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M F Carlier

Publications and source records attributed to M F Carlier.

At least 91 records · Page 5Linked to original sources
PubMed1988

Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4.

The binding of phosphate analogs to F-ADP-actin filaments and their effect on the dynamics of the polymer have been investigated. Orthovanadate binds to F-actin with the same affinity as phosphate and, at low saturation levels, stabilizes the filament structure in a Pi-like fashion; at higher concentration, it promotes destabilization of the filament. BeF-3 and A1F-4 bind to F-ADP-actin in competition with Pi, with a stoichiometry of 1 mol/mol of F-actin subunit and an affinity 3 orders of magnitude higher than Pi (KD = 2 and 25 microM for BeF-3 and A1F-4, respectively). BeF-3 and A1F-4 mimic Pi in stabilizing F-actin and slow down the rate of actin dissociation from filaments 150-fold. Only 1% of F-ADP-BeF3 subunits provide extensive stabilization of the filament. A quantitative analysis of the stabilization by BeF-3 is proposed. While Pi appears in rapid equilibrium with F-ADP-actin, BeF-3 binds to and dissociates from F-ADP-actin at very slow rates (k+ = 4 M-1 S-1; k = 8.10(-6) S-1). In addition, although functionally similar to the reconstituted F-ADP-Pi species, F-ADP-BeF3 has a different conformation as indicated by the 17% quenching of pyrenyl fluorescence linked to BeF-3 binding. We suggest that BeF-3 may be a good analog of the transition state F-ADP-P* and that Pi release following cleavage of ATP on F-actin might be rate-limited by the isomerization of F-ADP-P*.

Actins↗
PubMed1988

Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium.

In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (Pi) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of Pi and of its structural analogues AlF4- and BeF3- on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (KD = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF4- and BeF3- exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF3- to microtubules with a stoichiometry of 1 mol of BeF3- per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 microM. AlF4- and Pi compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, we propose that Pi and its structural analogues bind to the site of the gamma-phosphate of GTP in the E site and reconstitute a GDP-Pi-microtubule, from which tubulin subunits dissociate very slowly. We therefore understand that, following GTP cleavage on microtubules, Pi release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-Pi-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system [Carlier, M.-F., & Pantaloni, D. (1988) J. Biol. Chem. 263, 817-825] is underlined.

Aluminum↗
PubMed1988

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization.

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.

Actins↗
PubMed1988

Oscillations in microtubule polymerization: the rate of GTP regeneration on tubulin controls the period.

The essential reactions involved in the oscillatory kinetics of microtubule polymerization have been investigated. The rate of GDP dissociation from tubulin decreased cooperatively upon increasing tubulin concentration above 20 microM, consistent with the formation of GDP oligomers whose dissociation is rate limiting in nucleotide exchange. The apparent rate constant for nucleotide exchange at high tubulin concentration was 0.02 s-1 at 37 degrees C, which is the exact value needed in previous theoretical simulations to obtain oscillations with the real period of 70-80 s. A glass filter assay separating microtubules from oligomers and tubulin allowed nucleotide bound to non-microtubular tubulin during the oscillations to be monitored. In agreement with nucleotide exchange data, tubulin-bound GDP was found to oscillate in antiphase with microtubules. By varying the concentration of an enzymatic GTP-regenerating system, we could demonstrate that the period of the oscillations is directly controlled by the rate at which GTP is regenerated on tubulin, and oscillations can be observed under conditions where the dissociation of oligomers is no longer rate limiting. The possible physiological significance of GTP-regenerating systems in establishing synchrony in a microtubule population is evoked. The present data confirm and extend the model that we previously proposed to account for the oscillations.

Animals↗
PubMed1987

Actin polymerization and ATP hydrolysis.

F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP.Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP.Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

Actin Cytoskeleton↗
PubMed1987

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics.

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.

Animals↗
PubMed1987

Is microtubule assembly a biphasic process? A fluorimetric study using 4',6-diamidino-2-phenylindole as a probe.

The kinetics of microtubule assembly followed by turbidimetry usually describe an exponential process but hyperbolic or biphasic assembly curves have also been reported. A combination of turbidimetry, 4',6-diamidino-2-phenylindole (DAPI) fluorescence, electron microscopy, rapid sedimentation and wavelength dependence of scattered light have been used here to investigate these apparently biphasic kinetics of microtubule assembly. Experimental conditions have been developed under which the increase in DAPI fluorescence is a quantitative measurement of polymer formation which, in contrast to turbidity, does not depend on the size of the microtubules. Monophasic assembly curves were obtained when the increase in DAPI fluorescence was measured, whereas under the same conditions the turbidity change was a biphasic process. Biphasic turbidity curves are observed at low (less than 0.5 mM) magnesium ion concentration; the data are consistent with a rapid polymerization of tubulin into short polymers, followed by extensive length redistribution. The morphology of the early intermediates in microtubule assembly appears dependent on the concentration of Mg2+ ions.

Animals↗
PubMed1987

Measurement of Pi dissociation from actin filaments following ATP hydrolysis using a linked enzyme assay.

Using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase as a linked enzyme assay for determination of free inorganic phosphate, as described by Trentham et al. (1972, Biochem. J. 126, 635-644) we have been able to monitor the time course of Pi release from F-actin following ATP hydrolysis that accompanies ATP-actin polymerization. The rate constant for Pi dissociation from Mg-F-actin is 0.006 s-1 at 25 degrees C and pH 7.8, both in the presence of 1 mM Mg and 0.1 M KCl + 1 mM Mg. This result confirms the existence of ADP-Pi-F-actin as a major intermediate in the polymerization of ATP-actin (Carlier and Pantaloni, 1986, Biochemistry 25, 7789-7792). The method is potentially useful for other enzymes hydrolyzing triphosphate nucleotides, provided that the rate of Pi release is appreciably lower than 0.1 s-1.

Actins↗
PubMed1987

The mechanisms of ATP hydrolysis accompanying the polymerization of Mg-actin and Ca-actin.

The hydrolysis of ATP that accompanies actin polymerization occurs on the F-actin subsequent to the elongation step. For Mg-actin, the rate of ATP hydrolysis is similar to the rate of elongation at low concentrations of G-actin but increases more slowly as the G-actin concentration is increased. This behavior can be quantitatively modeled by assuming that ATP hydrolysis occurs predominantly, but not exclusively, on a single subunit of Mg-F-actin at the interface between an ATP-subunit cap and an ADP-subunit core. The rates of elongation of Ca-actin and Mg-actin are similar but the rate of ATP hydrolysis on Ca-F-actin is appreciably slower than the rate of elongation at all concentrations of Ca-G-actin. The data for Ca-actin can be modeled by assuming that ATP hydrolysis occurs essentially randomly on Ca-F-actin within a large ATP cap which can be as long as 2,000 subunits in a 10,000-subunit long filament.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Synchronous oscillations in microtubule polymerization.

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent greater than 50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.

Guanosine Diphosphate↗
PubMed1986

Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.

The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.

Actins↗
PubMed1986

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis.

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.

Actins↗
PubMed1986

Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.

The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders of magnitude higher than previously reported (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). As proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886), the Mg-actin complex undergoes a slow isomerization (Kis = 0.03-0.1) to a higher affinity state (K'D = 4-40 nM). The replacement of Ca2+ by Mg2+ at this high-affinity site causes a slow 10% increase in fluorescence that is 90% complete in about 200 s at saturating concentrations of Mg2+. Independently, Ca2+, Mg2+, and K+ bind to low-affinity sites (KD values of 0.15 mM for Ca2+ and Mg2+ and 10 mM for K+) which causes a rapid 6-8% increase in fluorescence (complete in less than 5 s). We propose that the activation step that converts Ca-G-actin to a polymerizable species upon addition of Mg2+ is the binding of Mg2+ to the low-affinity sites and not the replacement of Ca2+ by Mg2+ at the high-affinity site.

Actins↗
PubMed1986

Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

Actin Cytoskeleton↗
PubMed1985

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer.

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.

Actins↗
PubMed1985

The interaction between ATP-actin and ADP-actin. A tentative model for actin polymerization.

The involvement of interactions between ATP-actin and ADP-actin in actin polymerization has been studied. It has been found that ATP-actin and ADP-actin can copolymerize and that the rate of nucleation is enhanced when both ATP-actin and ADP-actin are present in solution. The fact that the heterologous interaction between ATP-actin (T) and ADP-actin (D) is stronger than either of the homologous reactions, T-T and D-D, agrees with the kinetic data in the accompanying paper (Carlier, M.-F., Pantaloni, D., and Korn, E.D. (1985) J. Biol. Chem. 260, 6565-6571) which show that filament ends having the DT conformation are more stable than those having the TT conformation. These data are incorporated into a model for actin polymerization in ATP in which the kinetic parameters for polymerization depend on the nature of the nucleotide (ADP or ATP) bound to the three terminal subunits of the actin filament.

4-Chloro-7-nitrobenzofurazan↗