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At least 91 records · Page 5Linked to original sources
PubMed1999

Viscoelastic properties of f-actin, microtubules, f-actin/alpha-actinin, and f-actin/hexokinase determined in microliter volumes with a novel nondestructive method.

A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1-100 nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10 microliters with temporal resolution >1 Hz. For 2-100 microM f-actin gels a final viscosity of 1.3-9.4 mPa s and a final elastic modulus of 2.229-2.254 GPa (corresponding to 1493-1501 m/s sound velocity) have been determined. For 10- to 100-microM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5-124 mPa s and a final elastic modulus of 2.288-2. 547 GPa (approximately 1513-1596 m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3 m/s (approximately 1.69 kPa) and decreased up to -7 m/s (approximately 49 kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to -12 m/s approximately 2.3-0.1 MPa, for 10- to 100-microM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2 mM ATP (when compared to 0.2 mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17 microM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26 GPa) was less than for f-actin polymerized in presence of 0.2 mM ATP (2.28 GPa).

Acoustics↗
PubMed1983

Structure of the actin molecule determined from electron micrographs of crystalline actin sheets with a tentative alignment of the molecule in the actin filament.

Electron microscopy and image processing of negatively stained crystalline sheets induced from Acanthamoeba actin have been used to yield a three-dimensional reconstruction of the actin molecule, including data to a maximum resolution of 15 A. This model shows actin to be an asymmetric, wedge-shaped molecule. A three-dimensional reconstruction of an averaged, polar actin filament from negatively stained polylysine-induced actin filament paracrystals has also been computed. We show two possible ways in which the wedge-shaped actin molecule from the sheets can be placed into such a filament reconstruction. In both, the major intermolecular contacts are formed on complementary surfaces of the actin subunit and follow the left-handed genetic helix of the filament, a feature also found in the filament reconstruction.

Actins↗
PubMed1986

Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.

The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.

Actins↗
PubMed1987

Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces.

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.

Actins↗
PubMed1996

Torsional rigidity of single actin filaments and actin-actin bond breaking force under torsion measured directly by in vitro micromanipulation.

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 +/- 1.2) x 10(-26) Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Actins↗
PubMed2004

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments.

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.

Actin Cytoskeleton↗
PubMed1993

A comparison of the atomic model of F-actin with cryo-electron micrographs of actin and decorated actin.

We compare the atomic model calculated from the crystal structure and the X-ray fiber diagram of orientated F-actin1) with the 3-D reconstructions produced from cryo-electron microscopy of actin2). Out to 30A resolution the two structures are essentially identical. Furthermore, by combining the atomic model with the reconstruction of S1-decorated actin filaments2) one can establish the nature of the actin binding site for myosin in the rigor complex. Each myosin head binds to two actin molecules on two distinct sites. Some of the actin residues involved in each of these binding sites can be identified. Furthermore, the atomic model of actin may be combined with the reconstruction of the S1 decorated thin filament to establish the tropomyosin binding site in the rigor complex. This result is compared with the model of tropomyosin-actin derived from an analysis of the X-ray fibre diagram of a reconstituted thin filament and are shown to be very similar.

Actins↗
PubMed2001

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization.

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1976

The polymerization of actin. II. How nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes.

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage, in the accumulation of actin in the periacrosomal region is the actin sequested in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrsomoal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state.

Acrosome↗
PubMed1982

A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction.

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.

Actins↗
PubMed1979

Comparative studies on actins from various sources. Fragments of actins from Ascaris muscle cleaved at cysteinyl residues in comparison with those of other actins.

Pure actins were obtained from various animal muscles: Vertebrata (skeletal, smooth, and cardiac muscles), Prochordata (smooth muscle), Nematoda (obliquely striated muscle), and Mollusca (striated, smooth and obliquely striated muscles). These actins were all identical in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All actins treated with 2-nitro-5-thiocyanobenzoic acid yielded four major (about 33,000, 26,000, 24,000, and less than 10,000 daltons) and three minor (22,000, 17,000, and 10,000 daltons) bands in addition to intact actin on gel electrophoresis. The results suggest that all actins from various types of muscle have cysteinyl residues at similar positions on the primary structure.

Actins↗
PubMed1994

The actin side-binding domain of gelsolin also caps actin filaments. Implications for actin filament severing.

Gelsolin is an actin filament-severing and -capping protein which is inhibited by polyphosphoinositides (PPI). Severing requires gelsolin binding to the side of the filaments through a site in segments 2 and 3 (S2-3) to position another site in segment 1 (S1) to sever filaments. In this paper, we report that S2-3, like S1, caps actin filaments. Since neither S1 and S2-3 caps as well as gelsolin, and neither severs actin filament, S2-3 may actively contribute to severing by capping filaments cooperatively with S1. We used deletional mutagenesis to locate the S2-3 sequence required for actin filament side binding, capping, and PPI binding and found that these sites are located close to the NH2 terminus of S2 (residues 161-172). S3, a segment which has no known function up to now and does not by itself bind actin, contributes to stable capping and may contain an additional PPI-binding site.

Actins↗
PubMed2006

Plasmodium motility: actin not actin' like actin.

Apicomplexan parasites such as Plasmodium and Toxoplasma display actomyosin-dependent motility in the absence of readily detectable actin polymers. Three recent studies indicate that parasite actin polymers, either harvested from parasites or formed from purified recombinant proteins, are exceptionally short ( approximately 100 nm). We propose that parasite motility could be directed by the transient formation of short actin filament scaffolds. Parasite actin polymers that support transmembrane receptors are pulled, by myosin interaction, backwards along the parasite periphery, resulting in forward movement.

Actin Cytoskeleton↗
PubMed1979

The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation.

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins--bovine aorta actin and chicken gizzard actin--differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably cloer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.

Actins↗
PubMed1992

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes.

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

Actin Cytoskeleton↗
PubMed1996

Dephosphorylation of a 34kd triton-insoluble F-actin pool protein is associated with phorbol ester-induced actin polymerization in human polymorphonuclear leukocytes.

Activation of human polymorphonuclear leukocytes (PMNs) by chemotactic peptide (FMLP) or phorbol ester (PMA) results in actin reorganization and PMN motility. Evidence suggests that PMA and FMLP activate PMN actin reorganization by different mechanisms. For example, the protein phosphatase inhibitor, okadaic acid (OA), inhibits PMA- but not FMLP-induced actin rearrangement, suggesting protein dephosphorylation is key to PMA but not FMLP actin changes and that PMN actin reorganization occurs by multiple mechanisms. Further support for multiple actin polymerization mechanisms is the recent description of distinct F-actin pools coexisting with G-actin in PMNs, Triton insoluble F-actin (TIF) and Triton soluble F-actin (TSF). These studies examine quantitative actin pool-specific actin polymerization in PMA- and FMLP-activated PMNs using quantitative SDS-PAGE and the phosphorylation of proteins in each actin pool using 32P orthophosphate (32P) labeling. The results show: (1) OA alone has no effect on actin pool content; (2) PMA induces actin growth only in the TIF pool similar to results with FMLP, and (3) OA pretreatment has no effect on FMLP actin polymerization, but inhibits PMA-induced changes. 32P results show that in basal PMNs, multiple phosphoproteins are found in the TIF including a protein of MW 34kd (pp34), the TSF pool contains a pp34 and a pp69 and the G-actin pool a pp34. PMA induces dephosphorylation of pp34 in the TIF (0.59 +/- 0.14 x basal, n = 3). OA prior to PMA prevents TIF pp34 dephosphorylation and actin shifts between the TIF, TSF, and G pools. OA alone results in phosphorylation of pp34 in all actin pools but no shift in actin content. The results show that (1) phosphoproteins exist in all three actin pools of PMNs-TIF-actin, TSF-actin, and G-actin; (2) both PMA and FMLP cause quantitatively identical actin polymerization in the TIF; and (3) in contrast, PMA but not FMLP TIF growth requires dephosphorylation of a pp34. This as yet unidentified phosphoprotein appears crucial to PMA- but not FMLP-induced actin polymerization.

Actins↗
PubMed1999

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.

Actins↗
PubMed1995

The state of actin assembly regulates actin and vinculin expression by a feedback loop.

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

3T3 Cells↗