Distinct Structural Changes Detected by X-Ray Fiber Diffraction in Stabilization of F-Actin by Lowering pH and Increasing Ionic Strength

Oda, Tatsuya; Makino, Kouji; Yamashita, Ichiro; Namba, Keiichi; Maéda, Yuichiro

doi:10.1016/s0006-3495(01)76063-8

Cited by 43 publications

(29 citation statements)

References 48 publications

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“…increased the yield of sedimented actin (Fig. 1), consistent with a lower Cc and higher rates of polymerization in more acidic conditions as described for RS-actin (47)(48)(49)(50)(51). In presence of JAS, in contrast to phalloidin, Pf-actin could be sedimented in a concentration-dependent manner (Fig.…”

Section: Resultssupporting

confidence: 83%

“…The idea that apicomplexan F-actin becomes more unstable after ATP hydrolysis than other eukaryotic actins is therefore intriguing. However, by stabilizing the ATP or ADP-P i state, we did not find evidence for this, while lowering the pH, which is also known to stabilize conventional actin filaments, resulted in at least some stabilization (47)(48)(49)(50)(51). In summary our findings show differences in the structure of Pf-actin from RS-actin, which we speculate could cause a reduced stability of Pf-actin filaments.…”

Section: Discussioncontrasting

confidence: 56%

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Malaria Parasite Actin Polymerization and Filament Structure

Schmitz¹,

Schaap²,

Kleinjung³

et al. 2010

Journal of Biological Chemistry

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A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and "zebra-striped" appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter-and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility. Plasmodium falciparum (Pf)5 is a protozoan causing human malaria and belongs to the apicomplexan group of intracellular parasites. Apicomplexan motility and host cell invasion requires the parasite's own acto-myosin motor system (1-3). The organization of the acto-myosin machinery under the cell surface of Apicomplexa has been described by a linear model (4) (for review, see Refs. 3, 5-7). In this model, actin filaments are linked via receptor-ligand interactions to a substrate or the host cell surface, and the action of myosin motors moves the parasite forward. Interestingly, the treatment of the closely related apicomplexan parasite Toxoplasma gondii (Tg) with the actin filament-stabilizing toxin jasplakinolide (JAS) increased both actin polymerization and the speed of gliding (8 -10). Based on these observations, a novel form of acto-myosin regulation has been proposed in which the availability of actin filaments regulates apicomplexan motility (8).Many recent studies have since focused on actin filament dynamics in these parasites. In contrast to other cell types it appears that in Apicomplexa, actin exists primarily in a monomeric form (8,10,11). This is consistent with the fact that the apicomplexan actin, in vitro and in vivo, only forms very short and unstable filaments (8,9,12,13). Unlike ...

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Section: Resultssupporting

confidence: 83%

Section: Discussioncontrasting

confidence: 56%

Malaria Parasite Actin Polymerization and Filament Structure

Schmitz¹,

Schaap²,

Kleinjung³

et al. 2010

Journal of Biological Chemistry

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“…The intercept value of K polym extrapolated to 1M cation is related to the intrinsic binding free energy of an actin subunit and associated cation with a filament end (27,28). These values are not identical for all cations evaluated, suggesting that filaments assembled with different cationic species have variable thermodynamic stabilities and salt-dependent conformational distribution(s) (6,7,20,29,30).…”

Section: Resultsmentioning

confidence: 99%

“…S1 and S2). This observation indicates that the actin filament thermodynamic stability depends on solution cations (18)(19)(20), since the C c of ADP-actin reflects the free energy associated with filament subunit incorporation (ΔG°0 polym ) according to ΔG°0 polym ¼ −RT ln K polym , where K polym is a macroscopic overall equilibrium constant for incorporation of monomers into filaments. We emphasize that K polym is an "observed" binding constant under given experimental conditions (e.g., salt concentration), defined only by the reaction between monomers and filament ends, and does not explicitly account for contributions from linked equilibria such as ion binding.…”

Section: Resultsmentioning

confidence: 99%

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Kang

Bradley

McCullough

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

148

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The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.ion-linkage | structural bioinformatics | persistence length | polyelectrolyte T he polymerization of the protein actin into double-stranded helical filaments powers many eukaryotic cell movements and provides cells with mechanical strength and integrity (1-4). Filament formation is favored when the total actin concentration exceeds the critical concentration (C c ) for assembly-defined as the monomer concentration at steady state for ATP-actin, or the dissociation constant for the reversible-equilibrium binding reaction of monomer binding to ADP-actin filament ends. Accordingly, the C c of ADP-actin is linked to the filament subunit interaction free energy such that lower C c values reflect greater thermodynamic stability (5).The effects of solution ionic conditions on the assembly and stability of actin filaments have been investigated for several decades (6-12). The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6, 7, 13-15), a behavior shared among characterized actins and their bacterial homologs (16). However, it is not firmly established if these salt effects on actin filament assembly and mechanics originate from nonspecific ion effects (e.g., electrostatic screening, counterion condensation, etc.) and/or specific ion binding interactions, potentially at discrete sites. Identification of saturable cation binding sites with different affinities favors specific and discrete binding sites on monomers (8-10, 17), but the location of these sites and their contributions to filament assembly and stiffness are unknown.Here we identify distinct cation-binding sites at subunit interfaces that regulate actin filament assembly and rigidity. Sit...

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“…It could rely on either the channel's activity (ie, Na + influx) or its colocalization with F-actin. 9 Both of these factors raise the F-actin:G-actin ratio in the cortical cytoskeleton 38,39 and thus increase cortical stiffness. Owing to the interdependency of the 2 mechanisms, it is conceivable that a combination of them might determine cortical stiffness.…”

Section: Discussionmentioning

confidence: 99%

Epithelial Sodium Channel Stiffens the Vascular Endothelium In Vitro and in Liddle Mice

et al. 2013

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Abstract-Liddle syndrome, an inherited form of hypertension, is caused by gain-of-function mutations in the epithelial Na + channel (ENaC), the principal mediator of Na + reabsorption in the kidney. Accordingly, the disease pathology was ascribed to a primary renal mechanism. Whether this is the sole responsible mechanism, however, remains uncertain as dysregulation of ENaC in other tissues may also be involved. Previous work indicates that ENaC in the vascular endothelium is crucial for the regulation of cellular mechanics and thus vascular function. The hormone aldosterone has been shown to concomitantly increase ENaC surface expression and stiffness of the cell cortex in vascular endothelial cells. The latter entails a reduced release of the vasodilator nitric oxide, which eventually leads to an increase in vascular tone and blood pressure. Using atomic force microscopy, we have found a direct correlation between ENaC surface expression and the formation of cortical stiffness in endothelial cells. Stable knockdown of αENaC in endothelial cells evoked a reduced channel surface density and a lower cortical stiffness compared with the mock control. In turn, an increased αENaC expression induced an elevated cortical stiffness. More importantly, using ex vivo preparations from a mouse model for Liddle syndrome, we show that this disorder evokes enhanced ENaC expression and increased cortical stiffness in vascular endothelial cells in situ. We conclude that ENaC in the vascular endothelium determines cellular

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Distinct Structural Changes Detected by X-Ray Fiber Diffraction in Stabilization of F-Actin by Lowering pH and Increasing Ionic Strength

Cited by 43 publications

References 48 publications

Malaria Parasite Actin Polymerization and Filament Structure

Malaria Parasite Actin Polymerization and Filament Structure

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Epithelial Sodium Channel Stiffens the Vascular Endothelium In Vitro and in Liddle Mice

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