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At least 541 records · Page 30Linked to original sources
PubMed1996

Actin' like actin?

The most biologically significant property of actin is its ability to self-associate and form two-stranded polymeric microfilaments. In living cells, these micro filaments form the actin cytoskeleton, essential for maintenance of the shape, passive mechanical properties and active motility of eukaryotic cells. Recently discovered actin-related proteins (ARPs) appear to share a common ancestor with conventional actin. At present, six classes of ARPs have been discovered, three of which have representatives in diverse species across eukaryotic phyla and may share functional characteristics with conventional actin. The three most ubiquitous ARPs are predicted to share a common core structure with actin and contain all the residues required for ATP binding. Surface residues involved in protein protein interactions, however, have diverged. Models of these proteins based on the atomic structure of actin provide some clues about how ARPs interact with each other, with conventional actin and with conventional actin-binding proteins.

Journal Article↗
PubMed1999

Effect of capping protein, CapZ, on the length of actin filaments and mechanical properties of actin filament networks.

We report on how physiological concentrations of capping protein shorten actin filaments and on the remarkably fluid nature of solutions of such short filaments even at the high concentrations that exist in cells. We measured the lengths of actin filaments formed by spontaneous polymerization of highly purified actin monomers by fluorescence microscopy after labeling with rhodamine-phalloidin. The length distributions are exponential with a mean of about 7 microm (2600 subunits). As observed previously with less quantitative assays, copolymerization with the actin capping protein, CapZ, reduces the length of the filaments. At cellular concentrations of capping protein, one filament forms for each molecule of capping protein and the population of filaments is uniformly short. Using CapZ to vary the length of actin filaments, we measured how their mechanical properties depend on length. The stiffness (elastic modulus) of actin filament networks depends steeply on the length, with long filaments contributing far out of proportion to their numbers to the stiffness. Even at physiological concentrations (300 microM), networks of filaments limited to lengths observed in cells with a 1 to 500 molar ratio of CapZ are more fluid and much less elastic than lower concentrations of longer actin filaments. Thus the high concentration of short actin filaments in cells must be crosslinked to produce the observed stiffness of the cortex.

Actins↗
PubMed1995

Effects of microcystin-LR on actin and the actin-associated proteins alpha-actinin and talin in hepatocytes.

Microcystin-LR (MCLR) is a commonly encountered blue-green algal hepatotoxin and a known inhibitor of cellular protein phosphatase types 1 and 2A. The toxin causes alterations in, and redistribution of, intermediate filaments, microtubules, and actin microfilaments (MFs) in affected cells. In this study, the effect of MCLR on the sequence of alterations in MFs and actin-associated proteins (AAPs) of isolated hepatocytes was examined in an effort to determine whether morphologic changes induced in MFs by microcystins are a result of prior dislocation of AAPs. We studied the effects of MCLR exposure on alpha-actinin and talin, two AAPs that play a role in the orientation of the MFs. Primary hepatocytes were incubated with 10 microns MCLR for 0-64 min. The distribution of actin, alpha-actinin, and talin were examined using fluorescence microscopy. MCLR induced similar changes in the distribution of actin and the AAPs. Actin filament redistribution was first observed after 12 min of MCLR exposure, and was characterized by detachment of MFs from the cell periphery, followed by condensation at distinct focal points and progressive collapse into the interior of affected cells. Changes in alpha-actinin and talin distribution were first observed after 20 min of toxin exposure. The AAPs appeared to detach from focal contacts on the cytoplasmic surface of the plasma membrane, condense into cytoplasmic aggregates, and ultimately collapse into a juxtanuclear bundle. The results of this study indicate that, in hepatocytes exposed to MCLR, the collapse of actin MFs occurs prior to the dislocation of alpha-actinin and talin. Changes in these actin associated proteins are not likely to account for the initial changes in actin MFs.

Actin Cytoskeleton↗
PubMed1993

Orientation of actin monomers in moving actin filaments.

We have visualized, under an optical microscope, the orientations of actin monomers in individual actin filaments undergoing Brownian motion in solution, actively sliding past myosin molecules, or immobile on a surface. For the visualization, two strategies have been adopted. One is to exploit the fluorescence polarization of a fluorescent probe firmly attached to actin. Using the probe phalloidin-tetramethylrhodamine, the fluorescence was clearly polarized along the filament axis, showing alignment of the probe molecules along the filament axis. Within our temporal resolution of 33 ms and spatial resolution of better than 1 micron (average over approximately 10(2) actin monomers), the orientation of the probe (hence of actin monomers) did not change upon interaction of the filament with heavy meromyosin; myosin-induced reorientation was estimated to be a few degrees at most. This first method, while highly sensitive to small reorientations of monomers off or toward the filament axis, does not report on reorientations around the axis. To detect rotation around the filament axis, we adopted the second strategy in which we attached small plastic beads to the actin filaments. Axial turns would be immediately apparent from the movement of the beads. Preliminary observations indicate that actin filaments can slide over a heavy meromyosin-coated surface without axial rotations. Since rotations have been implicated in different experiments, we are currently investigating the source of the apparent discrepancy. The attached bead also serves as a handle through which we can apply force, via optical tweezers, on the filament. By letting the sliding actin filament pull the bead against the optical force, we were able to estimate the sliding force and its fluctuation.

Actin Cytoskeleton↗
PubMed2001

Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

Actin Depolymerizing Factors↗
PubMed2004

bPAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin.

p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.

Actin Cytoskeleton↗
PubMed1998

Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation.

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

Actin Depolymerizing Factors↗
PubMed1988

Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein.

Oocytes, notably those of amphibia, accumulate large pools of nonfilamentous ("soluble") actin, both in the cytoplasm and in the nucleoplasm, which coexist with extensive actin filament arrays in the cytoplasmic cortex. Because the regulation of oogenically accumulated actin is important in various processes of oogenesis, egg formation, fertilization and early embryogenesis, we have purified and characterized the major actin-binding proteins present in oocytes of Xenopus laevis. Here we report that the major actin-binding component in the ooplasm, but not in the nucleus, is a polypeptide of Mr approximately 93,000 on SDS-PAGE that reduces actin polymerization in vitro in a Ca2+-dependent manner but promotes nucleation events, and also reduces the viscosity of actin polymers, indicative of severing activity. We have raised antibodies against the purified oocyte protein and show that it is different from villin, is also prominent in unfertilized eggs and early embryos and is very similar to a corresponding protein present in various tissues and in cultured cells, and appears to be spread over the cytoplasm. Using these antibodies we have isolated a cDNA clone from a lambda gt11 expression library of ovarian poly(A)+-RNA. Determination of the amino acid sequence derived from the nucleotide sequence, together with the directly determined sequence of the amino terminus of the native protein, has shown that this clone encodes the carboxy-terminal half of gelsolin. We conclude that gelsolin is the major actin-modulating protein in oogenesis and early embryogenesis of amphibia, and probably also of other species, that probably also plays an important role in the various Ca2+-dependent gelation and contractility processes characteristic of these development stages.

Actin Cytoskeleton↗