scholarly journals Formin's nucleation activity influences actin filament length

2021 ◽  
Author(s):  
Mark E Zweifel ◽  
Laura A Sherer ◽  
Biswaprakash Mahanta ◽  
Naomi Courtemanche
Keyword(s):  
Actin Polymerization ◽  
The Other ◽  
Polymerization Reaction ◽  
Actin Assembly ◽  
Filament Length ◽  
Reaction Proceeds ◽  
Cytoskeletal Dynamics ◽  
Nucleation Activity ◽  
Kinetics Of

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Although variations in elongation rates produce modest changes in the equilibrium lengths of formin-bound filaments, nucleation constitutes the primary mode of monomer consumption over the course of assembly. Sustained elongation of small numbers of formin-bound filaments therefore requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin's nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.

scholarly journals Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

2018 ◽  
Author(s):  
Sonal ◽  
Kristina A. Ganzinger ◽  
Sven K. Vogel ◽  
Jonas Mücksch ◽  
Philipp Blumhardt ◽  
...  
Keyword(s):  
Steady State ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Fine Tuning ◽  
Cellular Functions ◽  
Actin Cortex ◽  
Actin Turnover ◽  
Cytoskeletal Dynamics

ABSTRACTDynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

Métabolisme de l'acide indolyl-3-acétique dans les cultures de cellules d'Acer pseudoplatanus cultivés in vitro

1977 ◽  
Vol 55 (19) ◽  
pp. 2530-2534 ◽  
Author(s):  
F. Maillard ◽  
J.-P. Zrÿd
Keyword(s):  
Cell Wall ◽  
Acetic Acid ◽  
Culture Medium ◽  
Degradation Products ◽  
Cell Suspensions ◽  
The Other ◽  
Acer Pseudoplatanus ◽  
Kinetics Of

Incubation of cell suspensions of sycamore (Acer pseudoplatanus) with β-indoyl-3-acetic acid (IAA) first led to the formation of IAA-glycosides, then to that of IAA-aspartate. Great differences are observed between the kinetics of IAA transformed by two distinct strains: one, auxin dependent (S), the other, auxin independent (MB). Other degradation products are only found in the culture medium. The localization of IAA-degrading systems in the cell wall is postulated. The auxin requirement of the S strain is discussed.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

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 Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

1990 ◽  
Vol 110 (3) ◽  
pp. 681-692 ◽  
Author(s):  
A Shariff ◽  
E J Luna
Keyword(s):  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Heat Denaturation ◽  
Actin Assembly ◽  
Binding Studies ◽  
Actin Nucleation ◽  
Equilibrium Binding ◽  
Nucleation Activity

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

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 Homer3 regulates the establishment of neutrophil polarity

2015 ◽  
Vol 26 (9) ◽  
pp. 1629-1639 ◽  
Author(s):  
Julie Wu ◽  
Anne Pipathsouk ◽  
A. Keizer-Gunnink ◽  
F. Fusetti ◽  
W. Alkema ◽  
...  
Keyword(s):  
Cell Migration ◽  
Rna Interference ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Rac Gtpase ◽  
Gpcr Activation ◽  
Phosphoinositide 3 Kinase ◽  
Directional Cell Migration ◽  
Kinetics Of

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference–mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP3) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP3 and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.

A comparative study on the conditions of growth and sporulation of three strains of Clostridium petfringens type A

1996 ◽  
Vol 42 (3) ◽  
pp. 298-304 ◽  
Author(s):  
Mathilde Decaudin ◽  
Jean-Luc Tholozan
Keyword(s):  
Clostridium Perfringens ◽  
Slow Growth ◽  
Type A ◽  
High Volume ◽  
The Other ◽  
Additional Information ◽  
Mutant Strains ◽  
Dependent Strain ◽  
Kinetics Of

Different conditions of growth and sporulation of a strain of Clostridium perfringens type A (NCTC 8798) and two derived mutant strains, the lysozyme-germination dependent strain 8-6 and the revertant strain R3, have been determined. No sporulation was detected for the three strains in the Duncan and Strong (DS) medium; 100% sporulation was routinely obtained for the two mutant strains in the defined (D) medium. Factors promoting in vitro sporulation of C. perfringens type A were assayed: the volume of the culture, the type of preculture, and the addition of lysozyme in precultures. The paper also provides additional information on growth and sporulation of the mutant strains 8-6 and R3. Glucose concentrations up to 11 mM produced high percentages of sporulation. However, strain R3 still sporulated at 20% with 56 mM of glucose. A high volume of D medium led to slow growth kinetics and favoured sporulation. Faster kinetics of growth and the best percentage of sporulation were obtained with a young inoculum of the two mutant strains. On the other hand, the type of medium in the preculture (fluid thioglycollate (FTG) or basal carbonate yeast trypticase (BCYT)) did not influence the percentage of sporulation. However, while strain R3 was not affected by the addition of lysozyme in D medium, kinetics of growth were strongly influenced by this addition in strain 8-6, and the percentage of sporulation increased with a preculture in FTG medium and decreased when BCYT medium was used.Key words: Clostridium perfringens, medium, growth, sporulation.

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.

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