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Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity

Wang, Ze; Jiang, He; Yang, Zhi-Qiong; Chacko, Samuel
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 28/10/1997 EN
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It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDΔ24–53 lacking the residues 24–53 to myosin compared with the level of binding of CaDΔ54–85 missing the adjacent residues 54–85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24–53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaDΔ54–85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1–597, which lacks the major actin-binding site(s)...

Effects of actin filament cross-linking and filament length on actin- myosin interaction

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/11/1985 EN
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We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls)...

Actin filament organization in the fish keratocyte lamellipodium

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/06/1995 EN
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From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation- release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid- region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton...

A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics

Steinmetz, Michel O.; Goldie, Kenneth N.; Aebi, Ueli
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 11/08/1997 EN
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510.83227%
The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation– condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The “ragged” and “branched” filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state...

The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/11/1991 EN
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511.36844%
Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F- actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament...

Actin filament remodeling by actin depolymerization factor/cofilin

Pfaendtner, Jim; De La Cruz, Enrique M.; Voth, Gregory A.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
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511.88082%
We investigate, using molecular dynamics, how the severing protein, actin depolymerization factor (ADF)/cofilin, modulates the structure, conformational dynamics, and mechanical properties of actin filaments. The actin and cofilactin filament bending stiffness and corresponding persistence lengths obtained from all-atom simulations are comparable to values obtained from analysis of thermal fluctuations in filament shape. Filament flexibility is strongly affected by the nucleotide-linked conformation of the actin subdomain 2 DNase-I binding loop and the filament radial mass density distribution. ADF/cofilin binding between subdomains 1 and 3 of a filament subunit triggers reorganization of subdomain 2 of the neighboring subunit such that the DNase-I binding loop (DB-loop) moves radially away from the filament. Repositioning of the neighboring subunit DB-loop significantly weakens subunit interactions along the long-pitch helix and lowers the filament bending rigidity. Lateral filament contacts between the hydrophobic loop and neighboring short-pitch helix monomers in native filaments are also compromised with cofilin binding. These works provide a molecular interpretation of biochemical solution studies documenting the disruption of filament subunit interactions and also reveal the molecular basis of actin filament allostery and its linkage to ADF/cofilin binding.

Review of the mechanism of processive actin filament elongation by formins

Paul, Aditya S.; Pollard, Thomas D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/2009 EN
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We review recent structural and biophysical studies of the mechanism of action of formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addition to variable regulatory domains, formins typically have formin homology 1 (FH1) and formin homology 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains concentrate multiple profilin-actin complexes near the end of the filament. Actin transfers very rapidly from the FH1 domains onto the barbed end of the filament, allowing elongation at rates that exceed elongation by the addition of free actin monomers diffusing in solution. Binding of actin to the end of the filament provides the energy for the highly processive movement of the FH2 as a filament adds thousands of actin subunits. These biophysical insights provide the context to understand how formins contribute to actin assembly in cells.

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains – Implications for Actin Filament Nucleation

Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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510.83227%
Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem W domains that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is crosslinked to Cys-374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of Tβ4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with inter-subunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL crosslinked to actin Cys-374, and show it to be nearly identical to non-crosslinked W-actin structures. This result validates the use of crosslinking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity...

MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation

Chang, Eugene; Heo, Kyung-Sun; Woo, Chang-Hoon; Lee, Hakjoo; Le, Nhat-Tu; Thomas, Tamlyn N.; Fujiwara, Keigi; Abe, Jun-ichi
Fonte: American Society of Hematology Publicador: American Society of Hematology
Tipo: Artigo de Revista Científica
Publicado em 24/02/2011 EN
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Actin filament remodeling regulates several endothelial cell (EC) processes such as contraction, migration, adhesion, and shape determination. Mitogen-activated protein kinase (MAPK)–activated protein kinase 2 (MK2)–mediated phosphorylation of heat-shock protein 27 kDa (HSP27) promotes actin filament remodeling, but little is known about the regulation of this event in ECs. We found that tumor necrosis factor-α (TNF-α) SUMOylated MK2 at lysine (K)-339 affected EC actin filament organization and migration. Loss of the MK2 SUMOylation site (MK2-K339R) increased MK2 kinase activity and prolonged HSP27 phosphorylation, enhancing its effects on actin filament-dependent events. Both TNF-α–mediated EC elongation and steady laminar shear stress–mediated EC alignment were increased by MK2-K339R. Moreover, kinase-dead dominant-negative MK2 (DN-MK2) inhibited these effects. Cell migration is a dynamic process regulated by actin filament remodeling. Both wild-type MK2 (WT-MK2) and DN-MK2 significantly enhanced TNF-mediated inhibition of EC migration, and MK2-K339R further augmented this effect. Interestingly, the p160-Rho–associated coiled-coil kinase (ROCK) inhibitor Y-27632 reversed this effect by MK2-K339R, which strongly suggests that both excessive and insufficient levels of actin filament remodeling can block EC migration. Our study shows that MK2 SUMOylation is a new mechanism for regulating actin filament dynamics in ECs.

Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast*

Skau, Colleen T.; Courson, David S.; Bestul, Andrew J.; Winkelman, Jonathan D.; Rock, Ronald S.; Sirotkin, Vladimir; Kovar, David R.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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512.83703%
Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy...

Actin Filament Attachments for Sustained Motility In Vitro Are Maintained by Filament Bundling

Hu, Xiaohua; Kuhn, Jeffrey R.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/02/2012 EN
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510.61766%
We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg2+, Lys-Lys2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg2+ buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3...

Rotational Model for Actin Filament Alignment by Myosin

Miller, Callie J; Ermentrout, G Bard; Davidson, Lance A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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510.61766%
Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allow us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors...

Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes

Qian, Xiaojing; Mruk, Dolores D.; Wong, Elissa W. P.; Lie, Pearl P. Y.; Cheng, C. Yan
Fonte: Endocrine Society Publicador: Endocrine Society
Tipo: Artigo de Revista Científica
EN
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In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network)...

Actin filament dynamics in the actomyosin-VI complex is regulated allosterically by calcium-calmodulin light chain

Prochniewicz, Ewa; Pierre, Anaële; McCullough, Brannon R.; Chin, Harvey F.; Cao, Wenxiang; Saunders, Lauren P.; Thomas, David D.; De La Cruz, Enrique M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin light chains. We have used transient phosphorescence anisotropy (TPA) to monitor the microsecond rotational dynamics of erythrosin iodoacetamide-labeled actin with strongly-bound myosin VI (MVI) and to evaluate the effect of MVI-bound calmodulin (CaM) light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca2+ - and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of TPA decays. MVI with bound apo-CaM or Ca2+ - CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g. muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e. increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca2+ - dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin-binding site(s)...

Molecular Origins of Cofilin-linked Changes in Actin Filament Mechanics

Fan, Jun; Saunders, Marissa G.; Haddadian, Esmael J.; Freed, Karl F.; De La Cruz, Enrique M.; Voth, Gregory A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The actin regulatory protein cofilin plays a central role in actin assembly dynamics by severing filaments and increasing the concentration of ends from which subunits add and dissociate. Cofilin binding modifies the average structure and mechanical properties of actin filaments, thereby promoting fragmentation of partially decorated filaments at boundaries of bare and cofilin-decorated segments. Despite extensive evidence for cofilin-dependent changes in filament structure and mechanics, it is unclear how the two processes are linked at the molecular level. Here, we use molecular dynamics simulations and coarse-grained analyses to evaluate the molecular origins of the changes in filament compliance due to cofilin binding. Filament subunits with bound cofilin are less flat and maintain a significantly more open nucleotide cleft than bare filament subunits. Decorated filament segments are less twisted, thinner (considering only actin), and less connected than their bare counterparts, which lowers the filament bending persistence length and torsional stiffness. Using coarse-graining as an analysis method reveals that cofilin binding increases the average distance between the adjacent long-axis filament subunit, thereby weakening their interaction. In contrast...

Competitive displacement of cofilin can promote actin filament severing

Elam, W. Austin; Kang, Hyeran; De La Cruz, Enrique M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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514.09625%
Cofilin is an essential actin filament severing protein that functions in the dynamic remodeling of the actin cytoskeleton. Filament severing activity is most efficient at sub-stoichiometric cofilin binding densities (i.e. <1 cofilin per actin filament subunit), and peaks when the number density of boundaries (i.e. junctions) between bare and cofilin-decorated segments is maximal. A model in which local topological and mechanical discontinuities lead to preferential fragmentation at boundaries accounts for available experimental data, including direct visualization of cofilin and actin during real-time severing events. The boundary-severing model predicts that ligands (e.g. other actin-binding proteins) that compete with cofilin for actin filament binding and modulate cofilin occupancy on filaments will alter the bare-decorated segment boundary density, and thus, the filament severing activity of cofilin. Here, we directly test this model prediction by evaluating the effects of phalloidin and myosin, two ligands that compete with cofilin for filament binding, on the actin filament binding and severing activities of cofilin. Our experiments demonstrate that competitive displacement of cofilin lowers cofilin occupancy and promotes severing when initial cofilin occupancy is high (i.e. >50%). Even in the presence of competitive ligands...

Simultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells

McGrath, J. L.; Tardy, Y.; Dewey, C. F. Jr; Meister, J. J.; Hartwig, J. H.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica Formato: 428604 bytes; application/pdf
EN_US
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The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied previously to the study of actin dynamics in living cells. Traditionally, separate experiments estimate the mobility of actin monomer or the lifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fluorescence in PAF and FRAP experiments depends simultaneously on the diffusion coefficient of actin monomer, D, the fraction of actin in filaments, FF, and the lifetime of actin filaments, t (Tardy et al., 1995, Biophys. J. 69:1674–1682). Here we report the application of this mathematical model to the interpretation of PAF and FRAP experiments in subconfluent bovine aortic endothelial cells (BAECs). The following parameters apply for actin in the bulk cytoskeleton of subconfluent BAECs. PAF: D 5 3.1 6 0.4 3 1028 cm2/s, FF 5 0.36 6 0.04, t 5 7.5 6 2.0 min. FRAP: D 5 5.8 6 1.2 3 1028 cm2/s, FF 5 0.5 6 0.04, t 5 4.8 6 0.97 min. Differences in the parameters are attributed to differences in the actin derivatives employed in the two studies and not to inherent differences in the PAF and FRAP techniques. Control experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer...

A Mechanistic Model of the Actin Cycle

Bindschadler, M.; Osborn, E. A.; Dewey, C. F. Jr; McGrath, J. L.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica Formato: 364500 bytes; application/pdf
EN_US
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592.93117%
We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for enhancing Pi release from the ADP-Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of available actin. This limitation arises because as the cycle becomes more dynamic...

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1

Wong, Wilson; Skau, Colleen T.; Marapana, Danushka S.; Hanssen, Eric; Taylor, Nicole L.; Riglar, David T.; Zuccala, Elizabeth S.; Angrisano, Fiona; Lewis, Heather; Catimel, Bruno; Clarke, Oliver B.; Kershaw, Nadia J.; Perugini, Matthew A.; Kovar, David R.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
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511.98516%
Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actin's polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly...

Microtubules at wound sites on Nitella internodal cells passively coalign with actin bundles when exposed to hydrodynamic forces generated by cytoplasmic streaming

Foissner, I; Wasteneys, Geoffrey
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
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587.97438%
The organization of cortical microtubules at wound sites in Nitella pseudoflabellata (A. Br. and Nordst.) em. R.D.W. and N. flexilis (L.) Ag. internodal cells was examined in relation to the regeneration of actin filament bundles in order to identify the