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PubMed2003

ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments.

Proteins in the ADF/cofilin (AC) family are essential for rapid rearrangements of cellular actin structures. They have been shown to be active in both the severing and depolymerization of actin filaments in vitro, but the detailed mechanism of action is not known. Under in vitro conditions, subunits in the actin filament can treadmill; with the hydrolysis of ATP driving the addition of subunits at one end of the filament and loss of subunits from the opposite end. We have used electron microscopy and image analysis to show that AC molecules effectively disrupt one of the longitudinal contacts between protomers within one helical strand of F-actin. We show that in the absence of any AC proteins, this same longitudinal contact between actin protomers is disrupted at the depolymerizing (pointed) end of actin filaments but is prominent at the polymerizing (barbed) end. We suggest that AC proteins use an intrinsic mechanism of F-actin's internal instability to depolymerize/sever actin filaments in the cell.

Actin Depolymerizing Factors↗
PubMed1983

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin.

Physico-chemical properties and interaction with actin of an actin-depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.

Actin Depolymerizing Factors↗
PubMed1985

Enhancement of actin-activated myosin ATPase by an 84K Mr actin-binding protein in vertebrate smooth muscle.

A Ca2+-dependent actin-severing 84K Mr protein prepared from bovine aorta caused four-fold activation of smooth muscle actin-activated myosin ATPase at a 1/10(2) molar ratio to actin in the presence of tropomyosin and light chain kinase-calmodulin in a Ca2+-dependent manner, while it inhibited it markedly at a 1/5 molar ratio. Purified actin-tropomyosin filaments under the experimental ATPase conditions were distributed in a range of more than 10 micron in length and the addition of the 84K Mr protein changed the filament length to around 1 micron at a 1/10(2) molar ratio to actin or less than 50 nm at a 1/5 molar ratio in the presence of Ca2+. However, the apparent length of actin filaments alone does not appear to be responsible for the activation of ATPase activity, since in the absence of tropomyosin, the ATPase activation was much less in spite of actin filament length changes. These results indicate the possibility that the 84K Mr protein plays an important role with tropomyosin in at least in vitro smooth muscle actin-myosin interaction.

Actin Cytoskeleton↗
PubMed1990

Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells.

Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D. Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2 X 10(4) and 1.8 X 10(4) nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which co-localised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3 X 10(3) and 1.2 X 10(4) caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3 X 10(3) caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15min) implies a maximum time for the complete turnover of actin in the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1999

Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms.

Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.

Actin Depolymerizing Factors↗
PubMed2000

The lens membrane skeleton contains structures preferentially enriched in spectrin-actin or tropomodulin-actin complexes.

The spectrin-based membrane skeleton plays an important role in determining the distributions and densities of receptors, ion channels, and pumps, thus influencing cell shape and deformability, cell polarity, and adhesion. In the paradigmatic human erythrocyte, short tropomodulin-capped actin filaments are cross-linked by spectrin into a hexagonal network, yet the extent to which this type of actin filament organization is utilized in the membrane skeletons of nonerythroid cells is not known. Here, we show that associations of tropomodulin and spectrin with actin in bovine lens fiber cells are distinct from that of the erythrocyte and imply a very different molecular organization. Mechanical disruption of the lens fiber cell membrane skeleton releases tropomodulin and actin-containing oligomeric complexes that can be isolated by gel filtration column chromatography, sucrose gradient centrifugation and immunoadsorption. These tropomodulin-actin complexes do not contain spectrin. Instead, spectrin is associated with actin in different complexes that do not contain tropomodulin. Immunofluorescence staining of isolated fiber cells further demonstrates that tropomodulin does not precisely colocalize with spectrin along the lateral membranes of lens fiber cells. Taken together, our data suggest that tropomodulin-capped actin filaments and spectrin-cross-linked actin filaments are assembled in distinct structures in the lens fiber cell membrane skeleton, indicating that it is organized quite differently from that of the erythrocyte membrane skeleton.

Actins↗
PubMed1992

Quasielastic light scattering study of thermal excitations of F-actin solutions and of growth kinetics of actin filaments.

In the first part of this work we report quasielastic light scattering (QELS) studies of the internal dynamics of transient actin networks over a time range of 10(-6)-10(-2) s, scattering angles between zeta = 20 degrees and 150 degrees, and a concentration range of 0.015 (0.3) to 0.7 mg/mL (15 microM). We confirm our previous result that (1) the dynamic structure factor g(q,t) is determined by the thermally excited undulations of the actin filaments and (2) that the initial decay of g(q, t) scales as g(q, t) varies; is directly proportional to exp(-q alpha t) while the long time decay scales as g(q, t) varies; is directly proportional to exp [-(Aq alpha t) 2/3] with alpha = 2.75. The deviation of alpha from the theoretical value of alpha = 3 predicted for Rouse-Zimm chains is similar to that found for high molecular weight macromolecular solutions by QELS. A refined analysis of the dynamic structure factor showed that it can be interpreted in terms of three relaxation processes (besides the contribution of the residual monomer diffusion): (1) the dominant Rouse-Zimm dynamics, which comprises between 65 (at high concentrations) and 85% of the signal; (2) a fast relaxation process with a decay constant of gamma = 9 x 10(3) s-1, which contributes at all concentrations with the same amplitude; and (3) a nonexponential ultraslow contribution of the form g(us) varies; is directly proportional to exp [(-gamma ust)]1/4. The third contribution appears only at high concentrations and increases strongly with decreasing scattering angles. It is thus attributed to fluctuations of the mesh size of the transient actin network. In the second part we show that high sensitivity QELS may be applied to follow the actin polymerization process at low temperatures (10 degrees C). The apparent diffusion coefficient and the static scattering intensity of the actin filaments were determined as functions of polymerization time tpol. We show that the process consists of the rapid growth of a few filaments that become very long (approximately 10 microns; even at actin concentrations of 0.04 micrograms/mL) near the critical growth concentration of 0.012 micrograms/mL, as is expected for a growth process determined by nucleation. Finally, we studied actin networks polymerized in the presence of complexes of gelsolin with actin. By application of the CONTIN program we could determine the length distribution of the filaments.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1991

F-actin bundling protein from Physarum polycephalum: purification and its capacity for co-bundling of actin filaments and microtubules.

An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.

Actins↗
PubMed1992

G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.

The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2-terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non-conserved NH2-terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F-actin binding proteins, converts the peptide's inhibitory phenotype into an F-actin stimulatory property. These results allow the proposal of a simple model for G- to F-actin modulation.

Actins↗
PubMed1981

Spectrin-4.1-actin complex of the human erythrocyte: molecular basis of its ability to bind cytochalasins with high-affinity and to accelerate actin polymerization in vitro.

The spectrin-4.1-actin complex isolated from the cytoskeleton of human erythrocyte was found to be similar to muscle F-actin in several aspects: Both the complex and F-actin nucleate cytochalasin-sensitive actin polymerization; both bind dihydrocytochalasin B with similar binding contrasts; both can be depolymerized by DNase I with loss of cytochalasin binding activity. From these results, we conclude that the actin in the complex is in an oligomeric form. However, the presence of spectrin and band 4.1 in the complex not only stabilized the actin in the complex as evidenced by its resistance to depolymerization in low-ionic-strength conditions and to DNase I as compared with F-actin, but also altered the characteristics of the binding site(s) for cytochalasins believed to be located at the "barbed" (polymerizing) end of the oligomeric actin.

Actins↗
PubMed1997

Cooperative rigor binding of myosin to actin is a function of F-actin structure.

Many aspects of cooperative behavior within pure F-actin filaments have now been described. We have used two myosin fragments, heavy meromyosin (HMM) and Subfragment 1 (S1), to look at the rigor binding to different forms of F-actin. With Ca2+ bound at the high-affinity metal binding site in actin, there is a very large cooperativity in the binding of HMM, but no cooperativity for S1. With Mg2+ bound at the high affinity site, or with conditions that stabilize the conformation of subdomain-2 of actin, there is no cooperativity seen with either HMM or S1. These results show that the two heads of HMM can induce structural changes in F-actin that are not observed with the single head of S1. They also support the notion that the binding of myosin to F-actin induces a conformational change in subdomain-2 of actin, and that under certain conditions this conformational change can be cooperatively propagated through an actin filament.

Actins↗
PubMed1999

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking.

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.

Actinin↗
PubMed1984

On the mechanism for inactivation of cytochalasin binding activity associated with F-actin and spectrin-band 4.1-actin complex by sulfhydryl reagents.

The sulfhydryl group modifying reagent, p-hydroxymercuribenzoate, inhibited the cytochalasin binding activity of the actin nuclei in the spectrin-band 4.1-actin complex from the erythrocyte membrane and of muscle F-actin. Kinetic studies indicated that while the cytochalasin binding activity was immediately inhibited, the actin remained filamentous and depolymerized slowly over a period of 1 to 2 h. Scatchard analysis of the binding data revealed that initially only the KD was affected. However, prolonged incubation led to depolymerization of the F-actin and dissociation of the spectrin-band 4.1-actin complex, resulting in loss of binding sites. It thus appears that certain actin sulfhydryl group(s) are important for cytochalasin binding. However, the most reactive sulfhydryl group (cys-374) on actin does not appear to be involved.

Actins↗
PubMed1988

Immunoelectron microscopic localization of actin in neurites of cultured embryonic chick dorsal root ganglia: actin is a component of granular, microtubule-associated crossbridges.

Axons have been shown to contain substantial quantities of actin distributed along their length. However, the general lack of information on the structure and organizational state of this protein in axons has made it difficult to assign it a functional role. In the present study, we used electron microscopic immunocytochemistry (immunogold labeling) on neurites from cultured embryonic chick dorsal root ganglia to: (1) determine the distribution of actin in neurites: (2) identify actin-containing structures; and (3) reveal whether or not actin is associated preferentially with microtubules. Results show that actin is present throughout neurites but is organized primarily into short filaments that are localized almost exclusively to granular, microtubule-associated crossbridges. We propose that these short actin filaments are part of the framework of a supramolecular 'carrier complex' for the slow component b polypeptides. In addition, actin-containing crossbridges are often closely associated with the surfaces of membrane-bound organelles. This suggests that actin and microtubule-associated crossbridges are involved somehow in fast axonal transport, although the nature of their participation in this process still needs to be resolved.

Actins↗
PubMed1994

The covalent maleimidobenzoyl-actin-myosin head complex. Cross-linking of the 50 kDa heavy chain region to actin subdomain-2.

We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl-G-actin to the central 50 kDa segment of the myosin-S-1 heavy chain by analyzing the structure of the maleimidobenzoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-1 and different maleimidobenzoyl-G-actin derivatives. The cross-linking is shown to occur only on the stretch of residues 48-67 in actin subdomain-2 with Lys-50, residing on the outer part of the DNase-I-binding loop, as the most likely site of cross-linking. Because the maleimidobenzoyl-F-actin-S-1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F-actin-S-1 complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain-2.

Actins↗
PubMed1996

Expression of ubiquitin, actin, and actin-like genes in African swine fever virus infected cells.

Northern blot hybridisation was used to study the accumulation of specific cellular mRNAs (ubiquitin and actin) in Vero cells infected with African swine fever virus (ASFV). ASFV modulates the cytoplasmic levels of ubiquitin and actin mRNAs throughout infection. Before viral DNA replication, degradation of ubiquitin mRNAs is dependent on de novo protein synthesis, since treatment with cycloheximide (CH) allowed the accumulation of ubiquitin mRNAs, while treatment with cytosine arabinoside (araC) induced a reduction in ubiquitin transcripts. Nevertheless, viral DNA replication is essential to the final increase observed in ubiquitin mRNA degradation. Furthermore, ubiquitin transcription seems to be tightly related to viral gene transcription, since before viral DNA replication ubiquitin and viral transcripts accumulate at opposite rates. Concerning actin transcription, the first step in actin mRNA degradation does not depend on de novo protein synthesis, since treatment with CH induced a reduction in actin mRNA. The second step in actin mRNA degradation, similarly to ubiquitin, depends on viral DNA replication. Finally, in the present study it has also been shown that ASFV codifies for actin-like genes. This is the first report of a virus encoding an actin-like gene.

Actins↗
PubMed1983

The effect of serum vitamin D-binding protein on polymerization and depolymerization of actin is similar to the effect of profilin on actin.

The mechanism of the interaction between two genetically determined serum vitamin D-binding protein forms and the muscle skeletal actin was investigated. Vitamin D-binding protein was isolated in a good yield from human serum, using immunoaffinity chromatography. 16 mg of pure vitamin D-binding protein were obtained from 100 ml of serum. The interaction between purified vitamin D-binding protein and skeletal muscle actin was studied by viscosity, delta A (232 nm) measurements and by electron microscopy. The effect of vitamin D-binding protein on actin polymerization is characterized by the decrease of the nucleation and elongation rates and by the decrease of the final concentration of polymerized actin in the steady state. The depolymerizing effect is not the result of direct action on vitamin D-binding protein on F-actin but rather of an increased concentration of the complex of the former protein with G-actin. The characteristics of the vitamin D-binding protein and profilin interactions with actin are similar. Both proteins seem to react only with G-actin.

Actins↗
PubMed1989

Isolation of camel brain actin--comparison of its biochemical properties with those of camel skeletal muscle, heart muscle and rabbit skeletal muscle actins.

1. Actins were purified from camel brain, skeletal muscle and heart muscle and their properties were compared. 2. Individual actins were homogeneous and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 3. Isoelectric focusing analysis of camel skeletal muscle and heart muscle actin showed a single polypeptide of the alpha-species, while camel brain actin showed two polypeptides of the beta- and gamma-species typical of non-muscle actin. 4. Actins from camel skeletal muscle and heart muscle showed a greater degree of similarity to each other and to rabbit skeletal muscle actin and showed some differences from camel brain actin, as confirmed by amino acid analysis and one-dimensional peptide mapping.

Actins↗