PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

N-terminal stathmin-like peptides bind tubulin and impede microtubule assembly

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.     

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for the first time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of approximately 50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.     

Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.     

Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.     

Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.     

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.     

Immunological detection of microtubule poison-induced conformational changes in tubulin

The interaction of tubulin-microtubule poison complexes with anti-tubulin antisera has been investigated using radioimmunoassay. The binding of the major antiserum used in this study to tubulin does not interfere with the binding of colchicine to the tubulin or affect the decay of the colchicine-binding activity of the tubulin. Conversely, if colchicine is incubated with the tubulin, forming tubulin-colchicine complexes, the tubulin-colchicine complexes are less efficient competitors for antibody-binding sites than tubulin alone. This is the result of the formation of specific colchicine-tubulin complexes, since tubulin, incubated with lumicolchicine or isocolchicine, behaves as if the tubulin were incubated alone in the radioimmunoassay. When tubulin is incubated with other microtubule poisons, podophyllotoxin or vinblastine, the tubulin-drug complexes have diminished ability to compete with tubulin as did the tubulin-colchicine complexes. These changes observed in the binding of tubulin-microtubule poison complexes to anti-tubulin antisera in a tubulin radioimmunoassay suggest that the binding of colchicine, podophyllotoxin, or vinblastine to tubulin induces subtle conformational changes on the surface of the tubulin dimer involving antigenic determinant sites.     

Zinc-stimulated microtubule assembly and evidence for zinc binding to tubulin

• 1.1. Microtubule reassembly was studied in supernatant fluids from rat brain. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared to extracts from controls; 10 μM zinc stimulated reassembly of tubulin in extracts from zinc-deficient animals.
• 2.2. Low zinc concentrations (250–900 μM zinc in the presence of 1 mM EGTA) stimulated reassembly oftubulin in brain extracts from control rats; similar concentrations of nickel had no effect whilst cobalt was inhibitory. In the absence of EGTA 20–40 μM zinc stimulated reassembly in brain extracts from normal rats.
• 3.3. Zinc-induced changes in reassembly were associated with changes in the free sulphydryl group content of the assembled crude microtubule protein; increased assembly was associated with a higher free sulphydryl group content, decreased assembly with a lower content.
• 4.4. ⁶⁵Zn was found to bind to tubulin. This binding was partly inhibited by N-ethylmaleimide.

     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.     

Enhanced microtubule binding and tubulin assembly properties of conformationally constrained paclitaxel derivatives

Microtubule binding and tubulin assembly promotion by a series of conformationally restricted paclitaxel (PTX) derivatives was investigated. In these derivatives, the C-4 acetate of the taxane is tethered to the C-3' phenyl at ortho and meta positions with different length linkers. The apparent affinity of these derivatives for GMPCPP-stabilized microtubules was assessed by a competition assay, and their influence on microtubule polymerization was evaluated by measuring the critical concentration of GDP-tubulin in the presence of the respective molecule. In general, taxane derivatives with higher apparent affinity for microtubules induced tubulin assembly more efficiently. Among the derivatives, molecules with the shortest tether display the strongest affinity for microtubules. These derivatives exhibited enhanced microtubule stabilization properties and efficiently induced GDP-tubulin assembly into microtubules at low temperature of 12 degrees C and in the absence of Mg2+ ions in 0.1 M PIPES. Based on molecular dynamics simulations, we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.     

Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding

Rotenone, a widely used insecticide, has been shown to inhibit mammalian cell proliferation and to depolymerize cellular microtubules. In the present study, the effects of rotenone on the assembly of microtubules in relation to its ability to inhibit cell proliferation and mitosis were analyzed. We found that rotenone inhibited the proliferation of HeLa and MCF-7 cells with half maximal inhibitory concentrations of 0.2 +/- 0.1 microm and 0.4 +/- 0.1 microm, respectively. At its effective inhibitory concentration range, rotenone depolymerized spindle microtubules of both cell types. However, it had a much stronger effect on the interphase microtubules of MCF-7 cells compared to that of the HeLa cells. Rotenone suppressed the reassembly of microtubules in living HeLa cells, suggesting that it can suppress microtubule growth rates. Furthermore, it reduced the intercentrosomal distance in HeLa cells at its lower effective concentration range and induced multipolar-spindle formation at a relatively higher concentration range. It also increased the level of checkpoint protein BubR1 at the kinetochore region. Rotenone inhibited both the assembly and the GTP hydrolysis rate of microtubules in vitro. It also inhibited the binding of colchicine to tubulin, perturbed the secondary structure of tubulin, and reduced the intrinsic tryptophan fluorescence of tubulin and the extrinsic fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid complex, suggesting that it binds to tubulin. A dissociation constant of 3 +/- 0.6 microm was estimated for tubulin-rotenone complex. The data presented suggest that rotenone blocks mitosis and inhibits cell proliferation by perturbing microtubule assembly dynamics.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.     

Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.     

Relating nucleotide‐dependent conformational changes in free tubulin dimers to tubulin assembly

The complex dynamic behavior of microtubules (MTs) is believed to be primarily due to the αβ‐tubulin dimer architecture and its intrinsic GTPase activity. Hence, a detailed knowledge of the conformational variations of isolated α‐GTP‐β‐GTP‐ and α‐GTP‐β‐GDP‐tubulin dimers in solution and their implications to interdimer interactions and stability is directly relevant to understand the MT dynamics. An attempt has been made here by combining molecular dynamics (MD) simulations and protein–protein docking studies that unravels key structural features of tubulin dimer in different nucleotide states and correlates their association to tubulin assembly. Results from simulations suggest that tubulin dimers and oligomers attain curved conformations in both GTP and GDP states. Results also indicate that the tubulin C‐terminal domain and the nucleotide state are closely linked. Protein–protein docking in combination with MD simulations suggest that the GTP‐tubulin dimers engage in relatively stronger interdimer interactions even though the interdimer interfaces are bent in both GTP and GDP tubulin complexes, providing valuable insights on in vitro finding that GTP‐tubulin is a better assembly candidate than GDP‐tubulin during the MT nucleation and elongation processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 282–291, 2013.     

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

The nucleotide switch of tubulin and microtubule assembly: a polymerization-driven structural change

GTP-binding proteins from the tubulin family, including alphabeta-tubulin, gamma-tubulin, bacterial tubulin, and FtsZ, are key components of the cytoskeleton and play central roles in chromosome segregation and cell division. The nucleotide switch of alphabeta-tubulin is triggered by GTP hydrolysis and regulates microtubule assembly dynamics. The structural mechanism of the switch and how it modulates assembly are beginning to be understood. A conserved structural change between the active and inactive states, different from other GTPases, may be extracted from recent tubulin and FtsZ structures. From these and the biochemical properties of tubulin, the new concept emerges that, contrary to what was thought, unassembled tubulin-GTP is in the inactive, curved conformation as in tubulin-GDP rings, and it is driven into the straight microtubule conformation by the assembly contacts; binding of the GTP gamma-phosphate only lowers the free energy difference between the curved and straight forms.