Kinetic Analysis of Tubulin Assembly in the Presence of the Microtubule-associated Protein TOGp
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m605641200
ISSN1083-351X
AutoresClaude Bonfils, Nicole Bec, Benjamin Lacroix, Marie-Cé cile Harricane, Christian Larroque,
Tópico(s)Fungal and yeast genetics research
ResumoThe microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction. The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction. Microtubules are highly dynamic structures that switch between growing and shrinking phases both in vivo and in vitro. These cytoskeleton polymers are necessary for many functions within the cell including intracellular transport, motility, morphogenesis, and cell division. The intrinsic dynamic instability of microtubules is further modified in the cell by numerous protein factors that favor alternatively elongation, shortening, or anchoring of these polymers. Because the mitotic spindle plays a crucial role in cell division, it has been used for decades as an important target in cancer chemotherapy. Many tubulin poisons have been identified, some of them, taxanes and vinca alkaloids, have demonstrated therapeutic value. However, all tubulin poisons are not of clinical utility. This has led to extensive efforts to explore other targets that could affect spindle integrity. A promising approach is to identify the protein regulators that modulate tubulin polymerization and to investigate their mechanism of action. The dynamic instability of microtubules is controlled in vivo by several classes of cellular factors including depolymerizing kinesins (MCAK/XKCM1) (1Hunter A.W. Caplow M. Coy D.L. Hancock W.O. Diez S. Wordeman L. Howard J. Mol. Cell. 2003; 11: 445-457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 2Walczak C.E. Mitchison T.J. Desai A. Cell. 1996; 84: 37-47Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), stathmins (3Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar), and microtubule-associated proteins (MAPs). 2The abbreviations used are: MAP, microtubule-associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Pipes, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline. This last group is composed of structural MAPs (MAP2, tau) that were first identified in brain tissue and of a group of XMAP215-related proteins whose generic member was first characterized in Xenopus eggs (4Gard D.L. Kirschner M.W. J. Cell Biol. 1987; 105: 2203-2215Crossref PubMed Scopus (284) Google Scholar). TOGp (HUGO gene CKAP5), which is highly expressed in tumors and brain (5Charrasse S. Mazel M. Taviaux S. Berta P. Chow T. Larroque C. Eur. J. Biochem. 1995; 234: 406-413Crossref PubMed Scopus (74) Google Scholar), is the human homolog of XMAP215. TOGp promotes microtubule assembly both in solution and from nucleation centers (6Charrasse S. Schroeder M. Gauthier-Rouviere C. Ango F. Cassimeris L. Gard D.L. Larroque C. J. Cell Sci. 1998; 111: 1371-1383Crossref PubMed Google Scholar). It was evidenced that this MAP possesses a high affinity for polymer lattice and that it binds protofilaments by its N terminus (7Spittle C. Charrasse S. Larroque C. Cassimeris L. J. Biol. Chem. 2000; 275: 20748-20753Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This protein is involved in microtubule aster formation in mammalian mitotic cells (8Dionne M.A. Sanchez A. Compton D.A. J. Biol. Chem. 2000; 275: 12346-12352Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar); moreover, TOGp is required for centrosome integrity and spindle pole organization (9Cassimeris L. Morabito J. Mol. Biol. Cell. 2004; 15: 1580-1590Crossref PubMed Scopus (155) Google Scholar). The TOGp family has a wide distribution; it is present from yeasts to humans. In addition to the human TOGp and to the frog XMAP215 protein, members of this group have been independently discovered in Drosophila melanogaster (Msps), in Dictyostelium discoideum (DdCP224), and in Arabidopsis thaliana (Mor1) (10Cullen C.F. Deak P. Glover D.M. Ohkura H. J. Cell Biol. 1999; 146: 1005-1018Crossref PubMed Scopus (139) Google Scholar, 11Graöf R. Daunderer C. Schliwa M. J. Cell Sci. 2000; 113: 1747-1758Crossref PubMed Google Scholar, 12Whittington A.T. Vugrek O. Wei K.J. Hasenbein N.G. Sugimoto K. Rashbrooke M.C. Wasteneys G.O. Nature. 2001; 411: 610-613Crossref PubMed Scopus (358) Google Scholar). Other forms with more divergent protein structure were identified in Caenorhabditis elegans (Zyg-9) (13Matthews L.R. Carter P. Thierry-Mieg D. Kemphues K. J. Cell Biol. 1998; 141: 1159-1168Crossref PubMed Scopus (147) Google Scholar) and in yeasts. Two forms, Dis1 and Alp14, are present in fission yeast (14Ohkura H. Adachi Y. Kinoshita N. Niwa O. Toda T. Yanagida M. EMBO J. 1988; 7: 1465-1473Crossref PubMed Scopus (176) Google Scholar, 15Garcia M.A. Vardy L. Koonrugsa N. Toda T. EMBO J. 2001; 20: 3389-3401Crossref PubMed Scopus (119) Google Scholar), whereas one member, StuII, was characterized in Saccharomyces cerevisiae (16Wang P.J. Huffaker T.C. J. Cell Biol. 1997; 139: 1271-1280Crossref PubMed Scopus (142) Google Scholar). This evolutionary conserved protein family is implicated in microtubule polymer assembly and spindle formation. Microtubules are hollow cylindrical aggregates of 25-nm diameter composed of heterodimers of α- and β-tubulin. Each of these subunits binds 1 mol of GTP. GTP bound to α-tubulin is not exchanged, whereas β-tubulin-bound GTP is hydrolyzed to GDP soon after tubulin polymerization. A significant amount of the free energy of this hydrolysis goes into the microtubule via a conformational change of the tubulin dimer; its consequence is to destabilize the structure. Microtubules can spontaneously assemble in vitro from a solution of purified tubulin in the presence of GTP by a temperature jump from 0 to 37 °C. The kinetics of tubulin assembly are generally interpreted as a two-step nucleation elongation process. The theoretical interpretation of tubulin polymerization is based on the actin helical aggregation model (17Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (573) Google Scholar, 18Wegner A. Engel J. Biophys. Chem. 1975; 3: 215-225Crossref PubMed Scopus (185) Google Scholar). However, the polymerization of microtubules is much more complex than the assembly of actin filaments and necessitates kinetic as well as thermodynamic considerations (19Erickson H.P. Pantaloni D. Biophys. J. 1981; 34: 293-309Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Its mathematical analysis requires an infinite set of interrelated differential equations (20Hall D. Biophys. Chem. 2003; 104: 655-682Crossref PubMed Scopus (23) Google Scholar). In the case of actin, some approximations were introduced by Wegner and Engel (18Wegner A. Engel J. Biophys. Chem. 1975; 3: 215-225Crossref PubMed Scopus (185) Google Scholar), leading to simplify the process to two inter-related differential equations, which after integration give a numerical solution of the polymerization curves (21Tobacman L.S. Korn E.D. J. Biol. Chem. 1983; 258: 3207-3214Abstract Full Text PDF PubMed Google Scholar, 22Houmeida A. Bennes R. Benyamin Y. Roustan C. Biophys. Chem. 1995; 56: 201-214Crossref PubMed Scopus (5) Google Scholar). The actin model cannot be directly extrapolated to tubulin. Microtubule elongation is well documented both at the structural and mechanistic levels; in contrast, nucleation is still poorly understood, mainly because it is composed of weakly concentrated transient intermediates (23Caudron N. Arnal I. Buhler E. Job D. Valiron O. J. Biol. Chem. 2002; 277: 50973-50979Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Voter and Erickson (24Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar) introduced a two-dimensional nucleation mechanism that improves the fitting with the experimental kinetic curves. More recently Flyvbjerg et al. (25Flyvbjerg H. Jobs E. Leibler S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5975-5979Crossref PubMed Scopus (142) Google Scholar) formulated a new assembly model in which the final nucleus is the result of a series of intermediate aggregates formed by the step by step addition of a variable number of tubulin monomers. Quantitative parameters describing the mechanism of tubulin assembly can be deduced from the kinetics by application of the theoretical models. It is possible to calculate from the polymerization time curves a range of physicochemical parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunit turnover at microtubule ends can be deduced from tubulin GTPase activity. In this paper we have determined the influence of TOGp on these kinetic parameters. The results showed that this MAP was a strong activator of microtubule production, able to influence various steps of the reaction at low concentration. For comparison, parallel experiments were conducted with colchicine and taxol, two microtubule reactive drugs, and with tau, a classical MAP from brain tissue. Tubulin was prepared according to the purification procedure described by Williams and Lee (26Williams Jr., R.C. Lee J.C. Methods Enzymol. 1982; 85: 376-385Crossref PubMed Scopus (410) Google Scholar). TOGp was isolated from pig brain cytosol. Pig brains were obtained from the local slaughterhouse and transported to the laboratory on ice within 2 h after bleeding. Meninges and superficial blood vessels were removed from the brains at 4 °C. Two brains (160 g) were homogenized in 200 ml of PEM buffer (100 mm Pipes/NaOH, pH 6.6, 1 mm EGTA, 1 mm MgSO4, 1 μg/ml leupeptin) for 30 s in a Warring blender mixer. The tissue was further disrupted by means of a Tenbroeck homogenizer with a Teflon pestle (five strokes on ice). The homogenate was centrifuged at 125,000 × g for 75 min at 5 °C, and the supernatant was recovered. This fraction was brought to 32% saturation by adding progressively solid ammonium sulfate at room temperature. The precipitate was recovered by centrifugation at 5000 × g for 20 min. The pellet was resuspended in 250 ml of a H2O/PEM (v/v) mixture. The solution was dialyzed overnight at 4 °C against 4 liters of PEM buffer. A small protein precipitate was eliminated after centrifugation at 10,000 rpm for 20 min. The protein TOGp was purified from the supernatant in two chromatographic steps. Hydroxyapatite—The column (1.6 × 20 cm) was filled with Macro-Prep ceramic hydroxyapatite from Bio-Rad and equilibrated with PEM buffer. The column was loaded with the cleared supernatant and rinsed with PEM buffer. The proteins were eluted with two successive salt concentration gradients. First the KCl concentration was raised from 0 to 2 m in PEM buffer. Then the PEM buffer was replaced by phosphate buffer (10 mm potassium phosphate, pH 6.8, 1 mm EGTA, 1 mm MgSO4, 1 μg/ml leupeptin), and a second gradient from 10 to 600 mm potassium phosphate was applied to the column. Usually, TOGp eluted with ∼400 mm potassium phosphate. The protein fractions were analyzed by Western blotting, and the chromatographic fractions enriched in TOGp were pooled. DEAE-Sepharose—This chromatography was performed in TEM buffer (Tris/HCl, 20 mm, pH 8.2, 1 mm EGTA, 1 mm MgSO4, 1 μg/ml leupeptin) on a 1 × 10 cm column of DEAE-Sepharose Fast Flow from Amersham Biosciences. The protein fraction eluted from the hydroxyapatite column was dialyzed against 2 liters of TEM buffer for 5 h and loaded on the column, and unadsorbed proteins were eliminated by rinsing with the TEM buffer. A KCl concentration gradient from 0 to 0.1 m in TEM buffer was then applied. The protein fractions eluted with this gradient were immediately stored at -80 °C. The qualitative composition of each fraction was determined on SDS-PAGE, and Western blots revealed with anti-TOGp antibodies. We applied the purification procedure described by Cleveland et al. (27Cleveland D.W. Hwo S.Y. Kirschner M.W. J. Mol. Biol. 1977; 116: 207-225Crossref PubMed Scopus (713) Google Scholar) to the 125 000 g pig brain supernatant. Rabbit polyclonal anti-TOGp antibodies were prepared as previously mentioned (6Charrasse S. Schroeder M. Gauthier-Rouviere C. Ango F. Cassimeris L. Gard D.L. Larroque C. J. Cell Sci. 1998; 111: 1371-1383Crossref PubMed Google Scholar). Mouse monoclonal anti-tau (clone tau-2) and mouse monoclonal anti-β-tubulin antibodies (clone Tub 2.1) were from Sigma. Immunogold-conjugated goat anti-rabbit IgG (5- and 15-nm particles) were from British Biocell International (Cardiff, UK). Fluorescein isothiocyanate-conjugated goat anti-rabbit antibody and Texas-Red-conjugated goat anti-mouse antibody were from Cappel (MP Biomedicals, Strasbourg, France). Proteins were resolved in denaturing 6% acrylamide gels, in a discontinuous buffer system, as described by Laemmli (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212243) Google Scholar). Then they were electrotransferred for 1 h at 350 mA on a polyvinylidene difluoride membrane (Immobilon P from Millipore, Bedford, MA) in Tris/glycine buffer (48 mm Tris, 39 mm glycine, pH 9.2, 1.3 mm SDS and 20% methanol). Protein bands were stained nonspecifically by Amido Black. Membranes were blocked overnight in 6% nonfat milk in phosphate-buffered saline (PBS) at 4 °C. They were subsequently probed for 2 h with either anti-TOGp, anti-tubulin, or anti-tau antibodies, diluted 1/1000. The blots were rinsed and incubated with the appropriate secondary antibody (1/2000) conjugated with peroxidase. Bound antibodies were detected by the enhanced chemiluminescence reagent ECL from Amersham Biosciences. The protein extract was resolved in a 6% acrylamide gel under denaturing conditions. After the run, the gel was stained with 0.2% Coomassie Blue R250 dissolved in 2% acetic acid, 50% methanol (high performance liquid chromatography grade). It was destained in 30% methanol. The 160- and 130-kDa polypeptides were both sequenced. They were excised from the gel and hydrolyzed by trypsin according to Rosenfeld et al. (29Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1140) Google Scholar). The resulting digest was fractionated using reverse phase chromatography on C8 then C18 Aquapore (2 × 10 mm) columns from Applied Biosystems (Foster City, CA) and eluted by an acetonitrile gradient in 0.1% trifluoroacetic acid. The eluate was monitored by UV spectroscopy (220 nm). Purified peptides were sequenced on a Procise sequencer from Applied Biosystem using the pulsed liquid program. Proteins resolved by polyacrylamide gel electrophoresis were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Selected protein spots were excised from the gel and destained. After reduction and alkylation of disulfide bonds, the dried gel pieces were incubated with trypsin. The resulting peptides were extracted, purified with Millipore Zip-Tip C18 columns, and added to the α-cyano-4-hydroxycinnamic acid matrix. MALDI-TOF mass spectrometry was performed at the Institut de Gé;nomique Fonctionnelle (CNRS UPR 2580, Montpellier, France) using a Ultraflex apparatus from Bruker Daltonics (Billerica, MA). The peptide masses were matched with the theoretical peptide masses of all proteins in the Swiss-Prot data base using the MASCOT search engine. Tubulin polymerization was monitored turbidimetrically at 350 nm with a MC2 (Safas, Monaco) spectrophotometer equipped with a thermal-jacketed cuvette holder. The cuvette had a 10-mm path length and was 2 mm wide internally. The final volume of the sample was 200 μl. Experiments were run in PEM buffer, 3.4 m glycerol (25% v/v), 1 mm GTP, tubulin amount was varied from 5 to 20 μm, and MAPs or drugs were added in the medium as indicated elsewhere. The reaction mixture was prepared at 0 °C, and the reaction was started by placing the cuvette in the spectrophotometer cell compartment thermostatted at 37 °C. The GTPase activity was detected by using the PiPer phosphate assay kit from Molecular Probes (Eugene, OR). Briefly, inorganic phosphate is combined with maltose to give glucose 1-phosphate and glucose. Then glucose oxidase converts glucose to gluconolactone and H2O2. Finally horseradish peroxidase converts Amplex Red to Resorufin in the presence of hydrogen peroxide. Resorufin can be detected by measuring the increase in fluorescence or absorbance of the solution. Tubulin was polymerized at 37 °C in the conditions indicated above, except that the amount of GTP was lowered to 0.1 mm to diminish the deep red coloration of the blank. The medium was divided in ten 50-μl samples that were incubated at 37 °C. At various time intervals the polymerization was stopped by denaturing the proteins for 5 min in boiling water. The precipitate was removed by centrifugation, and the supernatant was mixed with an equal volume of the kit reagent. The reaction was developed for 3 h at 37°C, and the absorbance was measured at 570 nm. A dilution range was prepared in the same conditions from a50 μm potassium phosphate solution to standardize the assay. Tubulin assembly is usually described as a sequence of bimolecular reactions (for details, see Ref. 20Hall D. Biophys. Chem. 2003; 104: 655-682Crossref PubMed Scopus (23) Google Scholar) in which the polymer is built by successive additions of basal units of α- and β-tubulin dimers; for simplicity, these building blocks are frequently termed monomers. The initial bimolecular reactions are thermodynamically unfavorable (24Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar); small aggregates tend to dissociate. Once a certain size, n monomers (commonly named "critical nucleus"), is reached, the addition of the next monomer gives a polymer more stable than its precursor. From this step the elongation takes place by polymerization of subunits onto the microtubule ends. The reaction continues until the elongation process is compensated by the release of monomers at microtubule ends. At that time the polymer is in simple equilibrium with a fixed (critical) concentration of tubulin subunits. As indicated above, we followed the reaction of polymerization at 350 nm. It was shown previously (30Johnson K.A. Borisy G.G. J. Mol. Biol. 1977; 117: 1-31Crossref PubMed Scopus (166) Google Scholar) that there is a quite linear relationship between the turbidity and the total amount of microtubules. In consequence we considered the absorbance at 350 nm (A350 nm) as proportionally related to the mass concentration of tubulin polymer. Information concerning nucleation as well as elongation was drawn from the analysis of the sigmoid kinetics (30Johnson K.A. Borisy G.G. J. Mol. Biol. 1977; 117: 1-31Crossref PubMed Scopus (166) Google Scholar, 31Sternlicht H. Ringel I. J. Biol. Chem. 1979; 254: 10540-10550Abstract Full Text PDF PubMed Google Scholar). Two distinct parts can be seen on the curves; from time 0 to the first few minutes there is a lag phase corresponding principally to nucleation, then an exponential decay process takes place corresponding to elongation. Nucleation—This phase may be characterized by various parameters. The determination of the tenth time, t1/10 (the time necessary to produce 10% of the final amount of polymer) is of current use to estimate the lag time duration. Moreover, according to the theoretical models, we find that two parameters can be used to characterize the nucleus size. In this paper we termed these parameters p and q. The former is defined by Flyvbjerg et al. (25Flyvbjerg H. Jobs E. Leibler S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5975-5979Crossref PubMed Scopus (142) Google Scholar), on the basis of the "scaling" properties of the polymerization curves obtained with various amounts of tubulin. From the experimental results, these authors noticed that the increase in polymer concentration for the earliest times is proportional to tp. They formulated a theoretical model in which the parameter p was indicative of the number of successive steps in the nucleation process. The value of p can be easily determined by plotting log(A(t)/A∞) against log t. The second parameter, q, originates from the theory of helical aggregation of macromolecules of Oosawa and Kasai (17Oosawa F. Kasai M. J. Mol. Biol. 1962; 4: 10-21Crossref PubMed Scopus (573) Google Scholar). In the case of actin polymerization, it was demonstrated that the half-time of the reaction (t½) was proportional to [M0]q, [M0] being the initial monomer concentration. This relationship is valuable with other characteristic times, t1/20 or t1/10 (24Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar, 21Tobacman L.S. Korn E.D. J. Biol. Chem. 1983; 258: 3207-3214Abstract Full Text PDF PubMed Google Scholar, 25Flyvbjerg H. Jobs E. Leibler S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5975-5979Crossref PubMed Scopus (142) Google Scholar). Parameter q is obtained from the log-log plot of the tenth time of polymerization versus the total amount of monomer. Tobacman and Korn (21Tobacman L.S. Korn E.D. J. Biol. Chem. 1983; 258: 3207-3214Abstract Full Text PDF PubMed Google Scholar) indicate that it is equal to half of the nucleus size (n/2), whereas according to Voter and Erickson (24Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar) it is equal to (n + 1)/2. This apparent discrepancy is due in fact to different definitions of the critical nucleus in the two papers. In the former, the nucleus is defined as the first polymer that is itself more stable than its precursor, whereas in the second paper the nucleus is the least stable intermediate in the reaction. Nevertheless parameter q gives an objective estimation of the number of monomers in the critical nucleus. Elongation—Elongation develops after the lag phase following a procedure that is strongly similar to a first order chemical reaction. According to the pioneering work of Johnson and Borisy (30Johnson K.A. Borisy G.G. J. Mol. Biol. 1977; 117: 1-31Crossref PubMed Scopus (166) Google Scholar) the elongation reaction rate can be interpreted as the sum of the rates of polymerization and depolymerization as indicated in the equation dP/dt = -dM/dt = k+[M][E] - k- [E], where P represents the polymer, [M] is the concentration of free tubulin, [E] is the concentration of assembly competent microtubule ends, k+ is an apparent second order association rate constant corresponding to the sum of the rate constants for monomer addition at the two filament ends, and k- is an apparent first order dissociation rate constant corresponding to the sum of the rate constants for monomer dissociation at the two filament ends. At steady state the reactions of growth and shortening of microtubules are identical. At that time [M] is equal to [M∞], the critical concentration of tubulin. Consequently, k+ [M∞][E] = k-[E], and k- = k+[M∞]. It can be determined that the critical concentration [M∞] is equal to k-/k+ or to 1/K (K is the equilibrium association constant (30Johnson K.A. Borisy G.G. J. Mol. Biol. 1977; 117: 1-31Crossref PubMed Scopus (166) Google Scholar)). By replacing k- by its value in the differential equation, dP/dt =-dM/dt = k+[E]([M] - [M∞]). It can be assumed that [E] is constant during the elongation phase, the expression reduces to a pseudo-first order rate expression, the product k+ [E] can be replaced by a constant termed k, and the factor ([M] - [M∞]) represents the concentration of active tubulin named [Ma]. After integration, ln[Ma]/[Ma0] = -kt. The ratio [Ma]/[Ma0] is easily accessible from the measures of the absorbance at 350 nm. It is equal to (A∞ - At)/A∞), where At represents the absorbance at a given time, and A∞ is the absorbance maximum obtained at the plateau of the kinetic curve. By plotting ln(1 - At/A∞) as a function of time, the pseudo-first order rate constant of elongation, kobs, can be determined. Microtubules were prepared in a spectrophotometer cuvette (200 μl final volume) at 37 °C as indicated above in the microtubule assembly assays. Polymers prepared with native 200-kDa TOGp and control polymers with tubulin alone were incubated simultaneously. First Method—Microtubules were centrifuged at 36,000 × g for 30 min, the supernatant was discarded, and the pellet was resuspended in 200 μl of PEM buffer, 25% glycerol, 0.1 mm GTP. Anti-TOGp antibodies (5 μl) were added, and the mixture was incubated for 3 h at 30°C. The antibodies were eliminated by centrifugation at 36,000 × g for 30 min at 35 °C. The pellet was resuspended in 200 μl of PEM/glycerol/GTP buffer and mixed with 5 μl of immunogold-conjugated goat anti-rabbit IgG (5- and 15-nm gold-labeled antibodies were used alternatively). The incubation lasted 2 h at 30°C. The secondary antibody was eliminated by centrifugation at 30 °C, and the microtubules were suspended in 200 μl of fresh buffer and placed at 30 °C. Second Method—After tubulin aggregation at 37 °C, 4 μlof anti-TOGp antibodies were added to the solution, and the incubation was continued for 3 h at 30°C. The secondary antibody (15 μl) was then included, and the incubation was prolonged for 2 h. The polymers were separated from tubulin and antibodies by centrifugation in a 2-ml sucrose gradient (37–60%) for 1 h at 180,000 × g in a swinging rotor thermostatted at 30 °C. Microtubules were present in the first 0.1-ml fraction at the bottom of the gradient. Microtubules were diluted in PEM/glycerol/GTP buffer to a protein concentration of 0.2 mg/ml, deposited onto Formvar-carbon coated grids, and negatively stained with 2% uranyl acetate. Grids were examined using a Jeol 1200 EX electron microscope at an accelerating voltage of 80 kV. Primary cultures of hypothalamus cells were prepared by mechanoenzymatic dissociation of fetal (day 17) rat hypothalamus. Cells (106) were plated in 16-mm diameter culture dishes containing a 10-mm glass coverslip previously coated with poly-d-lysine (32Rage F. Benyassi A. Arancibia S. Tapia-Arancibia L. Endocrinology. 1992; 130: 1056-1062PubMed Google Scholar). Cultures were maintained at 37 °C in a 95% air, 5% CO2 atmosphere in minimum Eagle's medium containing 10% Nu serum, 0.6% glucose, 2 mm glutamine, 2.5 units/ml penicillin-streptomycin adjusted to pH 7.3. Two days after seeding, the cells on the coverslip were fixed for 10 min in cold methanol (-20 °C) and gradually rehydrated with PBS. Cells were then incubated for 60 min with a mixture of anti-TOGp rabbit antiserum (1/200) and anti-tubulin monoclonal antibody (1/200) in PBS containing 1 mg/ml albumin. After washing in PBS, incubation was carried out in the same solution containing both fluorescein isothiocyanate-conjugated anti-rabbit antibodies and Texas Red-conjugated anti-mouse antibodies. Stained cells were mounted in 0.25% Airvol 205 in PBS, and images were recorded using a 63× NA objective on a Leica inverted microscope. Purification of TOGp—The purification procedure is detailed under "Materials and Methods." The elution profile of the DEAE-Sepharose chromatography, which is the last step of the purification, is shown in Fig. 1. TOGp was eluted from the column by increasing the ionic strength of the buffer with 0.1 m KC
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