Biomedical subjects

M F Carlier

Publications and source records attributed to M F Carlier.

At least 37 records · Page 2Linked to original sources

PubMed1999

Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motility.

Shigella flexneri, an invasive bacterial pathogen, promotes formation of two cytoskeletal structures: the entry focus that mediates bacterial uptake into epithelial cells and the actin-comet tail that enables the bacteria to spread intracellularly. During the entry step, secretion of bacterial invasins causes a massive burst of subcortical actin polymerization leading the formation of localised membrane projections. Fusion of these membrane ruffles leads to bacterial internalization. Inside the cytoplasm, polar expression of the IcsA protein on the bacterial surface allows polymerization of actin filaments and their organization into an actin-comet tail leading to bacterial spread. The Rho family of small GTPases plays an essential role in the organization and regulation of cellular cytoskeletal structures (i.e. filopodia, lamellipodia, adherence plaques and intercellular junctions). We show here that induction of Shigella entry foci is controlled by the Cdc42, Rac and Rho GTPases, but not by RhoG. In contrast, actin-driven intracellular motility of Shigella does not require Rho GTPases. Therefore, Shigella appears to manipulate the epithelial cell cytoskeleton both by Rho GTPase-dependent and -independent processes.

Actins↗

PubMed1998

Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover.

The mechanism of control of the steady state of actin assembly by actin depolymerizing factor (ADF)/cofilin and profilin has been investigated. Using Tbeta4 as an indicator of the concentration of ATP-G-actin, we show that ADF increases the concentration of ATP-G-actin at steady state. The measured higher concentration of ATP-G-actin is quantitatively consistent with the increase in treadmilling, caused by the large increase in the rate of depolymerization from the pointed ends induced by ADF (Carlier, M.-F. , Laurent, V., Santolini, J., Didry, D., Melki, R., Xia, G.-X., Hong, Y., Chua, N.-H., and Pantaloni, D. (1997) J. Cell Biol. 136, 1307-1322). Experiments demonstrate that profilin synergizes with ADF to further enhance the turnover of actin filaments up to a value 125-fold higher than in pure F-actin solutions. Profilin and ADF act at the two ends of filaments in a complementary fashion to increase the processivity of treadmilling. Using the capping protein CapZ, we show that ADF increases the number of filaments at steady state by 1. 3-fold, which cannot account for the 25-fold increase in turnover rate. Computer modeling of the combined actions of ADF and profilin on the dynamics of actin filaments using experimentally determined rate constants generates a distribution of the different actin species at steady state, which is in quantitative agreement with the data.

Actin Depolymerizing Factors↗

PubMed1998

Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation.

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

Actin Depolymerizing Factors↗

PubMed1998

Bistability in the isocitrate dehydrogenase reaction: an experimentally based theoretical study.

The enzyme isocitrate dehydrogenase (IDH, EC 1.1.1.42) can exhibit activation by one of its products, NADPH. This activation is competitively inhibited by the substrate NADP+, whereas NADPH competes with NADP+ for the catalytic site. Experimental observations briefly presented here have shown that if IDH is coupled to another enzyme, diaphorase (EC 1.8.1.4), which transforms NADPH into NADP+, the system can attain either one of two stable states, corresponding to a low and a high NADPH concentration. The evolution toward either one of these stable states depends on the time of addition of diaphorase to the medium containing IDH and its substrate NADP+. We present a theoretical and numerical analysis of a model for the IDH-diaphorase bienzymatic system, based on the regulatory properties of IDH. The results confirm the occurrence of bistability for parameter values derived from the experiments. Depending on the total concentration of NADP+ plus NADPH and the concentration of IDH, the system can either admit a single steady state or display bistability. We obtain an expression for the critical time t*, before which diaphorase addition leads to the lower steady state and after which addition of the enzyme leads to the upper steady state of NADPH. The analysis is extended to the case where the second substrate of IDH, isocitrate, is consumed in the course of the reaction without being regenerated. Bistability occurs only as a transient phenomenon in these conditions.

Dihydrolipoamide Dehydrogenase↗

PubMed1998

Control of actin dynamics.

Actin-based motility processes are tightly linked to the rapid turnover of actin filaments. Factors that control the steady state of actin assembly, such as capping proteins and actin-depolymerizing factor/cofilin, directly affect motility. Actin-depolymerizing factor increases the treadmilling of actin filaments in vitro and in vivo. Cellular factors that are involved in linking initiation of barbed end assembly to cell signaling are being identified using Listeria monocytogenes and Saccharomyces cerevisiae as model systems.

Actin Cytoskeleton↗

PubMed1998

[Cytoskeletal actin and its associated proteins. Some examples in Protista].

Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin IB at the leading edge of E. histolytica. ABP-120 organizes F-actin in a network and myosin IB participates in the pseudopod formation. Similar approaches using T. vaginalis resulted in the discovery of an actin-binding protein that participate in the F-actin reorganization during adhesion of parasites to target cells. This protein is homologous to alpha-actinin from other eukaryotic cells. Finally, by using cell biology approaches, F-actin was observed in the cytoplasm as well as in the nucleus of Dinoflagellates. The recent developments in the molecular genetics of protozoa will provide new insights to understand the roles of actin-binding proteins during cytoskeleton activities.

Actin Depolymerizing Factors↗

PubMed1997

Kinetics of myosin subfragment-1-induced condensation of G-actin into oligomers, precursors in the assembly of F-actin-S1. Role of the tightly bound metal ion and ATP hydrolysis.

In a low ionic strength buffer and in the absence of free ATP, the interaction of G-actin (G) with myosin subfragment-1 (S1) leads to the formation of arrowhead-decorated F-actin-S1 filaments, through a series of elementary steps. The initial formation of GS and G2S complexes is followed by their condensation into short oligomers. The kinetics of formation of G-actin-S1 oligomers have been monitored in a stopped-flow apparatus using a combination of light scattering and fluorescence of NBD-labeled actin. Oligomers appear more stable and are formed at a faster rate from MgATP-G-actin than from CaATP-G-actin. The actin-bound ATP is hydrolyzed when oligomers are formed from MgATP-G-actin, not when they are formed from CaATP-G-actin. The formation of oligomers is energetically favored in the presence of cytochalasin D. All data are consistent with the view that the actin-actin interactions which take place upon condensation of GS and G2S into oligomers are very similar to lateral actin-actin interactions along the short pitch helix of actin filaments, which are involved in actin nucleation. These interactions trigger ATP hydrolysis on actin.

Actins↗

PubMed1997

Mechanism of myosin subfragment-1-induced assembly of CaG-actin and MgG-actin into F-actin-S1-decorated filaments.

The kinetics and mechanism of myosin subfragment-1-induced polymerization of G-actin into F-actin-S1-decorated filaments have been investigated in low ionic strength buffer and in the absence of free ATP. The mechanism of assembly of F-actin-S1 differs from salt-induced assembly of F-actin. Initial condensation of G-actin and S1 into oligomers in reversible equilibrium is a prerequisite step in the formation of F-actin-S1 . Oligomers have a relatively low stability (10(6) M-1) and contain S1 in a molar ratio to actin close to 0.5. Increased binding of S1 up to a 1:1 molar ratio to actin is associated with further irreversible condensation of oligomers into large F-actin-S1 structures of very high stability. In contrast to salt-induced assembly of F-actin, no monomer-polymer equilibrium, characterized by a critical concentration, can be defined for F-actin-S1 assembly, and end-to-end annealing of oligomers is predominant over growth from nuclei in the kinetics. Simultaneous recordings of the changes in light scattering, pyrenyl- and NBD-actin fluorescence, ATP hydrolysis, and release of Pi during the polymerization process have been analyzed to propose a minimum kinetic scheme for assembly, within which several elementary steps, following oligomer formation, are required for assembly of F-actin-S1. ATP hydrolysis occurs before polymerization of MgATP-G-actin but not of CaATP-G-actin. The release of inorganic phosphate occurs on F-actin-S1 at the same rate as on F-actin.

Actin Cytoskeleton↗

PubMed1997

Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules.

Stathmin is an important regulatory protein thought to control the dynamics of microtubules through the cell cycle in a phosphorylation-dependent manner. Here we show that stathmin interacts with two molecules of dimeric alphabeta-tubulin to form a tight ternary T2S complex, sedimenting at 7.7 S. This complex appears in slow association-dissociation equilibrium in the analytical ultracentrifuge. The T2S complex is formed under a variety of ionic conditions, either from GTP- or GDP-tubulin or from the tubulin-colchicine complex. The S16/25/38/63E mutated stathmin in contrast is in rapid equilibrium with tubulin in the T2S complex. The T2S complex cannot polymerize in microtubules nor in ring oligomers. Stathmin acts as a pure tubulin-sequestering protein via formation of the T2S complex. It does not act directly on microtubule ends to promote catastrophe nor enhance microtubule dynamics.

Animals↗

PubMed1997

Control of actin dynamics in cell motility.

Actin polymerization plays a major role in cell movement. The controls of actin sequestration/desequestration and of filament turnover are two important features of cell motility. Actin binding proteins use properties derived from the steady-state monomer-polymer cycle of actin in the presence of ATP, to control the F-actin/G-actin ratio and the turnover rate of actin filaments. Capping proteins and profilin regulate the size of the pools of F-actin and unassembled actin by affecting the steady-state concentration of ATP-G-actin. At steady state, the treadmilling cycle of actin filaments is fed by their disassembly from the pointed ends. It is regulated in two different ways by capping proteins and ADF, as follows. Capping proteins, in decreasing the number of growing barbed ends, increase their individual rate of growth and create a "funneled" treadmilling process. ADF/cofilin, in increasing the rate of pointed-end disassembly, increases the rate of filament turnover, hence the rate of barbed-end growth. In conclusion, capping proteins and ADF cooperate to increase the rate of actin assembly up to values that support the rates of actin-based motility processes.

Actin Depolymerizing Factors↗

PubMed1997

Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

Actin Cytoskeleton↗

PubMed1997

Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.

Animals↗

PubMed1996

Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay.

ATP and GTP are hydrolyzed during self-assembly of actin and tubulin, respectively. It is known that nucleotide is hydrolyzed on the polymer in two consecutive steps, chemical cleavage of the gamma-phosphate followed by the slower release of Pi. This last step has been shown to play a crucial role in the dynamics of actin filaments and microtubules. Thus far, evidence for a transient GDP-Pi state in microtubule assembly has been obtained using a glass fiber filter assay that had a poor time resolution [Melki, R., Carlier, M.-F., & Pantaloni, D. (1990) Biochemistry 29, 8921-8932]. We have used a new Pi assay [Webb, M. R. (1992) Proc. natl. Acad. Sci. U.S.A. 89, 4884-4887], in which the purine phosphorylase catalyzes the phosphorolysis of 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) into mercaptopurine and ribose phosphate, which is accompanied by an increase in absorbance. This enzyme-linked assay has been used to follow the release of Pi during polymerization of Mg-actin. A value of 350 s was found for the half-time for Pi release on F-actin, in good agreement with previous determinations. The release of Pi following GTP hydrolysis in microtubule assembly was followed using a stopped-flow apparatus. Rapid microtubule assembly was achieved using taxol. The use of a stopped-flow apparatus permitted the continuous recording, with a dead time of 0.8 ms, of both time courses of microtubule assembly and Pi release with greatly improved time resolution. The release of Pi developed with a short lag (35 and 2 s for G-actin and tubulin, respectively) following assembly and appeared 50-fold faster on microtubules than on actin filaments.

Actins↗

PubMed1996

Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly.

Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg2+ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a G-actin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.

Actins↗

PubMed1996

Kinetics of association of myosin subfragment-1 to unlabeled and pyrenyl-labeled actin.

The kinetics of reaction of myosin subfragment-1 (S1) with F-actin have been monitored by the changes in light scattering and in pyrenyl-actin fluorescence at 20 degrees C, pH 7.5, and physiological ionic strength. The association rate constant of S1 to F-actin decreases about 10-fold as the molar ratio of bound S1 increases from 0 to 1. This decrease in k+ is most likely due to the steric hindrance of available binding sites by initially bound S1. The apparent rate constant for association of S1 to bare filaments is 9 microM-1 s-1, a value 1 order of magnitude higher than the one previously estimated from experiments in which S1 was in excess over F-actin. The anticooperative binding kinetics of S1 to F-actin are consistent with the negative cooperativity displayed in the equilibrium binding curves of S1 to pyrenyl-F-actin. Fluorescence titration curves of partially labeled pyrenyl-F-actin by S1 are sigmoidal, consistent with a 4-fold higher affinity of S1 for unlabeled than for labeled action. This conclusion is strengthened by kinetic data of S1 binding to partially labeled F-actin, which exhibit a biphasic behavior due to the slower dissociation of S1 from unlabeled than from labeled actin.

Actins↗

PubMed1996

Tbeta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentration.

Thymosin beta 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins.

Actin Cytoskeleton↗

PubMed1996

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.

Actins↗

PubMed1996

The antiproliferative activity of the tetrapeptide Acetyl-N-SerAspLysPro, an inhibitor of haematopoietic stem cell proliferation, is not mediated by a thymosin beta 4-like effect on actin assembly.

Acetyl-N-SerAspLysPro (AcSDKP), known as a negative regulator of haematopoiesis, has been principally reported as an inhibitor of haematopoietic pluripotent stem cell proliferation. The tetrapeptide sequence is identical to the N-terminus of thymosin beta 4 (T beta 4), from which it has been suggested that it may be derived. Recently, evidence was shown that T beta 4 plays a role as a negative regulator of actin polymerization leading to the sequestration of its monomeric form. The structural similarity between the N-terminus of T beta 4 and AcSDKP has raised the possibility that AcSDKP may also participate in intracellular events leading to actin sequestration. The effect of T beta 4 on the proliferation of haematopoietic cells was compared to that of AcSDKP. The results revealed that T beta 4, like AcSDKP, exerts an inhibitory effect on the entry of murine primitive bone marrow cells into cell cycle in vitro. Qualitative electrophoretic analysis and quantitative polymerization assays were used to investigate the role of AcSDKP in actin polymerization. AcSDKP does not affect actin assembly at concentrations up to 50 microM, and does not compete with T beta 4 for binding to G-actin. These results suggest that AcSDKP is not involved in cell cycle regulation via an effect on the process of actin polymerization.

Actin Cytoskeleton↗