Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.

Vinblastine is one of several tubulin-targeting Vinca alkaloids that have been responsible for many chemotherapeutic successes since their introduction in the clinic as antitumour drugs. In contrast with the two other classes of small tubulin-binding molecules (Taxol and colchicine), the binding site of vinblastine is largely unknown and the molecular mechanism of this drug has remained elusive. Here we report the X-ray structure of vinblastine bound to tubulin in a complex with the RB3 protein stathmin-like domain (RB3-SLD). Vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly. Together with electron microscopical and biochemical data, the structure explains vinblastine-induced tubulin self-association into spiral aggregates at the expense of microtubule growth. It also shows that vinblastine and the amino-terminal part of RB3-SLD binding sites share a hydrophobic groove on the alpha-tubulin surface that is located at an intermolecular contact in microtubules. This is an attractive target for drugs designed to perturb microtubule dynamics by interfacial interference, for which tubulin seems ideally suited because of its propensity to self-associate.

Phosphoproteins of the stathmin family interact with the alphabeta tubulin heterodimer (tubulin) and hence interfere with microtubule dynamics. The structure of the complex of GDP-tubulin with the stathmin-like domain of the neural protein RB3 reveals a head-to-tail assembly of two tubulins with a 91-residue RB3 alpha helix in which each copy of an internal duplicated sequence interacts with a different tubulin. As a result of the relative orientations adopted by tubulins and by their alpha and beta subunits, the tubulin:RB3 complex forms a curved structure. The RB3 helix thus most likely prevents incorporation of tubulin into microtubules by holding it in an assembly with a curvature very similar to that of the depolymerization products of microtubules.

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

A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol a-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules.This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate reveals three bands of apparent molecular weights 40 000

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Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/ tubulin interaction leads to the formation of a 7.7 S complex with a 60-Å Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 M at pH 6.5. The affinity was lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.Stathmin (1, 2), also designated Op18, p18, p19, prosolin, and metablastin (3-6), is a ubiquitous cytosolic phosphoprotein highly conserved in vertebrates (7,8) and specifically abundant in neurons (9 -11). Expression and phosphorylation of stathmin are modulated in various situations related to the control of cellular activities, and it has been proposed that it may act as a relay integrating various intracellular signaling pathways (1). Expression of stathmin was shown to be regulated in vivo during development (7,(12)(13)(14), during tissue regeneration (15, 16), and in cell culture by cell/cell interactions (17). Stathmin is also up-regulated in many malignant cell types and tumors (5,18,19). Phosphorylation of stathmin is observed in response to hormones (20), cytokines (21), neurotransmitters (22), and growth and differentiation factors (23). Moreover, progression through the cell cycle appears to require multisite phosphorylation of stathmin (24). Actually, overexpression of a nonphosphorylatable mutant of stathmin resulted in a large population of cells blocked in G 2 /M with a high DNA content (24,25). Finally, stathmin is the generic element of a protein family whose other members most probably play distinct roles related to the control of neuronal differentiation or to the expression of neuron-specific traits (8, 26).The molecular mechanism(s) by which stathmin acts in these processes remain largely unknown. Two domains can be distinguished in the primary structure of stathmin, an N-terminal "regulatory" domain that contains the four phosphorylation sites that account for all of the electrophoretic f...

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.