Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin

Abstract

Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

Keywords: GMPCPP; T2S complex; Zn-sheets; computer simulation.

Fig. 1.

Overview of stathmin structures and tubulin polymers used in this work. (A, Upper) Schematic showing the human stathmin constructs used in this work. (Lower) The structure (3RYI) of the complex (T₂S complex) formed between the two tubulin heterodimers (blue or green) and stathmin (red) (23, 24). (Left) A view from the side of the protofilament-like structure; (Right) the view from the front (down the axis) of the protofilament. Note that part of stathmin (the N-terminal tail) is bound the exposed surface of α-tubulin; this is the surface that is exposed at the minus end and would bind to the α-tubulin monomer of an incoming tubulin dimer. (B) Chemically induced tubulin polymers with different conformations. 1: Tx-MT, Taxol stabilized tubulin polymers, which are made by stepwise addition of Taxol and consist primarily of MTs (12); 2: CPP-MT, GMPCPP induced tubulin polymers, which are formed by replacing GTP with GMPCPP and consist primarily of MTs (13, 14); 3: CPP-PF, protofilament-rich structures that are made by treating GMPCPP-MTs with calcium (13, 14); 4: DL-rings, Dolastatin-10–induced tubulin rings, which consist of curved protofilament-based rings and other structures similar to the “ram's horns” that form when tubulin depolymerizes (–18); 5: Zn-Sheets, Zinc-induced tubulin sheets, which are flat sheets formed by antiparallel associations between protofilaments and thus have two exposed protofilaments exposed at both lateral edges (19, 20).

Fig. 2.

Effect of stathmin on different tubulin polymers. (A) Cosedimentation assays and SDS/PAGE analysis showing the effect of 8.0 μM stathmin (St) on different tubulin (T) polymers as indicated (2.0 μM). Note that stathmin induces depolymerization of both CPP-PF and Zn-sheets, but has no effect on DL-rings or Tx-MT (see Fig. 1B and Fig. S2 for details of these different filament types). Depolymerization was stronger at pH 6.8 (pH 6.0 for Zn-sheets) than at pH 7.5. Data for Zn-sheets is shown only at pH 6.0 because Zn-sheets are not stable at higher pH (19, 20). Samples were incubated for 30 min and then sedimented at 165,000 × g for 20 min. Equal fractions of supernatant (s) and pellet (p) were separated by SDS/PAGE, followed by staining with Coomassie blue. Band intensities and data analysis were performed as described in SI Materials and Methods. (B and C) Concentration-dependent effect of stathmin on various tubulin polymers. Increasing concentrations of stathmin (0–14 μM) were incubated with tubulin polymers (2.0 μM). Samples were sedimented and the amount of depolymerized tubulin was measured as described above. Data represent the average of three independent experiments. Values are presented ± SD. (D) Analysis of the tubulin:stathmin stoichiometry for stathmin sedimenting with DL-rings. To determine the binding stoichiometry of stathmin to tubulin dimer in DL-rings, the amount of stathmin bound per sedimentable tubulin dimer at various stathmin concentrations was calculated. Data are the average of three independent experiments. Values are presented ± SD. These data show that stathmin binds to DL-rings with a 2:1 dimer:stathmin stoichiometry, similar to that seen with interactions between stathmin and tubulin dimers (3, 4).

Fig. 3.

Binding affinities of stathmin for tubulin polymers. (A) Binding of stathmin to Tx-MT and DL-rings. The binding of stathmin (2.0 μM) was measured as a function of tubulin polymer concentration (0–12 μM) by cosedimentation assay. The fraction of stathmin bound (in the pellet) was plotted against the concentration of unbound polymerized tubulin [calculated from the total polymerized tubulin assuming a 2:1 ratio (tubulin dimer:stathmin)] (SI Materials and Methods), and the data were fit to the bimolecular binding curve to obtain the apparent K_d. Data are an average of three independent experiments and error bars are ± SD. (B) Effects of full-length and ∆N-stathmin on depolymerization of Zn-sheets. Zn-sheet (2.0 μM) polymer was incubated with various concentrations of stathmin or ∆N-stathmin (0–14 μM) as indicated in Zn-Mes buffer containing 10 μM Taxol. Samples were sedimented and the amount of depolymerized tubulin was measured as described in Fig. 2. Data represent the average of three independent experiments ± SD. These data indicate that the N terminus of stathmin plays a significant role in its ability to depolymerize MTs. (C) Conceptual models for the catastrophe-promoting mechanism by which stathmin depolymerizes microtubules. The data in Figs. 2 and 3 suggest that stathmin can directly promote MT catastrophe by acting on laterally unbound protofilaments at MT tips and does so by some combination of the following three mechanisms (numbers in brackets refer to the figure above): [1], Binding of stathmin to protofilaments could inhibit lateral interactions between protofilaments by steric inhibition [1a] or by inducing curvature [1b] (4, 23, 24), thus destabilizing the tip and increasing the likelihood of catastrophe; [2], Stathmin could increase the GTPase of the tubulin subunits to which it is bound, promoting catastrophe by decreasing the size the GTP cap; [3], The N-terminal peptide of stathmin could promote protofilament severing by binding to the intradimer surface (Fig. 1) (28), promoting catastrophe by removing portions of the GTP cap. All three mechanisms would be expected to operate with equal effectiveness at both the plus and minus ends. A fourth mechanism would be unique to the minus end: [4], The N-terminal peptide of stathmin could bind to and cap the α-tubulin dimer exposed at the minus end, allowing stathmin to bind to the minus end with stronger affinity and preventing the incorporation of new tubulin dimers, thus providing a mechanism for the surprising asymmetry in stathmin’s ability to induce catastrophe. [5], In addition, it remains possible that binding of stathmin to the MT lattice could contribute to catastrophe, as previously suggested (6, 7). [6], Finally, an additional activity that is likely relevant in the cellular context is the demonstrated ability of stathmin to sequester tubulin dimers into the assembly incompetent T₂S complex. This activity reduces the amount of tubulin available for MT assembly, the overall polymer mass (–, –25), and therefore the likelihood that MTs exhibit persistent growth (29, 30).

Fig. 4.

Effect of stathmin on dynamic instability (DI) behavior in simulations of MT dynamics. Using our previously established computational model of MT dynamics (10), we tested the hypothesis that binding of stathmin to free protofilaments could account for stathmin’s observed effects on MT dynamics by examining how adding stathmin molecules with varied activities alter the behavior of the simulated MTs. (A) Effects observed when stathmin (1 μM free, activities as indicated) binds with moderate affinity (K_d = 1 μM) to laterally unbonded regions of PF. (B) Effects observed when stathmin (1 μM free, activities as indicated) binds with weak affinity (K_d = 25 μM) anywhere on the lattice. Examination of the data in A shows that binding of stathmin to PFs can dramatically increase catastrophe frequency, and that DI is more sensitive to effects of stathmin on lateral bond formation than on the hydrolysis rate or the dimer detachment rate. Comparison of A and B shows that weak binding of stathmin to the lattice is less effective than stronger binding of stathmin to PFs. (C) Possible mechanism for stathmin's activity asymmetry. Simulations were conducted as in A, except that in some cases stathmin was given a capping activity (simulated as decreased k_grow) or an increased affinity. These data show that the higher affinity and capping activity that are predicted to result from binding of the stathmin N terminus to α-tubulin increase stathmin’s catastrophe promoting ability, and thus could account for the experimental observation (7) that stathmin has stronger effects at the minus end than at the plus end. (D) Effect of 0.05 µM free stathmin on DI (this is the concentration expected when binding of stathmin (1 μM) to free tubulin dimers (10 μM) is taken into account; see SI Materials and Methods for calculations). These simulations assume that stathmin binds to laterally unbound PFs and that stathmin’s main activity is to prohibit lateral bond formation. These data show that stathmin molecules with affinity values similar to those measured in Fig. 3A can have strong effects on DI even in the presence of free tubulin. Data in all panels represent the average ± SD for three independent simulations of a single MT, each corresponding to more than 1 h of simulated time. The raw dynamic instability data corresponding to these bars graphs are provided in Table S1. F_cat, catastrophe frequency; F_res, rescue frequency; k_bond, rate constant for lateral bonding between tubular dimers; k_g, rate constant for the formation of the longitudinal bond between tubulin dimers; k_h, GTP hydrolysis rate constant; k_s, rate constant for breakage of the longitudinal bond between tubulin dimers; S_free, stathmin free; V_grow, macroscopically observed MT growth rate; V_short, macroscopically observed MT depolymerization rate.