MCAK preferentially binds to growing microtubule ends

(A) The representative kymographs of single-molecule GFP-MCAK (green) binding events on dynamic microtubules (red, tubulin: 16 μM) growing from GMPCPP microtubule seeds (blue) in the presence of 1 mM ATP, AMPPNP, ADP and APO (the nucleotide-free state). The binding events at the plus-end and lattice were indicated by a white arrowhead and a blue arrowhead, respectively. Vertical bar: 5 s; horizontal bar: 2 μm.

(B) The plot showing the representative intensity profiles of a single GFP-MCAK molecule (green) and a growing microtubule end (red). The peak position of a GFP-MCAK molecule was recorded as the binding location (dashed line in green).

(C) The spatial distributions of the binding sites of GFP-MCAK (green, n=152 events) along the long axis of microtubules. The averaged intensity profile of the growing microtubule ends (red) was shown as the positional reference.

(D) Statistical quantification of the apparent association constant (kon) of GFP-MCAK on the plus-end and lattice of dynamic microtubules in the presence of 1 mM ATP (n=66 microtubules from 3 assays), AMPPNP (n=29 microtubules from 2 assays), ADP (n=21 microtubules from 2 assays) and APO (n=36 microtubules from 3 assays). The statistical comparison was made versus the kon of MCAK on the corresponding location (i.e. plus end or lattice) in the ATP condition.

(E) Statistical quantification of RE/L in the presence of ATP, AMPPNP, ADP and APO. The statistical comparison was made versus the RE/L of the plus-end binding events in the ATP condition.

(F) Statistical quantification of the dwell time of GFP-MCAK on the growing end and lattice of dynamic microtubules in the presence of ATP (n=966 events from 3 assays for the plus end; n=702 events from 3 assays for lattice), AMPPNP (n=231 events from 2 assays for the plus end; n=142 events from 2 assays for lattice), ADP (n=327 events from 2 assays for the plus end; n=1184 events from 2 assays for lattice) and APO (n=71 events from 3 assays for the plus end; n=573 events from 3 assays for lattice). The statistical comparison was made versus dwell time of the binding events on the corresponding location (i.e. plus end or lattice) in the ATP condition.

(G) The mean-squared displacement (MSD) of GFP-MCAK was plotted against the time interval (t, 0.1s per frame). The diffusion coefficient (D) of 0. 023 μm2 s-1 was obtained using a linear fitting (<x2>= 2Dt). The error bar represented SEM (n=203 trajectories).

In panel D, E and F, all the data were presented as mean ± SEM. All the comparisons were performed using the two-tailed Mann-Whitney U test with Bonferroni correction, n.s., no significance; ***, p<0.001.

MCAK binds to the entire GTP cap of growing microtubule ends

(A) The representative kymograph showing the individual binding events of GFP-MCAK (1 nM, with 1 mM ATP, left), EB1-GFP (10 nM, middle) or XMAP215-GFP (1 nM, right) at growing microtubule ends (red, tubulin: 12 μM). The plots showed the representative intensity profiles of a single molecule (green) and a growing microtubule end (red). The peak position of individual molecules was determined using a Gaussian fit (dashed line in green). Vertical bar: 5 s; horizontal bar: 2 μm.

(B) The spatial distributions of the binding sites of GFP-MCAK (green, n=123 events), EB1-GFP (black, n=184 events) and XMAP215-GFP (blue, 142 events) along the long axis of microtubules. The averaged intensity profile of the growing microtubule ends (red) was shown as the positional reference. The region within the FWHM of the blue curve was considered to be the binding region of XMAP215. The MCAK molecules localized to the left of this region was considered to be proximally distributed (grey bar). The region within the FWHM of the black curve was considered to be the binding region of EB1. The MCAK molecules localized to the right of this region was considered to be distally distributed (orange bar).

(C) The averaged intensity profiles of the growing microtubule ends were used to quantify the localization of GFP-MCAK (green, 123 microtubules), EB1-GFP (black, 184 microtubules) and XMAP215-GFP (blue, 142 microtubules). Note that the morphologies of microtubule ends in three conditions were nearly identical. In panels B and C, the red arrows indicated the direction of microtubule growth.

MCAK strongly binds to GTPγS microtubules in a nucleotide-independent manner

(A) The representative projection images of GFP-MCAK binding to GTPγS (red arrowhead), GDP (purple arrowhead) and GMPCPP microtubules (cyan arrowhead) in the presence of 1 mM AMPPNP (left), 1 mM ATP (left middle), 1 mM ADP (right middle) and at the APO state (right). Note that the binding on GDP or GMPCPP microtubules was compared to that on GTPgS microtubules in the same flow cell. Scale bar: 5 μm.

(B) Statistical comparison of the normalized fluorescence intensity of GFP-MCAK on different microtubules in the presence of 1 mM AMPPNP (112 GTPgS microtubules, 56 GMPCPP microtubules, 56 GDP microtubules from 3 assays), 1 mM ATP (88 GTPgS microtubules, 60 GMPCPP microtubules, 28 GDP microtubules from 3 assays), 1 mM ADP (95 GTPgS microtubules, 51 GMPCPP microtubules, 44 GDP microtubules from 3 assays) or at the APO state (66 GTPgS microtubules, 30 GMP-CPP microtubules, 36 GDP microtubules from 3 assays). All data were normalized to the binding intensity of GFP-MCAK (1 nM) on GTPgS microtubules in the AMPPNP condition.

(C) The ratios of the binding intensity of GFP-MCAK on GTPgS microtubules to that on GDP or GMPCPP microtubules in various nucleotide conditions. The dashed line on the plot represented 1. The statistical comparisons were performed between the ratios and 1.

In panel B and C, All the data were presented as mean ± std. All the comparisons were performed using the two-tailed Mann–Whitney U test with Bonferroni correction, ***, p<0.001.

MCAK strongly binds to the ends of GMPCPP microtubules in a nucleotide state-dependent manner

(A) The representative projection images and intensity profiles showing GFP-MCAK binding (upper) at the ends of GMPCPP microtubules (lower) in the presence of 1 mM AMPPNP (left), 1 mM ATP (left middle), 1 mM ADP (right middle) or at the APO state (right). Red arrowhead: the end-binding of MCAK. Red bar: ends. Black bar: lattice. Scale bar=2 μm.

(B) Statistical quantification of the binding intensity of GFP-MCAK on the lattice and end of GMPCPP microtubules in the presence of 1 mM AMPPNP (47 microtubules from 3 assays), 1 mM ATP (40 microtubules from 3 assays), 1 mM ADP (45 microtubules from 3 assays) or at the APO state (52 microtubules from 3 assays). All data were normalized to the binding of GFP-MCAK (1 nM) on GTPgS microtubules in the AMPPNP condition. All the data were presented as mean ± std. The statistical analysis was performed using the two-tailed pair t-test by Bonferroni correction, ***, p<0.001.

(C) The ratios between the binding intensity of GFP-MCAK at the end and the intensity on the lattice of GMPCPP microtubules in various nucleotide conditions. The ratios were presented as mean ± std. The dashed line on the plot represents 1. The statistical comparisons were performed between the ratios and 1 using the two-tailed Mann-Whitney U test with Bonferroni correction, ***, p<0.001.

The preference for GDP·Pi-tubulins facilitates the binding preference of MCAK for growing microtubule ends

(A) The structural model of the MCAKsN+M-tubulin complex (PDB ID: 5MIO). The cartoon schematic in upper panel showed the domain organization of MCAK. Loop2 and α4 helix, two regions mediating the interaction between MCAK and tubulin, were enlarged and the key sites (K524, V298) were highlighted in red. The corresponding residues of tubulin that may interact with K524 and V298 were highlighted in green.

(B) The representative projection images of GFP-MCAKK524A and GFP-MCAKV298S binding to GTPγS (red arrowhead), GDP (purple arrowhead) and GMPCPP microtubules (cyan arrowhead) in the presence of 1 mM AMPPNP. Scale bar: 5 μm.

(C) Statistical quantification of the binding intensity of GFP-MCAKK524A (144 GTPgS microtubules, 72 GMPCPP microtubules, 62 GDP microtubules from 3 assays) and GFP-MCAKV298S (124 GTPgS microtubules, 65 GMPCPP microtubules, 59 GDP microtubules from 3 assays) on different microtubules. Note that all data were normalized to the binding intensity of GFP-MCAK on GTPgS microtubules in the AMPPNP condition. The data were presented as mean ± std.

(D) The ratios of the binding intensity of GFP-MCAKK524A or GFP-MCAKV298S on GTPgS microtubules to that on GDP or GMPCPP microtubules in the presence of 1 mM AMPPNP. Purple dashed line: The GTPgS/GDP ratio of GFP-MCAK. The cyan dashed line: the GTPgS/GMPCPP ratio of GFP-MCAK. The data were presented as mean ± std. The statistical comparisons were made versus the corresponding value of GFP-MCAK.

(E) The representative projection images and intensity profiles showing the binding of GFP-MCAKK524A and GFP-MCAKV298S to the end (red arrowhead in the upper panels) and lattice of GMPCPP microtubules (lower panels) in the presence of AMPPNP. Red bar: ends. Black bar: lattice. Scale bar=2 μm.

(F) Statistical quantification of the binding intensity of GFP-MCAKK524A (88 microtubules from 3 assays) and GFP-MCAKV298S (68 microtubules from 3 assays) on the lattice and end of GMPCPP microtubules in the presence of AMPPNP (lower panel). All the data were normalized to the binding of GFP-MCAK (1 nM) on GTPgS microtubules in the AMPPNP condition. All the binding intensity data were presented as mean ± std. The statistical analyses were performed using the two-tailed paired t-test by Bonferroni correction, n.s., no significance; ***, p<0.001. The inset (upper) showing the ratios of the end-binding intensity of GFP-MCAKK524A or GFP-MCAKV298S to the lattice-binding intensity on GMPCPP microtubules in the presence of AMPPNP. The ratios were presented as mean ± std. The statistical comparisons were made versus the ratio of GFP-MCAK (the dashed line shown in Fig. 4C).

(G) The representative kymographs of the single-molecule binding events of GFP-MCAKK524A (10 nM, green) and GFP-MCAKV298S (30 nM, green) on growing microtubules (red, tubulin: 16 μM) in the presence of 1 mM ATP. The end-binding events were indicated using white arrowheads and enlarged. For the original kymo-graphs, vertical bar: 2 s; horizontal bar: 2 μm. For the enlarged kymographs, vertical bar: 1 s; horizontal bar: 0.5 μm.

(H) Statistical quantification of the apparent association constant (kon-P) of GFP-MCAKK524A (33 microtubules from 3 assays) and GFP-MCAKV298S (23 microtubules from 2 assays) on growing microtubule ends in the presence of ATP (lower). The statistical comparisons were made versus the kon-P of GFP-MCAK. The inset (upper) showing the statistical quantification of RE/L of GFP-MCAKK524A and GFP-MCAKV298S. The RE/L of both mutants were compared to that of GFP-MCAK in the ATP condition. All the data were presented as mean ± SEM.

(I) Statistical quantification of the dwell time of GFP-MCAKK524A (19 binding events from 3 assays) and GFP-MCAKV298S (43 binding events from 3 assays) on growing microtubule ends in the presence of ATP. The dwell time were presented as mean ± SEM. The dwell time of GFP-MCAKK524A and GFP-MCAKV298S were compared to that of GFP-MCAK.

In panel C, D, F (the upper inset), H and I, all the comparisons were performed using the two-tailed Mann-Whitney U test with Bonferroni correction, n.s., no significance; *, p<0.05; **, p<0.01; ***, p<0.001.

Functional specification of MCAK and XMAP215 at growing microtubule ends

(A) The representative kymographs of dynamic microtubules (tubulin: 10 μM) in the control condition or in the presence of MCAK, XMAP215 or both. Vertical bar: 100 s; horizontal bar: 2 μm.

(B) The probability distribution of microtubule lifetime in the experimental conditions indicated in the panel A. The lines were gamma fitting curves. Control: 443 microtubules from 7 assays. 20 nM MCAK: 621 microtubules from 7 assays. 50 nM XMAP215: 237 microtubules from 3 assays. 20 nM MCAK+50 nM XMAP215: 283 microtubules from 3 assays.

(C) The plots of catastrophe frequency versus the lifetime of microtubules showing how the likelihood of catastrophe depended on the age of microtubules. The data were from the experiments shown in panel A.

Cartoon schematics depicting the working model of MCAK at growing microtubule-ends

(A) A hypothetic model for the binding cycle of MCAK, given its binding preference on the EB cap. T: MCAK‧ATP. D: MCAK‧ADP.

(B) The co-operation schematics of MCAK, EB1 and XMAP215 at growing microtubule ends. Pathway 1: the direct binding of MCAK to microtubule ends. Pathway 2: the indirect binding of MCAK to microtubule ends via EB1.

Single-molecule fluorescence analysis and the apparent off-rates (koff) of GFP-MCAK

(A) The fluorescence intensity distribution of GFP-MCAK (red, n=509 binding events) was fitted using a Gaussian function. The background intensity was subtracted. The intensity range (μ ± 2σ=300 ± 145 A.U.) was used to determine if a fluorescence spot represents a single dimer.

(B) The apparent off-rate (koff) of GFP-MCAK on growing microtubule ends in the presence of ATP (minus end, square; plus end, circle), AMPPNP (down triangle), ADP (up triangle) and APO (diamond). koff was calculated by fitting the dwell time of individual GFP-MCAK binding events to a single exponential function.

Projection of single-molecule fluorescence images

The representative images showing how to generate a summation image using 1000 frames of raw images. The GDP and GTPγS microtubule were indicated using green and yellow arrowheads, respectively. The concentration of GFP-MCAK here was 1 nM. The experiment was performed in the presence of 1 mM AMPPNP. Scale bar: 5 μm.

GFP-MCAKK525A and GFP-MCAKV298S are depolymerizing-deficient mutants

(A) The representative kymographs showing the depolymerizing effect of GFP-MCAK, GFP-MCAKK525A and GFP-MCAKV298S on GMPCPP-stabilized microtubules in the presence of 1 mM ATP. Vertical bar: 100 s, horizontal bar: 2 μm.

(B) Statistical quantification of the depolymerization rates of GFP-MCAK (n=21 microtubules from 3 assays), GFP-MCAKK525A (n=11 microtubules from 2 assays) and GFP-MCAKV298S (n=52 microtubules from 3 assays). The data were presented as mean ± std. The statistical analysis was performed using the two-tailed Mann-Whitney U test with Bonferroni correction, ***, p<0.001.

Structural models showing the kinesin-13-tubulin contact site and the TOG-tubulin contact site

The structural model showing that the TOG domain (PDB ID: 4FFB) and the motor domain of MCAK (PDB ID: 6BBN) shared, to some extent, the binding site on a tubulin dimer.