scholarly journals Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution.

1991 ◽  
Vol 115 (3) ◽  
pp. 677-687 ◽  
Author(s):  
M L Cano ◽  
D A Lauffenburger ◽  
S H Zigmond
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Average Length ◽  
Length Distribution ◽  
Fold Increase ◽  
Dissociation Rate ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Actin Depolymerization ◽  
Time Courses

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

scholarly journals Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin.

1994 ◽  
Vol 126 (5) ◽  
pp. 1231-1240 ◽  
Author(s):  
A R Menkel ◽  
M Kroemker ◽  
P Bubeck ◽  
M Ronsiek ◽  
G Nikolai ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Direct Interaction ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Permeabilized Cells ◽  
Cell Matrix ◽  
Actin Binding Domain ◽  
Major Structural Component

Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.

scholarly journals Emergence and maintenance of different sized actin filaments in a common pool of building blocks

2021 ◽  
Author(s):  
Deb Sankar Banerjee ◽  
Shiladitya Banerjee
Keyword(s):  
Growth Inhibition ◽  
Actin Filament ◽  
Actin Filaments ◽  
Length Distribution ◽  
Building Blocks ◽  
Nucleation And Growth ◽  
Actin Binding ◽  
Filament Length ◽  
Growth Promoters ◽  
Spontaneous Nucleation

Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the mean length of individual actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different size using the same set of molecular building blocks. Here we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth rate modulation by actin binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneities. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning F-actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.

Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3452-3452
Author(s):  
Elzbieta Pluskota ◽  
Dorota Szpak ◽  
Katarzyna Bialkowska ◽  
Kamila Bledzka ◽  
Edward F. Plow
Keyword(s):  
Actin Filaments ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Fold Increase ◽  
Actin Binding ◽  
Binding Studies ◽  
Vascular Integrity

Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain. Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments. Disclosures No relevant conflicts of interest to declare.

scholarly journals PakB binds to the SH3 domain of Dictyostelium Abp1 and regulates its effects on cell polarity and early development

2013 ◽  
Vol 24 (14) ◽  
pp. 2216-2227 ◽  
Author(s):  
Yidai Yang ◽  
Marc de la Roche ◽  
Scott W. Crawley ◽  
Zhihao Li ◽  
Emilia Furmaniak-Kazmierczak ◽  
...  
Keyword(s):  
Early Development ◽  
Actin Filaments ◽  
Cellular Localization ◽  
Critical Role ◽  
Leading Edge ◽  
Sh3 Domain ◽  
Cell Polarization ◽  
Actin Binding ◽  
Filamentous Actin ◽  
P21 Activated Kinase

Dictyostelium p21-activated kinase B (PakB) phosphorylates and activates class I myosins. PakB colocalizes with myosin I to actin-rich regions of the cell, including macropinocytic and phagocytic cups and the leading edge of migrating cells. Here we show that residues 1–180 mediate the cellular localization of PakB. Yeast two-hybrid and pull-down experiments identify two proline-rich motifs in PakB-1-180 that directly interact with the SH3 domain of Dictyostelium actin-binding protein 1 (dAbp1). dAbp1 colocalizes with PakB to actin-rich regions in the cell. The loss of dAbp1 does not affect the cellular distribution of PakB, whereas the loss of PakB causes dAbp1 to adopt a diffuse cytosolic distribution. Cosedimentation studies show that the N-terminal region of PakB (residues 1–70) binds directly to actin filaments, whereas dAbp1 exhibits only a low affinity for filamentous actin. PakB-1-180 significantly enhances the binding of dAbp1 to actin filaments. When overexpressed in PakB-null cells, dAbp1 completely blocks early development at the aggregation stage, prevents cell polarization, and significantly reduces chemotaxis rates. The inhibitory effects are abrogated by the introduction of a function-blocking mutation into the dAbp1 SH3 domain. We conclude that PakB plays a critical role in regulating the cellular functions of dAbp1, which are mediated largely by its SH3 domain.

scholarly journals Cofilin – a protein controlling dynamics of actin filaments

2017 ◽  
Vol 71 (1) ◽  
pp. 0-0 ◽  
Author(s):  
Zofia Ostrowska ◽  
Joanna Moraczewska
Keyword(s):  
Oxidative Stress ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Polymerization Process ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Filamentous Actin ◽  
Membrane Phospholipids ◽  
Cell Recovery ◽  
Number Of Ends

Cofilins are evolutionary conserved proteins present in all Eukaryotic cells. Their primary function is dynamic reorganization of actin cytoskeleton. Two cofilin isoforms are known: cofilin 1, present in all studied non-muscle cells and in embryonic muscle cells, and cofilin 2, which dominates in mature skeletal and cardiac muscles. Polypeptide chains of both isoforms fold into a structure homological to a conservative ADF (actin depolymerizing factor) domain, which is characteristic of actin depolymerizing factor. In cofilin molecule two actin-binding sites were found. One site binds monomeric and filamentous actin, the second one interacts only with the filament. Binding of cofilin to actin filament causes a change in the orientation of subunits, which results in filament severing. This increases number of ends which can either elongate or shorten the filament, depending on the conditions. Cofilin interactions with monomeric actin decreases availability of polymerization-competent actin subunits. Cofilin activity is controlled by phosphorylation, binding membrane phospholipids, local pH and oxidative stress. Under conditions of oxidative stress oxidation of cysteine residues leads to formation of dimers, which are able to cross-link actin filaments. Stable actin-cofilin rods save cellular ATP, which is not used during active polymerization process. This facilitates faster cell recovery from the stress. The final cellular reaction on the environmental stimuli is a resultant of cofilin activity and activities of other actin-binding proteins, which function either synergistically or antagonistically. Due to the central role in the regulation of actinfilaments dynamics, cofilin is involved in development of cancer, neurodegenerative diseases, congenital myopathies and cardiomyopathies.

scholarly journals Irreversible inhibition of rat glutathione S-transferase 1-1 by quinones and their glutathione conjugates. Structure-activity relationship and mechanism

1991 ◽  
Vol 276 (3) ◽  
pp. 661-666 ◽  
Author(s):  
B van Ommen ◽  
J H T M Ploemen ◽  
J J P Bogaards ◽  
T J Monks ◽  
S S Gau ◽  
...  
Keyword(s):  
Exponential Function ◽  
Active Site ◽  
Time Course ◽  
Fold Increase ◽  
Enzyme Inactivation ◽  
Cysteine Residues ◽  
Glutathione S Transferase ◽  
Irreversible Inhibition ◽  
Complete Inactivation ◽  
Time Courses

The irreversible inhibition of the rat glutathione S-transferase (GST) isoenzyme 1-1 by a series of halogenated 1,4-benzoquinones and their GSH conjugates was studied quantitatively by analysing the time course of enzyme inactivation. With increasing numbers of chlorine substituents, the rate of inhibition greatly increased. Incorporation of a GSH moiety in all cases increased the rate of inactivation compared with the non-substituted compound, and this was due to the increased affinity of the inhibitor for the active site. The ratio between the rates of inhibition for a given quinone with and without GSH substituent was largest for the three dichlorobenzoquinones, with the 2,6-isomer showing a 41-fold increase in rate of inhibition upon conjugation with GSH. The time courses of inhibition could be fitted either to a bi-exponential function (for the GSH conjugates and the higher chlorinated quinones) or to a mono-exponential function (all other quinones). It is concluded that the second component describes the affinity part of the reaction. GST 1-1 possesses two cysteine residues, with modification of one of these, probably located in the vicinity of the active site, having a major impact on the enzyme activity. Compounds with affinity towards the active site preferentially react with this residue. Non-specific quinones react equally with both cysteine residues. This was confirmed by the observation that complete inactivation of GST 1-1 by 2,5-dichlorobenzoquinone was achieved only after modification of two residues, whereas the corresponding GSH conjugate already completely inhibited the enzyme after modification of one residue.

scholarly journals Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ.

1991 ◽  
Vol 115 (6) ◽  
pp. 1629-1638 ◽  
Author(s):  
E L Bearer
Keyword(s):  
Actin Filament ◽  
Direct Observation ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Capping Protein ◽  
Calcium Dependent ◽  
Definition Of

Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.

scholarly journals Calponin induces actin polymerization at low ionic strength and inhibits depolymerization of actin filaments

1995 ◽  
Vol 312 (2) ◽  
pp. 587-592 ◽  
Author(s):  
T Kake ◽  
S Kimura ◽  
K Takahashi ◽  
K Maruyama
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Length Distribution ◽  
Inhibitory Effect ◽  
Molar Ratio ◽  
Equimolar Ratio ◽  
Rapid Elongation ◽  
Low Ionic Strength ◽  
Poisson Type

Calponin from chicken gizzard induced polymerization of actin in the presence of 10 mM KCl. Only 2 min after the addition of KCl in the presence of a 0.0625-0.25:1 molar ratio of calponin to actin, a Poisson-type length distribution (with an average length of approx. 0.7 micron) was observed with formed actin filaments. This result suggests that calponin-actin complexes served as nuclei for rapid elongation. Calponin caused a rapid polymerization of actin even in G-buffer (2 mM Tris/HCl, pH 8.0) which is usually used for depolymerization of actin filaments. Binding of calponin at a level of up to 1.25 mol per mol of actin was observed in the actin filaments formed in the presence of calponin at very low ionic strengths. When actin filaments were exposed to 3.3 mM KCl, by dilution with G-buffer, a rapid depolymerization occurred. Addition of calponin greatly retarded the depolymerization process and, in the presence of an equimolar ratio of calponin to actin, depolymerization hardly occurred. In the presence of calmodulin, this inhibitory effect on depolymerization was reversed by Ca2+, releasing calponin from actin filaments.

Sign in / Sign up

Export Citation Format

Share Document