of microfilaments and formation of both intranuclear and prominent cytoplasmic cofilin-actin rods in cofilin-transfected cells, suggesting that DMSO-treatment or heat shock triggers cofilin-actin interaction. We further found that a myosin ATPase inhibitor (BDM) induced a reduction in cytoplasmic staining with phalloidin in cofilin-transfected cells. The results suggest that myosin activity might be involved in the regulation of cofilin-actin interactions in vivo.  相似文献   

13.
Actin bundles are common cytoskeletal structures but ones which are usually polymorphic, varying from bundle to bundle. Two-dimensional arrays of actin filaments crosslinked by actin-bundling proteins are more tractable structures to analyze than are the three-dimensional bundles found in cells. The first step in analyzing these two-dimensional "rafts" is to determine the spatial relationships between neighboring filaments. It is difficult to discern such relationships by inspection of the electron micrographs of rafts, but easy by examination of the Fourier transforms. We provide theory and examples of the analysis of transforms of rafts, and show that different bundling proteins give rise to different kinds of rafts.  相似文献   

14.
It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments.  相似文献   

15.
Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.  相似文献   

16.
Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.  相似文献   

17.
18.
The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.  相似文献   

19.
In a previous paper, we studied elementary models for polymerization, depolymerization, and fragmentation of actin filaments (Edelstein-Keshet and Ermentrout, 1988, Bull. Math. Biol. 60, 449-475). When these processes act together, more complicated dynamics occur. We concentrate on a particular case study, using the actin-fragmenting protein gelsolin. A set of biological parameter values (drawn from the experimental literature) is used in computer simulations of the kinetics of gelsolin-mediated actin filament fragmentation.  相似文献   

20.
During exercise, dynamic hyperinflation-induced intrinsic positive end-expiratory pressure (PEEPi) and decreased dynamic lung compliance (CL,dyn) of patients with chronic obstructive pulmonary disease (COPD) increase the elastic work of inspiration (Wi) more than would be predicted from the increase in tidal volume (VT). This contributes significantly to their exertional breathlessness. In 10 stable patients with COPD, the dynamic Wi was measured during incremental bicycle exercise to exhaustion. The total Wi was then partitioned into the portion required to overcome PEEPi (Wi,PEEPi) and nonPEEPi elastic load (Wi,nonPEEPi). The latter is used to overcome the increase in the total respiratory system elastance during inflation. From resting breathing to peak exercise, Wi more than doubled (p<0.001). This increase was largely due to Wi,PEEPi, which significantly rose from 1.7+/-0.3 to 5.3+/-0.8 L x cm H2O(-1) (p<0.001). In comparison, Wi,nonPEEPi increased from only 3.0+/-0.4 to 5.1+/-0.5 L x cm H2O(-1) (p<0.01). Consequently, Wi,PEEPi as a fraction of total Wi increased from 35.5+/-5.6 to 51.0+/-3.3% (p<0.02). In addition, the measured Wi,nonPEEPi at peak exercise, when expressed as a percentage of its value during resting breathing, was 25% more than that predicted from the increase in VT alone. Assuming a constant chest wall compliance, this can be attributed to the exercise-induced decrease in CL,dyn, which was 0.27+/-0.04 and 0.17+/-0.02 L x cm H2O(-1) (p<0.01), respectively, during resting breathing and peak exercise. In conclusion, the dynamic hyperinflation-induced intrinsic positive end-expiratory pressure is more important than the increase in tidal volume in raising the work of inspiration during exercise in patients with chronic obstructive pulmonary disease; the decrease in dynamic lung compliance plays a definite but less important role.  相似文献   

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