A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

Proteins

Full length human MCAK-his6 and MCAK-his6-EGFP in wild type and mutated forms were expressed in Sf9 cells (Invitrogen, Carlsbad, CA, USA) and purified using nickel affinity chromatography as described previously (Helenius et al., 2006). MCAK concentrations are given as monomer concentrations. Porcine brain tubulin was purified as described previously (Castoldi & Popov, 2003). Single cycled, fluorescently labelled microtubules and double cycled microtubules were prepared as described previously (Patel et al., 2016). The concentration of microtubules is given as the concentration of polymerised tubulin.

Microtubule depolymerisation

Microtubule depolymerisation rates were determined by measuring the length of immobilised, GMPCPP-stabilised, rhodamine labelled, single cycled microtubules over time after the addition of 40 nM MCAK as described previously (Patel et al., 2016).

ATPase assays

ATPase rates in solution were measured using 3 µM MCAK and monitoring the production of ADP by HPLC as described previously (Friel, Bagshaw & Howard, 2011; Friel & Howard, 2011; Patel et al., 2016). For assays with tubulin or double-cycled microtubules 0.1 µM MCAK was used and the production of ADP was monitored by linking it to the oxidation of NADH (De La Cruz & Ostap, 2009). For both assays the change in concentration of ADP per second was divided by the concentration of MCAK to give the ATPase activity per second per motor domain.

Single molecule TIRF assays

Single molecules of MCAK-GFP were observed on immobilised, GMPCPP-stabilised, rhodamine labelled, single cycled, microtubules using TIRF microscopy as described previously (Patel et al., 2016). Kymographs for individual microtubules were used to measure the time individual MCAK-GFP molecules spent at the microtubule end and on the lattice.

ADP dissociation assays

The dissociation of mantADP from MCAK was measured as described previously (Patel et al., 2014), in solution or with the addition of 10 µM tubulin or 5.7 µM double cycled microtubules (chosen to have a comparable number of microtubule ends as the ATPase assay with microtubules). The fluorescence traces were fitted to a single exponential, or double exponential if required, with an additional linear function to account for the photobleaching of mant.

The phosphomimic mutant T537E reduces depolymerisation activity and microtubule-stimulated ATPase activity

Firstly, we confirmed the effect of the substitution T537E on MCAK’s depolymerisation activity in vitro. It has been shown previously at high concentration (500 nM) that the mutation T537E decreases depolymerisation activity 4-fold relative to wild-type MCAK (Sanhaji et al., 2010). We measured depolymerisation activity at 40 nM, the concentration at which the fastest microtubule depolymerisation for wild-type MCAK is observed (Helenius et al., 2006). Under these conditions the phosphomimic mutant had a depolymerisation rate 50-fold slower than the wild-type (0.06 ± 0.06 µm min⁻¹ and 3.04 ± 0.53 µm min⁻¹ (mean ± standard deviation), respectively) (Fig. 1B). Thus, confirming that the phosphomimic substitution dramatically decreases MCAK’s depolymerisation activity.

We next measured the ATPase activity of T537E in the absence of tubulin, in the presence of unpolymerized tubulin and in the presence of microtubules. The ATPase rate of the T537E mutant in solution was not significantly different to wild type (5.33 ± 0.33 × 10⁻³s⁻¹ compared with 4.47 × 10⁻³ ± 2.60 × 10⁻³s⁻¹, p = 0.6002) (Fig. 1C). Similarly, the ATPase in the presence of unpolymerized tubulin was not significantly affected by the phosphomimic mutation: 0.194 ± 0.055 s⁻¹ compared with 0.299 ± 0.047 s⁻¹ for wild-type MCAK, p = 0.0658 (Fig. 1C). These data indicate that the phosphomimic mutant folds correctly as it turns over ATP at a similar rate to wild-type both in solution and in the presence of unpolymerized tubulin. These data also suggest that T537E can still interact with unpolymerized tubulin, as its ATPase rate is accelerated by unpolymerized tubulin to the same degree as wild type. By contrast, there is a dramatic difference in the microtubule-stimulated ATPase activity of T537E relative to wild-type MCAK. The microtubule-stimulated ATPase rate of T537E is 14-fold slower than wild type MCAK: 0.335 ± 0.081 s⁻¹ and 4.75 ± 0.055 s⁻¹, respectively (Fig. 1C). The ATPase rate for T537E was in fact similar to the ATPase for both wild-type and T537E in the presence of unpolymerized tubulin. The difference between the ATPase rates in the presence of unpolymerized tubulin compared to the presence of microtubules for wild-type MCAK has previously been shown to be due to the acceleration of ADP dissociation caused specifically by microtubule ends (Friel & Howard, 2011). Therefore, the reduction in the ATPase of T537E to a similar level to that in the presence of unpolymerized tubulin suggests this mutation may have specifically impaired MCAK’s ability to recognise the microtubule end.

The mutation T537E abolishes long microtubule end residence events

The ATPase activity of wild-type MCAK is maximally accelerated by microtubule ends (Friel & Howard, 2011; Hunter et al., 2003). The residue T537 is near the α4 helix of the MCAK motor domain (Fig. 1A). Residues in the α4 helix are critical to MCAK’s ability to recognise the microtubule end (Patel et al., 2016). This proximity to the α4 helix together with the observation that the mutation T537E specifically impairs the microtubule stimulated ATPase of MCAK suggests that this phosphomimic mutation may also interfere with MCAK’s ability to distinguish the microtubule end from the lattice. We used TIRF microscopy to observe the interaction of single molecules of MCAK-GFP and T537E-GFP with microtubules. Wild type MCAK makes short diffusive interaction with the microtubule lattice. However, when it reaches the microtubule end ADP dissociation is accelerated, leading to nucleotide exchange and ATP. MCAK binds tightly and displays longer microtubule end residence events (Fig. 2A). The MCAK mutant T537E displays similar short diffusive interactions with the microtubule lattice but, by contrast with the wild-type, does not show long end binding events (Fig. 2B). Quantification of microtubule end residence times for wild-type MCAK and T537E shows that 32% of molecules for the wild type but only 0.4% of molecules for T537E stayed at the microtubule end for longer than 2 s (Figs. 2C and 2D). The lattice residence time for T537E is unchanged relative to wild type (0.42 ± 0.01 s and 0.48 ± 0.02 s, respectively) and neither the association or dissociation rates are significantly different (Fig. S1 and Table S1). These data indicate that the affinity for the microtubule lattice is not significantly changed for this mutant. By contrast, the end residence time for T537E was decreased to 0.64 ± 0.02 s compared to 2.03 ± 0.13 s for the wild type. In agreement with this, the rate of dissociation from the microtubule end is increased relative to wild-type (Table S1). To investigate the possibility that the mutation had impaired MCAK’s ability to reach the microtubule end, we calculated the number of end interaction events per unit time for individual microtubules. This gave values of 0.71 ± 0.29 nM⁻¹ s⁻¹ and 0.97 ± 0.27 nM⁻¹ s⁻¹ for wild-type and T537E, respectively (Table 1). These data indicate that a similar number of MCAK molecules reach the microtubule end for both wild-type and mutant and that the phosphomimic mutation does not impair MCAK’s ability to reach the microtubule end. Rather, in the case of T537E, the average time a molecule remains at the microtubule end is significantly reduced. Taken together, these data imply that the molecular mechanism underlying the attenuation of depolymerisation activity due to this phosphomimic mutation is loss of ability to distinguish between the microtubule end and the microtubule lattice.

The microtubule end does not accelerate ADP dissociation from T537E

ATP turnover by MCAK is maximally accelerated by the microtubule end due to microtubule end specific acceleration of ADP dissociation. To test whether the difference in T537E interaction with the microtubule end had affected the ability of the microtubule end to accelerate ADP dissociation, we measured the rate of dissociation of ADP tagged with the small fluorophore mant (mantADP). In solution and in the presence of unpolymerised tubulin, the rate constant for ADP dissociation was not significantly affected by this mutation (WT 0.102 ± 0.013 s⁻¹, T537E 0.114 ± 0.013 s⁻¹, WT + Tb 0.114 ± 0.024 s⁻¹, T537E + Tb 0.120 ± 0.019 s⁻¹) (Table 1 and Fig. 2E). This is in agreement with the ATPase activities under these conditions which are not significantly changed.

In the presence of microtubules, the change in fluorescence associated with mantADP dissociation from wild type MCAK is best described by a double exponential function. The two phases can be explained as a microtubule-end stimulated fast phase and a slower phase corresponding to molecules which do not encounter the microtubule end (i.e. remain in solution, in contact with unpolymerised tubulin or in contact with the microtubule lattice over the course of the experiment). The fast phase of mantADP dissociation from wild type MCAK measured here has a rate constant of 4.11 ± 0.41 s⁻¹ and the slower phase a rate constant of 0.341 ± 0.051 s⁻¹. The rate constant for the slower phase is in close agreement with the rate constant for mantADP dissociation from MCAK in solution or in the presence of unpolymerized tubulin. This is in agreement with this phase representing mantADP dissociation from molecules which do not contact the microtubule end. By contrast with WT-MCAK, the change in fluorescence associated with mantADP dissociation from T537E was well described by a single exponential function with a rate constant of 0.369 ± 0.090 s⁻¹. The faster phase was lost for the phosphomimic mutant indicating that the microtubule end cannot accelerate mantADP dissociation from T537E. In fact, the rate constant for the single phase observed for mantADP dissociation from T537E was not significantly different from the slower phase of mantADP dissociation from wild type MCAK (p = 0.6679, Fig. 2F and Table 1). Further, the microtubule-stimulated mADP dissociation rate constant is also similar to the rate constant in solution and in the presence of unpolymerized tubulin for both wild-type MCAK and T537E. Therefore, despite single molecule TIRF data showing that MCAK T537E can reach the microtubule end (Fig. 2B and Table 1), the kinetics of ADP dissociation for this mutant are consistent with the kinetics of ADP dissociation for wild-type MCAK when in solution, in the presence of unpolymerized tubulin or on the microtubule lattice. These data suggest that the phosphomimic mutant T537E has lost the ability to distinguish between the microtubule lattice and the microtubule end.