scholarly journals Effects of lithium ions on actin polymerization in the presence of magnesium ions

1991 ◽  
Vol 274 (2) ◽  
pp. 421-425 ◽  
Author(s):  
R Colombo ◽  
A Milzani ◽  
P Contini ◽  
I Dalle Donne
Keyword(s):  
Actin Polymerization ◽  
Time Course ◽  
Monomer Concentration ◽  
Biological Action ◽  
Dissociation Rate ◽  
Actin Assembly ◽  
Salt Composition ◽  
Lithium Ions ◽  
Absolute Value

In spite of the abundant literature, questions on the biological action of Li+ are far from being answered. In the present paper we demonstrate that modification of the salt composition of the medium for actin polymerization, by gradually replacing K+ with Li+, leads to a dose-related change in the time course of actin assembly. The presence of Li+ influences actin polymerization in vitro by enhancing nucleation and decreasing critical monomer concentration at steady state. Furthermore, Li+ stabilizes actin polymers mainly by lowering the absolute value of the dissociation rate constant (K-) and shifting (towards lower values of actin monomer concentrations) the range of G-actin concentrations in which filament-subunit flux can occur. The influence of Li+ on actin and tubulin polymerization in vitro suggests that cytoskeletal structures could be some of the cytoplasmic targets of this ion.

scholarly journals WASP-interacting Protein Is Important for Actin Filament Elongation and Prompt Pseudopod Formation in Response to a Dynamic Chemoattractant Gradient

2006 ◽  
Vol 17 (10) ◽  
pp. 4564-4575 ◽  
Author(s):  
Scott A. Myers ◽  
Laura R. Leeper ◽  
Chang Y. Chung
Keyword(s):  
Actin Polymerization ◽  
Time Course ◽  
Leading Edge ◽  
Delayed Response ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Interacting Protein

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.

scholarly journals Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

2010 ◽  
Vol 190 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Huaqing Cai ◽  
Satarupa Das ◽  
Yoichiro Kamimura ◽  
Yu Long ◽  
Carole A. Parent ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Time Course ◽  
Specific Inhibitor ◽  
Ras Proteins ◽  
Extracellular Signaling ◽  
Upstream Regulator ◽  
Pkb Phosphorylation ◽  
G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

scholarly journals Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.

1981 ◽  
Vol 88 (3) ◽  
pp. 654-659 ◽  
Author(s):  
T D Pollard ◽  
M S Mooseker
Keyword(s):  
Electron Microscopy ◽  
Growth Rates ◽  
Rate Constants ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Monomer Concentration ◽  
Dissociation Rate ◽  
Filament Bundles ◽  
Dissociation Rate Constants ◽  
Pointed End

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.

scholarly journals Negative Regulation of Yeast Eps15-like Arp2/3 Complex Activator, Pan1p, by the Hip1R-related Protein, Sla2p, during Endocytosis

2007 ◽  
Vol 18 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Jiro Toshima ◽  
Junko Y. Toshima ◽  
Mara C. Duncan ◽  
M. Jamie T.V. Cope ◽  
Yidi Sun ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Actin Assembly ◽  
Related Protein ◽  
Loss Of Function ◽  
Actin Organization ◽  
Coiled Coil Region ◽  
Kinase Phosphorylation

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites ∼25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification–tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2ΔCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals Mechanism of the interaction of human platelet profilin with actin.

1991 ◽  
Vol 113 (5) ◽  
pp. 1081-1089 ◽  
Author(s):  
P J Goldschmidt-Clermont ◽  
L M Machesky ◽  
S K Doberstein ◽  
T D Pollard
Keyword(s):  
Actin Polymerization ◽  
Human Platelet ◽  
Muscle Actin ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Binding Assays ◽  
Spontaneous Polymerization ◽  
A Cell ◽  
In Vitro Effects

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.

scholarly journals Activation of the Cdc42 Effector N-Wasp by the Shigella flexneri Icsa Protein Promotes Actin Nucleation by Arp2/3 Complex and Bacterial Actin-Based Motility

1999 ◽  
Vol 146 (6) ◽  
pp. 1319-1332 ◽  
Author(s):  
Coumaran Egile ◽  
Thomas P. Loisel ◽  
Valérie Laurent ◽  
Rong Li ◽  
Dominique Pantaloni ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Shigella Flexneri ◽  
Terminal Segment ◽  
Actin Assembly ◽  
Bacterial Surface ◽  
Terminal Domain ◽  
Infected Cells ◽  
Vitro Analysis

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA–N-WASP–Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH2-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.

scholarly journals Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

1997 ◽  
Vol 139 (5) ◽  
pp. 1243-1253 ◽  
Author(s):  
R.J. Eddy ◽  
J. Han ◽  
J.S. Condeelis
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Movement ◽  
Capping Protein ◽  
End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

scholarly journals Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ying Xie ◽  
Jialin Sun ◽  
Xiao Han ◽  
Alma Turšić-Wunder ◽  
Joel D. W. Toh ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Growth ◽  
Actin Polymerization ◽  
Stress Conditions ◽  
Scaffolding Protein ◽  
Actin Assembly ◽  
Intrinsically Disordered ◽  
Actin Cables

Abstract A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation.

scholarly journals In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

1997 ◽  
Vol 138 (1) ◽  
pp. 95-103 ◽  
Author(s):  
Terry Lechler ◽  
Rong Li
Keyword(s):  
Actin Polymerization ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Direct Role ◽  
Permeabilized Cell ◽  
Permeabilized Cells ◽  
Actin Organization

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

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