Biomedical subjects
Publications and source records attributed to M F Carlier.
A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.
The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.
Under controlled levels of fragmentation by sonication in the presence of ATP, actin filaments reached different length distributions corresponding to different steady states of polymerization. When the sonication was interrupted, a spontaneous decrease in the number of filaments and corresponding increase in length was observed. The kinetics of the process of length redistribution subsequent to fragmentation was studied for the simpler case of F-actin filaments at equilibrium in the presence of ADP. Analysis of the data shows that a diffusion-like random walk mechanism of length redistribution quantitatively accounts for the observations much better than the previously proposed end-to-end reannealing of filaments. The involvement of this process in the kinetics of actin polymerization and in establishing the length distribution at steady state is discussed.
F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.
The interaction between tubulin and microtubule-associated proteins (MAPs) in solutions of cycled microtubule protein has been studied by using radioactively labeled MAPs. Kinetic data of MAP association to microtubules in the polymerization process indicate that an oligomer P of tubulin and MAPs is the polymerizing species. Analysis of MAP binding to microtubules formed from solutions in which the ratio MAPs/tubulin was varied shows evidence for a polymorphism of tubulin-MAP oligomers. When the ratio MAPs/tubulin is decreased by addition of dimeric tubulin to 3 times cycled microtubule protein, an oligomer P' less rich in MAPs than P and unable to incorporate in microtubules is formed. The data further show that while tau, MAP1, and MAP2 can bind to oligomer P, only MAP1 and MAP2 can bind to oligomer P'. Therefore, the interactions of tau factor and of MAP1 and MAP2 with tubulin follow different patterns.
Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with 125I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution of both tau and spectrin-like proteins in eukaryotic cells is in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.
This paper reports an experimental study of the interference of GTP hydrolysis in the mechanism of microtubule assembly, following the model and theory previously published [Hill, T. L. & Carlier, M.-F. (1983) Proc. Natl. Acad. Sci. USA 80, 7234-7238]. Results from dilution experiments show that microtubules depolymerize faster below the critical concentration than expected with a reversible polymerization model. The experimental plot of flux versus tubulin concentration exhibits a slope discontinuity at the critical concentration, in agreement with the theory. Theoretical points calculated by the Monte Carlo method can be fitted qualitatively to the data. A consequence of this peculiar dynamic behavior of microtubules is that the ratio of tubulin dissociation and association rate constants measured, respectively, below and above the critical concentration does not yield the true value of the critical concentration. It is emphasized that the presence of GTP at microtubule ends is necessary to maintain the stability of the polymer.
Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.
The assembly of microtubule protein has been investigated under conditions where the microtubule seeds concentration was varied. The plot of the apparent first order elongation rate constant with microtubule number concentration strongly deviated from the linearity predicted by Oosawa's condensation model and reached a limit at a high concentration of seeds. The increase in seeds concentration was quantitatively assessed from the histograms of microtubule length distribution and from the parallel increases in the rate of depolymerization at 4 degrees C and steady state rate of GTP hydrolysis at microtubule ends. The limit elongation rate constant was independent of elongating sites and tubulin concentrations and was attributed to the true first order rate constant of a conformation change of tubulin preceding its assembly in microtubules. It is hypothesized that polymerizable tubulin would represent a small fraction only of the tubulin molecules in equilibrium with unpolymerizable species. Microtubule assembly would follow the shift of this equilibrium which is fast as compared to the rate of polymerization at low concentration of elongating sites but becomes rate limiting at higher concentrations.
A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state subunit flux at one end of the polymer. The GTP/GDP features just mentioned result in a nonlinear plot of the flux versus tubulin concentration. Microtubules are predicted to exhibit a different kinetic behavior below and above the critical concentration, which can be considered as a transition between two regimes.
GTP hydrolysis associated with polymerization is a distinctive feature of microtubule assembly. This reaction may be fundamentally linked to the dynamic properties of microtubules in vivo. Kinetic analysis of the connection between microtubule assembly and associated GTP hydrolysis indicates that these two events are kinetically uncoupled, GTP hydrolysis occurring after tubulin incorporation in the microtubule. As a consequence, the combination of the diffusional incorporation of GTP in microtubules at steady-state and of subsequent GTP hydrolysis results in the formation of a steady-state GTP cap at microtubule ends. The interplay between GTP and GDP at microtubule ends is examined. Inhibition by GDP of steady-state GTP hydrolysis at microtubule ends and of microtubule elongation is understood within a tight reversible binding of GDP at microtubule ends generating 'inactive' elongation sites. Nucleotides are freely exchangeable at microtubule ends. This result indicates that the nature of the nucleotide present at microtubule ends must be considered in a model for microtubule assembly. These data are pooled in order to define the general features of a model describing microtubule assembly and treadmilling in terms somewhat different from previously proposed models.
A quantitative analysis of the interplay between guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) in microtubule assembly and accompanying GTP hydrolysis has been performed when tubulin was polymerized in the presence of microtubule-associated proteins (MAPs) which display an interfering GTPase activity. The use of adenylyl beta-imidodiphosphate, which specifically inhibits the MAPs GTPase activity, and of vinblastine (or podophyllotoxin), which specifically inhibits GTP hydrolysis due to tubulin, made possible a study of the extensive GTP hydrolysis associated to microtubule assembly. The results indicate that GDP binds to microtubule ends with an affinity comparable to GTP, thus strongly inhibiting both the elongation process and the steady-state GTP hydrolysis at microtubule ends. GDP shifts the equilibrium between tubulin and microtubules toward disassembly. The MAPs which are released from the microtubules during the GDP-driven depolymerization cluster on the remaining microtubules. The resulting increased stability of microtubules is quantitatively consistent with the decrease in the critical concentration of the polymerizing species GTP-tubulin.
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Several types of experiments were designed to elucidate the mechanism of initiation of tubulin assembly in the presence of microtubule-associated proteins (MAPs): (1) The evolution of the proportion of the double ring species with temperature was examined in the ultracentrifuge, under nonpolymerizing conditions (guanosine diphosphate = 0.5 mM). A net dissociation of rings into dimers occurred when temperature increased. The transition took place above 20 degrees C. (2) The kinetic parameters of the exchange at equilibrium between free tubulin dimers and tubulin in rings were studied at different temperatures, using the technique of isotopic exchange at equilibrium with radioactively labeled tubulin. The values found for the half-time of the exchange reaction varied between or approximately 100 min at 0 degrees C and 10 min at 2 degrees C. (3) The relative participation of unlabeled rings and labeled dimers to microtubules in the time course of assembly was studied at 22 degrees C. The time dependence of microtubule specific radioactivity showed that tubulin incorporated in the initial stages of assembly came predominantly from rings (85-90%). This result indicates that oligomers directly issued from rings are the first intermediates in the assembly process and suggests that at the beginning of polymerization incorporation of isomers or fragments of rings proceeds at a faster rate than their dissociation into dimers. (4) Polymerization experiments in the presence of MAPs with increasing concentrations of tubulin dimers indicated that MAPs are in rapid equilibrium with the microtubules and are distributed along the wall in a more or less loose lattice depending on the relative concentrations of tubulin and MAPs in the solution.
The correlation between the time courses of pure tubulin assembly and accompanying guanosine 5'-triphosphate (GTP) hydrolysis has been studied at different tubulin concentrations in the range where the rate of assembly varies with a strong cooperativity. One GTP molecule was found hydrolyzed per molecule of tubulin dimer incorporated in the microtubule. This hydrolysis was not strictly coupled to polymerization and occurred in a subsequent step. Consequently, in the first stages of assembly, tubulin-GtP complex is the transient major constituent of microtubules. Kinetic data of GTP hydrolysis have been treated within a model of two consecutive first-order reactions: [tubulin-GTP]free k1 leads to [tubulin-GTP]MT k2 leads to [tubulin-GDP]MT + [Pi] GTP hydrolysis proceeded at an intrinsic rate k2 = 0.25 min-1 independent of tubulin concentration. Simultaneous measurements of polymerization, GTPase activity, and incorporation of [3H]GTP followed by unlabeled GTP chase indicated that before its hydrolysis GTP bound to microtubules was exchangeable while after hydrolysis GDP remained locked in the E site. The possibility is discussed that after assembly tubulin undergoes a conformation change which could trigger GTP hydrolysis and sequestration of GDP.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.