In vitro depolymerization dynamics of brain endogenous microtubules

A subcellular fraction containing fragments of endogenous microtubules stabilized in 50% glycerol was separated by diferential centrifugation of rat brain homogenates. The pellets were suspended in glycerol-deficient media, and microtubule depolymerization was monitored by measuring the decrease of sedimentable tubulin. Concomitantly, the number and size of microtubules in the suspensions were followed via electron microscopy. Depolymerization was accompanied by a proportional decrease in the number of microtubules, whereas the average size did not change significantly. After approximately 20 min, a subpopulation of microtubules became stable and did not suffer further depolymerization. These results indicate that upon dilution some microtubules completely depolymerize, whereas others remain stable in the glycerol-deficient medium. The degree of depolymerization depended on both the volume of the resuspension media and on the final glycerol concentration. The results suggest that the depolymerization of the remaining microtubules is prevented by stabilizing factors released from depolymerizing microtubules. Tubulin dimers are not one of these factors, since depolymerization was not altered by the addition of colchicine or by changing the concentration of free tubulin in the medium.     

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.     

Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes.

The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final-state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final-stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin-colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine-tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine-liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-associated proteins from Antarctic fishes

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.     

Taxol stabilization of microtubules in vitro: dynamics of tubulin addition and loss at opposite microtubule ends

We have investigated the effects of taxol on steady-state tubulin flux and on the apparent molecular rate constants for tubulin addition and loss at the two ends of bovine brain microtubules in vitro. These microtubules, which consist of a mixture of 70% tubulin and 30% microtubule-associated proteins (MAPs), undergo a net addition of tubulin at one end of each microtubule (A end) and a precisely balanced net loss of tubulin at the opposite end (D end) at steady state in vitro. They do not exhibit to a detectable extent the "dynamic instability" behavior described recently for MAP-free microtubules, which would be evident as an increase in the mean microtubule length and a decrease in the number of microtubules in the suspensions [Mitchison, T., & Kirschner, M. (1984) Nature (London) 312, 237-242]. We used a double-label procedure in which microtubules were labeled with tritium and carbon-14 at A ends and carbon-14 at D ends to distinguish the two ends, combined with a microtubule collection procedure that permitted rapid and accurate analysis of retention of the two labels in the microtubules. We found that taxol slowed the flux of tubulin in a concentration-dependent manner, with 50% inhibition occurring between 5 and 7 microM drug. The effects of taxol on the apparent molecular rate constants for tubulin addition and loss at the two microtubule ends were determined by dilution analysis at an intermediate taxol concentration. The results indicated that taxol decreased the magnitudes of the dissociation rate constants at the two ends to similar extents, while exerting little effect on the association rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin-microtubule interactions in the algaNitella: analysis of the mechanism by which microtubule depolymerization potentiates cytochalasin's effects on streaming

Summary In the characean algaNitella, depolymerization of microtubules potentiates the inhibitory effects of cytochalasins on cytoplasmic streaming. Microtubule depolymerization lowers the cytochalasin B and D concentrations required to inhibit streaming, accelerates inhibition and delays streaming recovery. Because microtubule depolymerization does not significantly alter³H-cytochalasin B uptake and release, elevated intracellular cytochalasin concentrations are not the basis for potentiation. Instead, microtubule depolymerization causes actin to become more sensitive to cytochalasin. This increased sensitivity of actin is unlikely to be due to direct stabilization of actin by microtubules, however, because very few microtubules colocalize with the subcortical actin bundles that generate streaming. Furthermore, microtubule reassembly, but not recovery of former transverse alignment, is sufficient for restoring the normal cellular responses to cytochalasin D. We hypothesize that either tubulin or microtubule-associated proteins, released when microtubules depolymerize, interact with the actin cytoskeleton and sensitize it to cytochalasin.Abbreviations APW artificial pond water - Ca_c cytoplasraic free calcium concentration - DMSO dimethyl sulfoxide - MT microtubule-minus - MT+ microtubule-plus.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

Structural plugs at microtubule ends may regulate polymer dynamics in vitro

Microtubules contain in their lumens distinct structures (plugs) that influence their dynamic behavior in vitro. As observed by electron microscopy, plugs are stain-occluding structures 10-30 nm in length that occur along the lengths and at the ends of microtubules. Plugs occur at a frequency of 20-40% at the ends of microtubules assembled from cycled microtubule protein containing MAPs. While the composition of plugs is not known, preliminary evidence suggests that they are accretions of tubulin, that they are labile, and that they are more common in preparations containing MAPs. When polymers are induced to depolymerize by endwise subunit dissociation, the frequency of plugged microtubule ends increases transiently, suggesting that plugs temporarily stabilize microtubules. The functional significance of plugs may be that they prevent the sudden complete loss of microtubules through catastrophic disassembly. It is possible that plugs, by slowing the rate of disassembly, enable a polymer to add GTP-tubulin subunits, thereby forming a stabilizing GTP-cap. These observations suggest that plugs may stabilize polymers and account for the frequent transitions from shortening to growing phases that characterize dynamic instability.     

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.     

Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.     

Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex

Substoichiometric concentrations of tubulin-colchicine complex (TC) inhibits microtubule assembly through a copolymerization reaction between tubulin and TC. We have determined the rates and extent of TC incorporation into bovine brain microtubules and developed a theory that models copolymerization. Our analysis suggests that while the apparent association rate constants for tubulin and TC are similar, the apparent dissociation rate constants for TC are a factor of five or more larger than those of tubulin. Copolymer composition showed only slight changes during assembly despite changes in the solution phase and showed little dependence at high TC upon the initial tubulin concentration. The theory was based on coupled Oosawa-Kasai equations that allow for the co-assembly of two components, tubulin and TC. An expression was derived that relates copolymer composition to reaction mixture composition and to the affinity of microtubule ends for tubulin and TC. This expression predicts copolymer composition at TC concentrations less than 10 microM and correlates composition with assembly inhibition. We perceive copolymerization as a facilitated incorporation of TC requiring the presence of tubulin. TC incorporation was dependent on the ratio of total tubulin to the dissociation constant for TC bound to microtubule ends. The copolymerization reaction is thus characterized by an interplay of two effects (a) where tubulin facilitates the incorporation of TC into the microtubule, and (b) where TC inhibits the assembly of tubulin into microtubules.     

Microtubule depolymerization and phosphorylation of c-Jun N-terminal kinase-1 and p38 were involved in gambogic acid induced cell cycle arrest and apoptosis in human breast carcinoma MCF-7 cells

Gambogic acid (GA), an ingredient isolated from Garcinia hanburyi, has potent anticancer activity both in vitro and in vivo. In the present study, we examined the effects of GA on intracellular microtubules and reconstituted microtubules in vitro. Immunofluorescence microscopy revealed that 2.5 muM GA caused microtubule cytoskeleton disruption and microtubule depolymerization in human breast carcinoma MCF-7 cells, thereby reducing the amount of polymer form of tubulin and increasing the amount of monomer form of tubulin. We further confirmed that GA could depolymerize microtubule associated protein (MAP)-free microtubules and MAP-rich microtubules in vitro. Thus we suggested that GA-induced G2/M phase cell cycle arrest may be attributed to its depolymerization of microtubules. We also revealed that phosphorylation levels of p38 and c-Jun N-terminal kinase-1 (JNK-1) were increased markedly by GA, resulting in apoptosis of MCF-7 cells. Taken together, our results suggested that GA depolymerized microtubules and elevated the phosphorylation levels of JNK1 and p38, which caused G2/M cell cycle arrest and apoptosis in MCF-7 cells.     

Full-length dimeric MCAK is a more efficient microtubule depolymerase than minimal domain monomeric MCAK

MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.MCAK cosedimentation assays to compare the activity of full-length MCAK, which is a dimer, with MD-MCAK, which is a monomer. Full-length MCAK exhibits higher ATPase activity, more efficient microtubule end binding, and reduced affinity for the tubulin heterodimer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.     

Dependency of microtubule-associated proteins (MAPS) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules

Summary Microtubule-associated proteins (MAPS) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution.The addition of estramustine phosphate to microtubules reconstituted of MAPS prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4° C was dependent on intact bindings between the tubulin and MAPs.Abbreviations Pipes 1,4-Piperazinediethanesulfonic acid - EDTA Ethylenedinitrilo Tetraacetic Acid - MAPs Microtubule-Associated Proteins - SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis     

Tubulin-colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end

The effects of colchicine and tubulin-colchicine complex (TC) on microtubule depolymerization were studied using the axoneme-subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141-150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5-100 microM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4:1-1:1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC-affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.     

Dynamics of microtubules from erythrocyte marginal bands.

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.     

Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.