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Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips

Nature Cell Biology volume 15, pages 1079–1088 (2013)Cite this article

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Abstract

During vertebrate mitosis, the centromere-associated kinesin CENP-E (centromere protein E) transports misaligned chromosomes to the plus ends of spindle microtubules. Subsequently, the kinetochores that form at the centromeres establish stable associations with microtubule ends, which assemble and disassemble dynamically. Here we provide evidence that after chromosomes have congressed and bi-oriented, the CENP-E motor continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends. Using a combination of single-molecule approaches and laser trapping in vitro, we demonstrate that once reaching microtubule ends, CENP-E converts from a lateral transporter into a microtubule tip-tracker that maintains association with both assembling and disassembling microtubule tips. Computational modelling of this behaviour supports our proposal that CENP-E tip-tracks bi-directionally through a tethered motor mechanism, which relies on both the motor and tail domains of CENP-E. Our results provide a molecular framework for the contribution of CENP-E to the stability of attachments between kinetochores and dynamic microtubule ends.

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Figure 1: Loss of chromosome alignment in metaphase cells with inhibited CENP-E kinesin.
Figure 2: Motility of single molecules of CENP-E on microtubule wall.
Figure 3: Single molecules of CENP-E visualized at dynamic microtubule tips.
Figure 4: Motion of bead cargo in association with microtubule disassembly.
Figure 5: Investigation of the role of CENP-E in depolymerization-driven chromosome motions in vivo.
Figure 6: Analysis of possible mechanisms of CENP-E tip-tracking.
Figure 7: Mathematical model of CENP-E motility and comparison with the results of the in vitro study.
Figure 8: Tethered motor mechanism for processive and bi-directional microtubule tip-tracking and the testing of its predictions.

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Acknowledgements

We thank members of the Grishchuk and Ataullakhanov laboratories for stimulating discussions and technical assistance, A. Korbalev for help with the model videos, M. Porter for a kind gift of axonemes, M. Ostap, M. Lampson and J. R. McIntosh for critical reading of the manuscript, and E. Ballister and M. Lampson for assistance with live cell imaging. We thank University of California, San Diego, Neuroscience Microscopy Shared Facility (P30 NS047101) and J. R. McIntosh for help with the initial phase of this work (GM 033787). This work has been supported by grants from the NIH to E.L.G. (R01-GM098389) and to D.W.C. (R01-GM29513), and grants to F.I.A from Russian Fund for Basic Research (12-04-00111-a, 13-04-40188-H and RFBR 13-04-40190-H) and Presidium of Russian Academy of Sciences (Mechanisms of the Molecular Systems Integration and Molecular and Cell Biology programmes). B.V. has been supported by a postdoctoral fellowship from the Human Frontiers Science Program. E.L.G. is a Kimmel Scholar; her work is supported in part by the Pennsylvania Muscle Institute. D.W.C. receives salary support from the Ludwig Institute for Cancer Research.

Author information

Author notes

  1. Yumi Kim

    Present address: Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94704, USA,

  2. Nikita Gudimchuk and Benjamin Vitre: These authors contributed equally to this work

Authors and Affiliations

  1. Physiology Department, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    Nikita Gudimchuk, Anatoly Kiyatkin & Ekaterina L. Grishchuk

  2. Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA

    Benjamin Vitre, Yumi Kim & Don W. Cleveland

  3. Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow 119991, Russian Federation

    Fazly I. Ataullakhanov

  4. Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow 117513, Russian Federation

    Fazly I. Ataullakhanov

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Contributions

N.G. performed in vitro experiments and mathematical modelling; B.V. and A.K. performed in vivo experiments; B.V. and Y.K. purified proteins; N.G., B.V., F.I.A., D.W.C. and E.L.G. designed research, analysed data and wrote the paper.

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Correspondence to Ekaterina L. Grishchuk.

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Integrated supplementary information

Supplementary Figure 1 Motility of single CENP-E molecules on stable microtubules in TIRF and bead assays.

(a) Representative kymographs of GFP-labeled CENP-E molecules (1-4 nM) on Taxol-stabilized microtubules. The majority of TR CENP-E molecules that land on microtubules show unidirectional and processive motility. full-length CENP-E molecules associate with microtubules on average for a much shorter time (compare with kymographs from scans in the same chambers but away from microtubules on the left panels). The unidirectionally walking molecules (red arrows), as well as the diffusing ones (yellow), are also readily observed. (b) Mean squared displacement (MSD) of diffusing full-length CENP-E molecules as a function of time, bars are SEM. Red line is a linear fit, which corresponds to the diffusion coefficient 0.16±0.01 μm2/sec (Mean±SEM) (c,d) Histograms of the velocity and residence time of different CENP-E proteins, as determined in TIRF assay. “All full-length CENP-E” correspond to data from all visible events, including short-lived interactions, diffusion and unidirectional walking. (e) Schematics of the bead motility assay. Red cones symbolize a focused trapping laser beam. (f) Example distance vs. time curves for CENP-E-coated beads moving on Taxol-stabilized microtubules; some beads coated with full-length CENP-E showed diffusive motions, similar to behavior of bead-free full-length CENP-E molecules, see panel a. (g) Fraction of motile beads as a function of their binned integral GFP brightness. Data are Mean±SEM, based on 20 independent experiments with TR CENP-E (n = 360 beads), and 4 independent experiments with full-length CENP-E (n = 63 beads). Lines are fits to Poisson distribution. (h) Activity of full-length CENP-E relative to TR CENP-E in different assays (see Materials and Methods). Data are reported as Mean±SEM; n = 24 independent experiments for bead assay, n = 5 independent experiments for TIRF assay. Higher relative activity in the bead assay implies that full-length CENP-E is more active when it carries a bead cargo than when it is bead-free.

Supplementary Figure 2 Transient association between TR CENP-E and the microtubule tips.

Typical kymographs of TR CENP-E on microtubules, observed with two-color TIRF. microtubules are in red: HiLyte647 or dimly labeled Rhodamine tubulin was used. GFP-labeled motors are green. In some images the microtubule nucleating seeds are also green because they were grown using larger concentration of Rhodamine labeled tubulin, and excitation and imaging were carried out differently than with the HiLyte647 tubulin, see Materials and Methods. (a) TR CENP-E motor reaches and pauses shortly at the stable microtubule tip; under the same assay conditions the truncated kinesin-1 (K560-GFP) falls off without pausing, see text and Table S2 for more details. (b) TR CENP-E reaches and detaches quickly from the ends of depolymerizing microtubules. (c) TR CENP-E reaches and detaches quickly from the ends of polymerizing microtubules. In many of these kymographs the slope of CENP-E-GFP lines is less on red than on green microtubule segments, indicating that this motor walks slower on the GMPCPP-containing lattice (seeds) than on the GDP-containing microtubule walls (dynamic microtubule extensions).

Supplementary Figure 3 Lasting association between full-length CENP-E and the dynamic microtubule tips.

(a) The two-color TIRF microscopy image shows dynamic microtubule polymers (red) grown from the coverslip-attached seeds (in green, asterisks) in the presence of 4 nM GFP-labeled full-length CENP-E (green). Decoration of the plus tips is evident (arrows). (b) Example kymographs of full-length CENP-E complexes tracking the dynamic microtubule tips. The first panel shows a robust tracking by a bright dot, which contained 3-4 dimeric molecules (see also Supplementary Video S7). All other images are for 1-2 dimeric motors. Last two panels correspond to Supplementary Videos S5 and S4, respectively. (c) full-length CENP-E velocity on the GMPCPP-containing microtubules is slightly slower than on the GDP-microtubules made with Taxol (unpaired t-test, n = 18 molecules for each group). Data are Mean±SEM (d) Kymographs showing the excursions of full-length CENP-E away from the microtubule tip during the tracking. (e) A kymograph of a TR CENP-E motor molecule moving on Taxol-stabilized microtubule. The intensity of the moving dot changes in two steps, as seen from data in panel f. (f) Integral intensity (black line) of the moving TR CENP-E molecule shown in panel e. This curve was generated by smoothing the raw intensity data with a sliding average with window containing 7 data points, then subtracting the background. Red lines mark the intensity of 1 and 2 GFP molecules, as identified with the histogram on panel g. (g) Histogram of the intensity of GFP-labeled motors generated from the linescans, such as shown in panel f, for n = 39 TR CENP-E molecules. Distance between two adjacent peaks (arrows) corresponds to the intensity of a single GFP molecule, see Materials and Methods.

Supplementary Figure 4 Quantification of the motility of full-length CENP-E at dynamic microtubule tips.

(a, b) Histograms of the association time between full-length CENP-E and the polymerizing (P, n = 45) and depolymerizing (D, n = 41) microtubule tips, respectively. (c, d) Histograms of the distances traveled by full-length CENP-E with the P and D-tip, respectively. Errors in all panels represent SEM. Data from 8 independent experiments.

Supplementary Figure 5 microtubule stability in monopolar spindles treated with different inhibitors.

Representative images of HeLa cells treated as indicated, see Fig. 5b and main text for details. Numbers in yellow indicate time in seconds after the start of imaging. Red bar indicates the addition of drugs 20 sec after the start of imaging. The following number of cells were analyzed for each conditions; 50 cells for DMS0 treated cell (control), 45 for CENP-E inhibitor treated cells, 50 for nocodazole treated cells and 40 cells were treated with both nocodazole and CENP-E inhibitor. Images were collected from 2 independent experiments. Nocodazole was used at 10 μM and CENP-E inhibitor (GSK-923295) at 200 nM.

Supplementary Figure 6 microtubule destabilization assay in cells with monopolar spindles.

(a) Representative immunofluorescence images of monastrol-arrested HeLa cells with or without nocodazole treatment, which causes microtubule depolymerization. Prior to nocodazole application CENP-E was depleted from the kinetochores by adding GSK-923295. This removal occurs in the microtubule-dependent manner, consistent with induction of rigor CENP-E binding to kinetochore microtubules and its subsequent passive transport to the spindle poles via poleward microtubule flux. DNA was visualized with H2B mCherry (white, left column), CENP-E was stained using HPX-1 anti CENP-E antibody (red), kinetochores were stained with anti-centromere antibodies (ACA, in green), and microtubules were stained with anti-tubulin antibodies (DM1a, white, second from the right column). Merged images show a combination of ACA, CENP-E-GFP and tubulin signals. Scale bar here and on other panels is 5 μm. (b) Box and whiskers diagram shows total monopolar spindle tubulin intensity in the presence or absence of CENP-E inhibitor without nocodazole (no microtubule depolymerization); data are based on 90 cells for the control and 83 cells for the experimental condition (+ CENP-E inhibitor) acquired from 3 independent experiments. The two-tailed unpaired student t test indicates no significant differences between the two conditions. Here and in panels below, the lower bars in these boxes represent the first quartile values, the second and third quartile are represented by the plain box separated by the median (horizontal bar) and the value for fourth quartile is represented by the upper bars. (c) Box and whiskers diagram showing diameter of the monopolar spindles in the presence or absence of CENP-E inhibitor without nocodazole. Data are based on 90 cells for the control and 83 cells for the experimental condition (+CENP-E inhibitor) from 3 independent experiments. The two-tailed unpaired student t test indicates significant differences between the two conditions: ***p<0.001. (d) Analogous quantification as in panel b but for cells in the presence or absence of CENP-E inhibitor 20 min after addition of nocodazole. Data are based on 95 cells for the control and 93 cells for the experimental condition (+CENP-E inhibitor) from 3 independent experiments. The two-tailed unpaired student t test indicates significant differences between the two conditions. ***p<0.001.

Supplementary Figure 7 Reduced stability of kinetochore-microtubule attachments in two cell lines following CENP-E depletion from the kinetochores.

(a) Timeline of the microtubule destabilization assay with monopolar spindles and representative images of fixed HeLa cells. Insets show two-color enlargements of the kinetochores on peripheral chromosomes (same images as in Fig. 5 panel e). See legend to Fig. 5 for details. Scale bar, 5 μm. (b) Schematics of the timing of RNAi and drug treatments (see Materials and Methods) and representative images of DLD-1 cells depleted of endogenous CENP-E and rescued (or not) with a GFP-tagged full-length CENP-E. Insets show two-color enlarged images of the chromosomes (green) and microtubules (white) at the periphery of the chromosome sphere. Similar results were obtained when CENP-E was depleted from the kinetochores with GSK-923295 or via RNAs approach.

Supplementary Table 1 Interactions between CENP-E and microtubule lattice, as determined with single molecule TIRF and bead assays. Here and in other tables the numbers are Mean±SEM, N, where N is the total number of observed events. For the beads assay only data from the microscopy chambers in which no more than 30% beads were motile are included. When bead motility is that infrequent, more than 80% of moving beads are likely to be driven by single motor molecules53. The velocity of single truncated CENP-E molecules reported here is consistent with findings in28,30, but it is faster than in6 due to the differences in assay conditions.
Full size table
Supplementary Table 2 Interactions between CENP-E and microtubule tips. Data reported here were obtained with complexes containing 1-2 CENP-E dimers. The association time with stable tips was examined using Taxol-stabilized microtubules. The association time and travel distance with polymerizing tips were determined from single exponential fits of the respective histograms (Supplementary Fig. S4 panels a,c, red curves). The association time and travel distance with the depolymerizing tips (Supplementary Fig. S4 panels b,d) and the association times with stable microtubule tips are reported as mean values. Time and distance of full-length CENP-E motility with microtubule disassembly are likely to be underestimated because they were frequently limited by a short microtubule length. NA– not applicable.
Full size table
Supplementary Table 3 Model parameters. Our mathematical model contains 11 independent parameters: 5 kinetic parameters to describe the rate of the transitions between different states, as illustrated in Fig. 7a,b, and 6 mechanical parameters. For further details about specifying the values of model parameters and model calibration see Mathematical Model of CENP-E Motility in Supplementary Note.
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Loss of chromosome alignment in cells with inhibited CENP-E.

HeLa cells expressing Tubulin–EYFP and Histone H2B–RFP (natural colours are inverted in this video for better visualization) were released from the metaphase arrest in the presence (upper panel) or absence (lower panel) of the CENP-E inhibitor GSK-923295 at the beginning of these sequences, played at 4 fps, 480x faster than recorded. In bi-polar spindles with rigor-locked CENP-E, several chromosomes lose their alignment and become stuck at the poles, leading to a prolonged cell cycle arrest. (MOV 555 kb)

Truncated CENP-E carries a bead along Taxol-stabilized microtubules.

At the start of this sequence, a 0.5 μm bead coated with TR CENP-E is held with a weak optical trap near a coverslip-attached Taxol-stabilized microtubule. A piezo-stage is then repositioned to promote the bead’s contact with the microtubule. When the bead starts walking, the trap is turned off to record the velocity and run length of the bead motions. In such assays, the TR-coated beads moved similarly to the beads coated with FL CENP-E, demonstrating that the non-motor domains have little effect on CENP-E traditional transport along a microtubule wall. Video was recorded in DIC at 7 fps and 100 millisec exposure, and played 2x faster than recorded. Scale bar, 2 μm. (MOV 1066 kb)

Truncated CENP-E motors moving on a dynamic microtubule.

Two TR CENP-E dimers sequentially land on the microtubule lattice and quickly walk to its growing plus end, where they detach readily. The microtubule then starts shortening, while another TR motor lands, walks and falls off the disassembling tip. Images were acquired with two-colour TIRF, so the microtubule is dimmer towards the plus end where it is farther away from the coverslip. The coverslip-attached Rhodamine-labeled microtubule seed is green, because Rhodamine is excited with a 488 nm laser. The 640 nm laser was used to visualize ‘red’ Hilyte-647 tubulin. Time-lapse image sequence was acquired at 2 fps, played 5x faster than recorded. Scale bar, 2 μm. (MOV 20587 kb)

Motility of a full-length CENP-E dimer on a disassembling microtubule.

A single FL CENP-E dimer reaches and then tracks with a disassembling microtubule end, exhibiting processive motility in both plus- and minus-end directions. Motions were recorded as in Supplementary Video S3 but continuously at 100 millisec per channel, and played 5x faster than recorded. Scale bar, 2 μm. (MOV 1402 kb)

Motility of full-length CENP-E complexes on a dynamic microtubule.

A FL CENP-E tetramer lands on the microtubule lattice, reaches a growing tip and stays tip-associated during the phases of growth and shortening. Another FL CENP-E complex, likely to be a single dimer, joins the first complex as it tracks with the disassembling end, until both get stuck to the coverslip surface (see Supplementary Fig. S3b, left panel, for the respective kymograph). Motions were recorded as in Supplementary Video S4, and played 3.3x faster than recorded. Scale bar, 2 μm. (MOV 3200 kb)

Motility of a full-length CENP-E dimer on a growing microtubule.

Arrow points to a single FL CENP-E dimer walking to a growing microtubule tip. Subsequently, the arrow’s position is fixed; the GFP-labelled protein continues to move slowly with microtubule polymerization, while displaying visible diffusion near the tip (see Fig. 8c for the respective kymograph). This sequence was recorded using only a 488 nm laser, which excites both green and red fluorescence, subsequently separated using different emission. The coverslip-attached microtubule seed is seen as brighter red, because of a higher proportion of Rhodamine-labeled tubulin. Video is played 2x faster than recorded. Scale bar, 2 μm. (MOV 858 kb)

Long-lasting association of full-length CENP-E with a dynamic microtubule tip.

A complex of 3–4 FL CENP-E dimers tracks a dynamic microtubule tip for several phases of microtubule growth and shortening. Motions were recorded as in Supplementary Video S6, and played 20x faster than recorded. Scale bar, 2 μm. (MOV 3733 kb)

Lack of coupling between beads coated with truncated CENP-E and depolymerizing microtubule end.

Motor-coated bead (0.5 μm) walks to the plus end of a microtubule, which was grown from a coverslip-attached axoneme and stabilized temporarily with a GMPCPP-containing, Rhodamine-labelled cap (arrowhead). With bright illumination, the Rhodamine-labelled microtubule cap is destroyed (images in red), triggering microtubule depolymerization and subsequent bead detachment. Time-lapse images were acquired with low-light DIC, so individual microtubules are hard to discern. Video was recorded at 1 fps with 60 millisec exposure, played 7.5x faster than recorded. (MOV 2258 kb)

Processive motion of the bead coated with full-length CENP-E towards the microtubule plus and then minus end.

This video shows an analogous experiment to that in Supplementary Video S8 but with the bead coated with FL CENP-E. The bead is seen moving on the microtubule wall towards the microtubule plus end, but when the microtubule begins to shorten, the bead moves in the minus-end direction in conjunction with microtubule disassembly. Arrow marks the initial position of the bead. Played 15x faster than recorded. (MOV 4136 kb)

Live imaging of microtubule destabilization assay in cells with monopolar spindles.

This video shows maximal projections of 3 confocal slices (0.5 μm per step) of HeLa cells expressing Mis12–GFP (kinetochore marker). Cells were treated with monastrol to create the mitotic configuration in which chromosomes are gathered in a rosette around the unseparated poles. Microtubule depolymerization was induced by nocodazole at the beginning of these two sequences and motions of the kinetochores were recorded every 10 sec for 5 min. The inward chromosome motion is greatly reduced in the cell treated with CENP-E inhibitor. Video is played 150x faster than recorded and repeated 3 times. Scale bar, 5 μm. (MOV 1015 kb)

Computer model of the motility of a full-length CENP-E dimer on a growing microtubule.

Motor domains are shown in blue and orange, CENP-E tails are pink and the coiled coil is grey. To simplify calculations, the motor walks along the same protofilament as the diffusing tail. Fluctuations in the length of microtubule protofilaments reflect fast tubulin exchange at the microtubule tip. This sequence shows the molecular events at a millisecond timescale, when microtubule elongation and motor walking appear much slower relative to the tubulin exchange and the diffusion and binding/unbinding of the tail. After reaching the end of the protofilament track, the motor domains dissociate rapidly (with the rate of tubulin exchange) but remain tethered to the microtubule lattice via the diffusing tail. The tail’s residency time on the microtubule lattice is 0.5 sec, whereas the motor re-binding to the lattice takes only 1 millisec, so the motor is highly likely to resume the plus-end-directed motion before the tail loses its microtubule association. Calculations were carried out with parameter values specified in Table S3 and with a 20 μsec time step; only 1 out of every 50 frames is shown. Video is played at 10 fps, so 1 sec of the video corresponds to 10 millisec of the ‘model’ time. (MOV 735 kb)

Computer model of the motility of full-length CENP-E dimer on a dynamic microtubule.

This simulation is for a longer time than in Supplementary Video S11, and it is played ‘faster’ to show the relatively slow processes of microtubule elongation, shortening and CENP-E tip-tracking. For simplicity, the elongating and shortening microtubule ends were modeled similarly, but with different constants of tubulin exchange. The CENP-E motor repeatedly walks to the end of the protofilament and detaches, but it remains tethered and quickly resumes its walking. This ‘tethered motor’ tip-tracking mechanism requires both the motor and tail CENP-E domains, but it does not require that the CENP-E can discriminate between the microtubule wall and its growing or shortening ends. The video was generated as Supplementary Video S11, but only 1 out of every 2,500 frames is shown at 10 fps. (MOV 1235 kb)

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Gudimchuk, N., Vitre, B., Kim, Y. et al. Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips. Nat Cell Biol 15, 1079–1088 (2013). https://doi.org/10.1038/ncb2831

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