Biomedical subjects

D Pantaloni

Publications and source records attributed to D Pantaloni.

At least 37 records · Page 2Linked to original sources

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

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↗

PubMed1995

Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins.

Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.

Actin Cytoskeleton↗

PubMed1995

Kinetics of the interaction of myosin subfragment-1 with G-actin. Effect of nucleotides and DNaseI.

The kinetics of interaction of monomeric pyrenyl-labeled G-actin with myosin subfragment-1 (S1 (A1) and S1(A2) isomers) has been examined in the stopped-flow at low ionic strength. The data confirm the previously reported existence of binary GS and ternary G2S complexes. The increase in pyrenyl-actin fluorescence which monitors the G-actin-S1 interactions is linked to the isomerization of these complexes following rapid equilibrium binding steps. The rates of isomerization are approximately 200 s-1 for GS and approximately 50 s-1 for G2S at 4 degrees C and in the absence of ATP. DNaseI and S1 bind G-actin essentially in a mutually exclusive fashion. Both GS and G2S are dissociated by MgATP and MgADP. The kinetics and mechanism of ATP-induced dissociation of G2S are quantitatively close to the ATP-induced dissociation of F-actin-S1, which indicates the G2S is a good model for the F-actin-S1 interface. GS and G2S display different kinetic behaviors in response to nucleotides, GS being less efficiently dissociated than G2S by MgATP. This result suggests that different mechanical properties of the cross-bridge might correlate with different orientations of the myosin head and different actin/myosin binding ratios.

Actins↗

PubMed1995

Binding of divalent cation and nucleotide to G-actin in the presence of profilin.

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s-1 to 2 s-1, the rate of MgADP dissociation is increased from 0.2 s-1 to 24 s-1. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 x 10(6) to 10(7) M-1) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-G-actin. Profilin binding to actin lowers the affinity of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly in vivo.

Actins↗

PubMed1995

Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface.

The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts.

Actins↗

PubMed1994

Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations.

We report the use of two independent new methods to measure the flexural rigidity of microtubules. Microtubules were grown off axonemal pieces adhering to a glass coverslip. In the first method, a hydrodynamic flow was applied to microtubules and the flexural rigidity was derived from the analysis of the bending shape of the microtubules at equilibrium in the flow. In the second method, the flexural rigidity was derived from the thermal fluctuations of the free end of axoneme-bound microtubules. With both methods, the flexural rigidity of standard GDP microtubules was estimated to be 0.85 +/- 0.2 x 10(-23) newtons x m2 which corresponded to a persistence length of 2 +/- 0.2 mm. Binding of ligands known to affect the biochemical properties of microtubules affected their rigidity. The structural analogs of inorganic phosphate AlF4- and [BeF3-, H2O], which bind to the site of the gamma-phosphate of GTP on GDP microtubule and reconstitute the GDP-Pi microtubule intermediate state of GTP hydrolysis, cause an approximately 3-fold increase in microtubule flexural rigidity and persistence length. Taxol and taxotere, antitumoral microtubule-stabilizing drugs, in contrast cause a decrease in flexural rigidity and appear to affect the three-dimensional superstructure of microtubules, which can no longer be considered as semi-flexible rods. The relationship between the mechanical properties of microtubules and their biological function is discussed.

Animals↗

PubMed1994

Myosin subfragment-1-induced polymerization of G-actin. Formation of partially decorated filaments at high actin-S1 ratios.

Myosin subfragment-1-induced polymerization of G-actin into arrowhead-decorated F-actin-myosin subfragment-1 (S1) filaments has been studied at low ionic strength and in the absence of ATP, using a combination of light scattering, fluorescence of 4-nitrobenz-2-oxa-1,3-diazol-7-yl- or pyrenyl-labeled actin, sedimentation, and electron microscopy techniques. When G-actin is in excess over myosin subfragment-1, the initial formation of fully decorated F-actin-S1 filaments, in which the actin:S1 molar ratio is 1:1, is followed by further incorporation of G-actin subunits in the polymer concomitant with the redistribution of the myosin heads along the polymer, leading to partially decorated filaments containing less than one S1/actin, in equilibrium with G-actin. This process leads to an overshoot in the light-scattering polymerization curves at high actin:S1 ratios. The concentration of G-actin at equilibrium with partially decorated filaments is a nonlinear function of the molar fraction of S1 in the polymer, indicating that actin-actin-S1 interactions are energetically more favorable than actin-actin or actin-S1-actin-S1 interactions.

Actins↗

PubMed1994

Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin.

In motile non-muscle cells, G-actin sequestering proteins, capping proteins and profilin regulate actin assembly in response to extracellular signals. The regulation of actin sequestration/assembly is performed via the control of the concentration of free G-actin at steady-state. The increase in free G-actin mediated by capping proteins results in an increased sequestration of actin. When barbed ends are uncapped upon cell stimulation, the participation of profilin-actin in filament assembly causes a decrease in free G-actin, which results in the depletion of the pool of sequestered actin and concomitant increase in the F-actin pool.

Actin Depolymerizing Factors↗

PubMed1994

Interaction of G-actin with thymosin beta 4 and its variants thymosin beta 9 and thymosin beta met9.

Thymosin beta 4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin beta 9 and thymosin beta met9 are minor variants of thymosin beta 4. The possible function of these peptides has been investigated by comparing the actin binding properties of these beta-thymosins. Thymosin beta 9 and thymosin beta met9 were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 microM (as compared to 2 +/- 0.3 microM for thymosin beta 4); like thymosin beta 4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin beta 4 and thymosin beta met9 with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta 4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin beta 4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of approximately 1.5 microM-1 s-1 and 2 s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin beta 4 function in vivo is discussed.

Actins↗

PubMed1993

How profilin promotes actin filament assembly in the presence of thymosin beta 4.

The role of profilin in the regulation of actin assembly has been reexamined. The affinity of profilin for ATP-actin appears 10-fold higher than previously thought. In the presence of ATP, the participation of the profilin-actin complex to filament elongation at the barbed end is linked to a decrease in the steady-state concentration of globular actin. This surprising effect is made possible by the involvement of the irreversible ATP hydrolysis accompanying actin polymerization. As a consequence, in the presence of thymosin beta 4 (T beta 4), low amounts of profilin promote extensive actin assembly off of the pool of actin-T beta 4 complex. When barbed ends are capped, profilin simply sequesters globular actin. A model is proposed for the function of profilin in actin-based motility.

Actin Cytoskeleton↗

PubMed1993

Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work.

Actins↗

PubMed1991

Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and tau 218-235 synthetic peptide as model systems.

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.

Amino Acid Sequence↗

PubMed1991

Myosin subfragment-1 interacts with two G-actin molecules in the absence of ATP.

The interaction between G-actin and myosin subfragment-1 (S1) has been monitored by pyrenyl-actin fluorescence and light scattering. In low ionic strength buffer and in the absence of ATP the polymerization of G-actin induced by myosin subfragment-1 is preceded by the formation of binary GS and ternary G2S complexes in which S1 interacts tightly in rapid equilibrium (K greater than 10(7) M-1) with one and two G-actin molecules, respectively. Pyrenyl fluorescence of G-actin is enhanced 4-fold in GS and 3-fold in G2S. At concentrations of G-actin and S1 in the micromolar range and above, G2S is the predominant species at G-actin/S1 ratios equal to or greater than 1. The isomer of myosin subfragment-1 carrying the A1 light chain, S1(A1), forms a tighter ternary complex than the isomer S1(A2). Actin-bound ATP is not hydrolyzed upon formation of GS and G2S. In the presence of one molar equivalent or more of myosin subfragment-1/mol of G-actin, in low ionic strength buffer containing no nucleotides, G-actin polymerizes faster in the presence of S1(A1) than in the presence of S1(A2). The interaction of S1 with G-actin is inhibited by the binding of ATP or ADP to S1, ATP having a higher affinity for S1 than ADP. The possible structural similarity of the G2S complex to the F-acto-S1 complex in the rigor state and the potential significance of a ternary (actin)2-myosin interaction for actomyosin-based motility are discussed.

Actins↗

PubMed1990

Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly.

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [gamma-32P,3H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-Pi-microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s-1 for the cleavage of the gamma-phosphate and 0.02 s-1 for the liberation of Pi, assuming a simple random model. Apparent rate constants for GTP hydrolysis and Pi release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed.

Absorption↗

PubMed1989

Cold depolymerization of microtubules to double rings: geometric stabilization of assemblies.

The kinetic pathway of microtubule depolymerization at 0 degrees C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0 degree C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were alpha-beta dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 microM, is much faster than nucleotide exchange on alpha-beta-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on alpha-beta-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the gamma-phosphate in GTP or GDP-Pi.

Animals↗