MAP4 and CLASP1 Operate As a Safety Mechanism to Maintain a Stable Spindle Position in Mitosis

ARTICLES

MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis

Catarina P. Samora1,3, Binyam Mogessie1,3, Leslie Conway2, Jennifer L. Ross2, Anne Straube1,4 and Andrew D. McAinsh1,4

Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein–dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These ﬁndings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasis.

Establishing the correct cell-division plane through precise positioning positioning machinery and determining the mechanisms that control of the mitotic spindle is crucial for multicellular-organism their activity is essential to further understand this process. development1,2. Mitotic-spindle positioning is mediated by astral Multiple proteins operating upstream of force generators have been microtubules, which are nucleated at spindle poles and grow out identified, largely through genetic approaches in worms and flies. Par to the cell cortex. Astral-microtubule plus ends engage the cortical proteins and a non-canonical G-protein signalling system seem to be machinery to generate the pulling and pushing forces necessary to involved in activating dynein–dynactin and recruiting microtubule alter or maintain spindle position in the cell3. Cortical force generators binding proteins14–17. However, the mechanisms controlling astral- contain dynein–dynactin4, a microtubule-minus-end-directed motor microtubule interactions at the cortex downstream of force generators complex required for correct spindle positioning in multiple systems5–9. are poorly understood. Microtubule-associated proteins (MAPs) are Such motors, if tethered at the cortex, generate pulling forces on the obvious candidates. Depletion of the plus-end tracking protein EB1 spindle by walking towards the microtubule minus end. Experiments and the tumour suppressors APC and VHL cause defects in spindle in Caenorhabditis elegans indicate that such pulling events are often positioning18–20. As these phenotypes can be explained by reduced coupled to microtubule depolymerization10. astral-microtubule numbers, they do not illuminate the mechanisms Growing evidence indicates that force generators are not uniformly by which force generators interact with astral microtubules. Thus, bound or activated across the surface of the cell cortex. In C. elegans the proteins that specifically effect astral-microtubule behaviour and embryos, force generators become enriched at the posterior cortex, force-generating events at the cell cortex remain to be identified. generating a force gradient that causes asymmetric positioning of the spindle11. In human cells, cortical cues are transmitted through RESULTS retraction fibres that anchor mitotic cells to the extracellular substrate MAP4 is required for correct spindle architecture and define the spatial positioning of actin regulators and thus the MAP4 is the only member of the MAP2/Tau family of microtubule- activity of cortical force generators12. Theoretical models based on binding proteins present in non-neuronal cells, although its function such experiments propose that active force generators are enriched in mitosis is unclear21–24. To revisit this we used RNA interference where retraction fibres protrude from the cell cortex13, enabling to specifically and efficiently deplete MAP4 in human cells. Three cells to sense their external environment and divide in the correct independent short interfering RNA (siRNA) oligonucleotides (MAP4-1, orientation. Identifying the components of the cortical-spindle MAP4-3, MAP4-4 siRNA) depleted MAP4 protein by more than

1Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK. 2Physics Department, University of Massachusetts, Amherst, Massachusetts 01003, USA. 3These authors contributed equally to this work. 4Correspondence should be addressed to A.S. or A.D.M. (e-mail: [email protected] or [email protected])

Received 28 March 2011; accepted 14 June 2011; published online 7 August 2011; DOI: 10.1038/ncb2297

a siRNA bc Zoom Zoom Zoom

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37 3 1 4 Anti-MAP4 3 siRNA Control 50 MAP4- MAP4- MAP4- MAP4- Anti-α-tubulin MAP4 α-tubulin Merge defQuantification of mitotic spindle types Geometry of bipolar spindle Bipolar Elliptical 100 Monopolar 100 Aberrant 90 Multipolar 90 80 80 siRNA

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MAP4- 30 30 α-tubulin 20 20 Percentage of cells γ-tubulin Percentage of cells 10 10 CREST α-tubulin 0 0 3 1 4 3 1 4 siRNA siRNA Control Control MAP4- MAP4- MAP4- MAP4- MAP4- MAP4- ghLength of the mitotic spindle Width of the mitotic spindle ijTotal amount of α-tubulin Complementation with incorporated in spindle mouse MAP4 35 Control 70 Control siRNA l 35 Control siRNA siRNA MAP4 siRNA Control siRNA MAP4 siRNA 30 60 (elliptical) 30 MAP4 + eGFP 25 siRNA 50 MAP4 siRNA Control siRNA + 25 (aberrant) eGFP–MAP4 20 40 ll 20 MAP4 siRNA 15 15 30 + eGFP 10 10 20 MAP4 siRNA + lll eGFP–MAP4 5 10 Percentage of cells 5 Percentage of cells Percentage of cells 0

0 0 10 20 30 40 50 60 70 80 90 0 100 5 6 7 8 9 10 345678 9 10 11 12 13 14 15 16 17 18 Percentage of cells Spindle length (µm) Spindle width (µm) Relative α-tubulin levels (a.u.) with elliptical spindle

Figure 1 MAP4 is required for correct spindle architecture. poles. (e) Percentages of cells with bipolar (black), monopolar (light (a) Immunoblots of whole-cell lysates transfected with control or grey) or multipolar (red) spindles in cells transfected with control or MAP4-4, MAP4-3 or MAP4-1 siRNA and probed with antibodies MAP4-3, MAP4-1 or MAP4-4 siRNA. (f) Percentages of elliptical- (black) against MAP4 and α-tubulin. (b) Immunofluorescence microscopy or aberrant- (red) shaped spindles in cells treated with siRNAs as indicated. images of metaphase cells and a magnified single microtubule (g,h) Distribution of spindle length (g) and width (h) in metaphase cells stained with 4,6-diamidino-2-phenylindole (DAPI; DNA, blue), MAP4 treated with control (black) or MAP4 (red) siRNA. (i) Distribution of (green) and α-tubulin (microtubules, red) antibodies. The images of protein levels of α-tubulin in cells transfected with control and MAP4-1 metaphase cells correspond to z -projections and the image of the single siRNA measured relative to γ-tubulin, after background correction. I–III: microtubule is from a single focal plane. (c) Quantification of MAP4 representative images of α-tubulin for control siRNA, MAP4 siRNA levels in cells transfected with control and MAP4 siRNA. Levels were elliptical and MAP4 siRNA aberrant cells, respectively. (j) Percentages determined from three-dimensional reconstructions of cells and calculated of cells with elliptical bipolar spindles after complementation of MAP4 relative to α-tubulin, after background correction. (d) Representative siRNA cells with mouse eGFP–MAP4. In all experiments, n 150 cells = immunofluorescence microscopy images of cells treated with MAP4 from three independent experiments. Triangles in histograms represent siRNA and stained with α-tubulin (red), γ-tubulin (green) and CREST mean values. Error bars represent s.e.m. Scale bars, 10 µm. Uncropped (blue) antisera showing an elliptical cell and a cell with hyper-focused images of blots are shown in Supplementary Fig. S5.

90% as judged by immunoblotting of cell extracts with anti-MAP4 control cells the majority of spindles (96%) have an elliptical shape antibodies (Fig. 1a). Immunofluorescence using anti-MAP4 and anti- with approximate dimensions of 12 µm (length) 8 µm (width), × α-tubulin antibodies showed that MAP4 associates with all microtubule treatment of cells with MAP4-1, MAP4-3 or MAP4-4 siRNA frequently populations within the mitotic spindle including astral microtubules resulted in spindles with an aberrant geometry (Fig. 1d–h), consistent (Fig. 1b). MAP4 appears as dots along the length of microtubules with observations in Xenopus extracts23. These spindles were slightly without any preference for the ends (Fig. 1b). The intensity of the longer and narrower, sometimes bent and microtubules at the poles MAP4 signal was reduced by more than 95% following treatment were more focused (Fig. 1b,d,g,h). The total amount of tubulin with MAP4-1, MAP4-3 or MAP4-4 siRNA (Fig. 1b,c). Whereas in incorporated in spindles was unchanged, indicating that geometrical

2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLES alteration is a consequence not of altered microtubule number, but and dynamics, we constructed a cell line that stably expresses the rather of their organization (Fig. 1i). Expressing mouse MAP4 in microtubule plus-end tracking protein EB3 fused to the red fluorescent cells treated with MAP4-3 siRNA rescued the number of elliptical protein tdTomato. This enabled us to follow the life history of spindles to 75% (Fig. 1j), confirming that this phenotype was not due individual astral-microtubule plus ends growing from nucleation sites to off-target siRNA effects . at the spindle pole to the cell cortex (Fig. 3a,b). We found that neither the frequency of astral-microtubule nucleation, nor that of microtubule Three-dimensional positioning of the mitotic spindle ends reaching the cell cortex, changed following MAP4 depletion requires MAP4 (Table 1). Furthermore, cytoplasmic-microtubule growth speed and To investigate the effect of MAP4 depletion in a living system, we the microtubule dwell time at the cell cortex before undergoing imaged HeLa cells stably expressing histone H2B–eGFP (enhanced catastrophe were independent of MAP4 depletion (Table 1, Fig. 3b,c). green fluorescent protein) and monomeric red fluorescent protein Quantification of the relative astral-microtubule signal intensity in fixed (mRFP)–α-tubulin to mark chromosomes and microtubules. Time- and α-tubulin-stained cells further confirmed that the number of astral lapse sequences revealed that spindles had hyper-focused poles microtubules in MAP4-depleted cells was not reduced (Supplementary immediately after nuclear breakdown, but formed an elliptical spindle Fig. S2). This indicates that MAP4 depletion does not obviously alter the in most cases after 9 min (Supplementary Movies S1, S2). Chromosome number or dynamics of astral-microtubule plus ends in the cytoplasm. congression was slightly delayed, and fixed-cell imaging confirmed that the number of cells in metaphase with unaligned chromosomes MAP4 prevents astral microtubules undergoing side-on sliding increased to 52% after MAP4 siRNA treatment, compared with at the cell cortex 13% after control siRNA treatment (Supplementary Fig. S1a–e). All Whereas our plus-end marker did not enable us to visualize cells did eventually undergo anaphase with a small increase in the depolymerization-coupled events, we observed movements of astral- number of segregation errors (Supplementary Fig. S1f,g). Importantly, microtubule plus ends along the cell cortex, indicating force-generation spindles were unusually motile and incorrectly positioned within the events that could reposition the spindle. To investigate this, we cell (Fig. 2a). In control cells, the frequency of spindle displacement determined the behaviour of astral microtubules reaching the cell

(>1 µm per 3 min frame) or rotation (>45◦ per 3 min frame) in the cortex. Microtubule ends dwelt at the cortex for a few seconds 1 xy plane (horizontal plane through the cell equator) was 0.07 min− and before either undergoing catastrophe (indicated by EB3 signal loss) 1 0.06 min− respectively, with very few cells undergoing rotations in the or continuing to grow along the cell cortex, thereby converting z axis (vertical plane) (Fig. 2b–d). In contrast, MAP4 siRNA treatment from an ‘end-on’ to a ‘side-on’ contact. This transition occasionally 1 resulted in spindles that frequently rotated (0.21 min− ) and displaced resulted in fast movements along the cell cortex that were indicative of 1 1 (0.2 min− ) in the xy plane and rotated in the z axis (0.25 min− ) motor-driven sliding (Fig. 3b,d and Supplementary Movie S5). This (Fig. 2b–d and Supplementary Movies S3, S4). To confirm the spindle was confirmed using a stable cell line expressing mCherry–α-tubulin mispositioning defects, cells were treated with control or MAP4 siRNA that enabled visualization of entire astral microtubules, albeit with and stained with anti-α-tubulin and anti-γ-tubulin antibodies (Fig. 2e). poor signal-to-noise ratio. We observed both the engagement of To determine the extent of z-axis spindle rotations, we measured the microtubules end-on and side-on at the cortex and sliding movements angle α of the spindle axis relative to the surface of the coverslip (Fig. 2f). of a laterally attached microtubule (Supplementary Movie S6). To

The average angle α was 8.6◦ in control and 31.2◦ in MAP4 siRNA- quantitatively compare astral-microtubule behaviour in control and treated cells, reflecting a random orientation18. Spindle orientation was MAP4-depleted cells, we selected cells with horizontally positioned rescued to 17.5◦ in MAP4-depleted cells co-transfected with mouse spindles and both poles visible in the same optical section, a pole-to- eGFP–MAP4 (Fig. 2g). We then determined the centre of the cell and cortex distance (p) greater than 2 µm and little or no spindle movement the centre of the spindle (the midpoint of the vector that joins the two during the observation time. This is important because MAP4 depletion spindle poles) and calculated the distance d between these two points, causes spindle displacements (see Fig. 2) that potentially distort the to derive a measure of spindle displacement in the xy plane (Fig. 2h). analysis. In MAP4-depleted cells frequency, speed and run length This value was small in control cells (0.49 µm), reflecting the stable of cortical-movement events increased, compared with control cells positioning of the spindle in the cell centre (Fig. 2i). MAP4 depletion (Table 1 and Supplementary Fig. S2c and Movie S7). Movements faster 1 caused an average spindle displacement of 1.44 µm from the cell than 1 µms− and longer than 2.5 µm increased more than threefold 1 centre (Fig. 2h,i), which could be partially rescued by expressing mouse after MAP4 depletion (0.18 and 0.08 min− per pole in control; 0.55 1 eGFP–MAP4 (d 0.9 µm) (Fig. 2i). We also observed similar spindle- and 0.28 min− per pole in MAP4-depleted cells). The frequencies = positioning defects in retinal pigment epithelial cells immortalized with and lengths of these movements are probably underestimated because hTERT (hTERT-RPE1) following MAP4 depletion (Fig. 2j–m). These imaging was restricted to a single focal plane to enable the fast sampling data confirm that MAP4 depletion leads to defects in the orientation rates required to measure microtubule dynamics. Nevertheless, these and positioning of the mitotic spindle. observations indicate that excess force generators are recruited to astral microtubules in the absence of MAP4, leading to increased end-on MAP4 does not affect astral-microtubule nucleation or dynamics to side-on conversion and depolymerization-uncoupled microtubule in the cytoplasm sliding along the cell cortex. Previous work has shown that spindle orientation defects are often a One prediction of this model is that, as a spindle pole approaches the consequence of reducing the number of astral microtubules18,19. To cortex (decrease in distance p), the phenotype would be exacerbated explore the effect of MAP4 depletion on astral-microtubule number because a higher proportion of astral microtubules would arrive at

a –3′ 0′ 3′ 6′ 9′ 12′ 33′ 36′ Control siRNA 0′ 3′ 6′ 9′ 12′ 24′ 27′ 30′ siRNA 33′ 36′ 39′ 45′ 105′ 108′ 120′ 195′ MAP4

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0.10 0.10 Control siRNA d V 0.1 z = –3 µm z = 0 µm z = 3 µm m steps z µ rotation (min 0.05 0.05 rotation (min α z xy siRNA displacement (min 0.2

0 xy 0 0 V Surface MAP4 MAP4 MAP4 MAP4 Control siRNA Control siRNA Control siRNA α-tubulin γ-tubulin CREST gh i Control siRNA MAP4 siRNA 50 Control siRNA 80 40 MAP4 siRNA 60 MAP4 siRNA 30 + eGFP–MAP4 )

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hTERT-RPE1 cells 0 Anti-MAP4 0 50 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 siRNA Anti-α-tubulin Control Displacement (µm)

MAP4

Figure 2 Loss of MAP4 leads to spindle position and orientation defects. as the distance between the centre of the cell (green cross) and the (a) Successive frames from live-cell movies of HeLa cells expressing middle of the spindle axis (white circle). (i) Distribution of spindle position H2B–eGFP/mRFP–α-tubulin (to mark chromosomes and microtubules relative to the cell centre in HeLa cells after control and MAP4 siRNA respectively) in control siRNA cells (top row) and MAP4 siRNA cells treatment or MAP4 siRNA complementation with mouse eGFP–MAP4. (lower rows). (b–d) Frequency of xy rotation (b) xy displacement (c) (j) Immunoblots of whole hTERT-RPE1 cell lysates transfected with control and z rotation (d) in cells transfected with control and MAP4 siRNA. or MAP4 siRNA and probed with antibodies against MAP4 and α-tubulin. The quantification was derived from live-cell movies (n 100 cells per (k) Statistical box diagrams of mitotic-spindle angle α in hTERT-RPE1 = condition). (e) Different z sections of fixed cells stained for α-tubulin, cells treated with control and MAP4 siRNA. Diagrams were plotted as γ-tubulin and CREST illustrating rotations in the z plane in MAP4-depleted explained in g.(l) Representative immunofluorescence microscopy images cells. Arrowheads indicate the position of the spindle poles. (f) Schematic of hTERT-RPE1 cells treated with control or MAP4 siRNA and stained for representation of mitotic-spindle angle α measurement in HeLa and α-tubulin, γ-tubulin and CREST, illustrating spindle displacements during hTERT-RPE1 cells. (g) Statistical box diagrams of mitotic-spindle angle metaphase. (m) Distribution of spindle position relative to cell centre in α in control, MAP4 siRNA and mouse eGFP–MAP4-complemented HeLa hTERT-RPE1 cells after control and MAP4 siRNA treatment. Displacement cells. The box spans from 25 to 75% of the data and the whiskers from 5 to d was calculated as described in h. Triangles in histograms represent mean 95% of the data. The means are shown as white squares. (h) Representative values. Error bars represent s.d. In all experiments, n 90 cells from three = immunofluorescence microscopy images of HeLa cells treated with control independent experiments (g–i) and n 50 cells from two independent = or MAP4 siRNA and stained for α-tubulin, γ-tubulin and CREST, illustrating experiments (k–m). Scale bars, 10 µm. Uncropped images of blots are spindle displacements during metaphase. Displacement d was calculated shown in Supplementary Fig. S5.

Table 1 Parameters of astral-microtubule dynamics. Control siRNA Control MAP4 Control DHC MAP4 DHC Control MAP4 + + + + + siRNA siRNA siRNA CLASP1 CLASP1 siRNA siRNA

Cell interior Nucleation frequency (min 1 per pole) 34.5 6.3 30.7 5.9 30.1 6.6 32.5 6.5 31.4 5.3 28.5 9.5 − ± ± ± ± ± ± Cytoplasmic growth speed (µms 1) 0.35 0.07 0.38 0.09 0.44 0.11 0.38 0.08 0.41 0.12 0.41 0.08 − ± ± ± ± ± ± Cortical contact frequency (min 1 per pole) 10.6 2.6 11.8 3.3 12.3 2.19.9 2.6 10.8 4.0 10.4 2.1 − ± ± ± ± ± ± At the cell cortex Static dwell time at cortex (s) 3.5 1.84.0 2.1 3.5 1.9 3.8 1.7 3.2 1.73.7 2.1 ± ± ± ± ± ± Frequency of cortical 0.36 0.14 0.73 0.35 0.43 0.11 0.34 0.18 0.92 0.32 0.45 0.20 ± ± ± ± ± ± movements 1 µm (min 1 per pole) ≥ − 1 Frequency of fast ( 1 µms− ) cortical 0.18 0.08 0.55 0.25 0.29 0.14 0.18 0.04 0.62 0.19 0.28 0.15 1≥ ± ± ± ± ± ± movements (min− per pole) Frequency of long ( 2.5 µm) cortical 0.08 0.06 0.28 0.19 0.10 0.03 0.07 0.01 0.31 0.10 0.11 0.10 1 ≥ ± ± ± ± ± ± movements (min− per pole)

Cytoplasmic growth speeds were measured for at least 50 microtubules from seven to ten cells. Astral-microtubule nucleation and cortical contact frequencies were measured during 2 min observation time from seven to ten cells. Static dwell times were measured for at least 173 microtubules from seven to ten cells. All cortical movement frequencies were measured from 20 to 34 cells from at least three independent experiments. Values are presented as mean s.d. ± the cortex at a shallower angle, thus promoting side-on engagement. MAP4 suppresses dynein–dynactin-mediated force generation To test this, we quantified the number of cortical-microtubule sliding The main force generators regulating spindle positioning are cortically events in MAP4-depleted cells at various values of p. The frequency localized dynein motors25–27. To test whether reducing dynein activity of movements along the cell cortex increased markedly as p decreased would rescue the spindle mispositioning defect following MAP4 (Fig. 3f and Supplementary Movie S8). These microtubule ends moved depletion, we treated cells with siRNA against dynein heavy chain significantly faster than the cytoplasmic growth speed, supporting the (DHC) either alone or in combination with MAP4 siRNA and idea that motor-driven microtubule sliding underlies these movements confirmed depletion by immunoblotting (Fig. 4a). Inhibition of the (Fig. 3d). In control cells, the frequency of movements along the cell dynein ATPase by sodium orthovanadate28,29 or prolonged treatment cortex also increased as p decreased but was much lower than in with DHC siRNA caused spindle displacements and z rotations MAP4-depleted cells (Fig. 3f). Moreover, most side-on movements (Supplementary Fig. S3a,b), confirming a role for dynein during spindle occurred at cytoplasmic growth speeds (Fig. 3d; compare to Table 1). positioning in mammalian cells. However, depletion of dynein for 48 h This demonstrated that a lateral contact with the cell cortex per se is had only minor effects on spindle position and z orientation and, when not sufficient to promote rapid microtubule sliding, but this requires combined with MAP4 siRNA, rescued the spindle displacement d as increased recruitment or activation of force generators. well as the spindle angle α to control values (Fig. 4b–d). The frequent and rapid microtubule movements observed in MAP4-depleted cells Cortical sliding events generate forces that misposition were largely abolished following co-depletion of DHC (Table 1, the spindle Supplementary Fig. S2c and Movies S9, S10). This indicates that in If these fast cortical sliding events were responsible for the spindle a wild-type situation MAP4 prevents dynein motors from generating movements in MAP4-depleted cells, we would predict that sliding excess force and thus maintains the spindle in the correct orientation events occurring farther from the spindle axis would be more likely to and position. Furthermore, dynein depletion could rescue the defects cause pole movements. To test this, we measured the pole-to-cortex in chromosome alignment, mitotic timing and spindle-pole focusing distance (p), the distance to the astral-microtubule plus end (l) and the of MAP4-depleted cells (Supplementary Fig. S3d,e,f). These data distance back to the pole (h) to calculate the angle (σ ) (see the schematic indicate that MAP4 controls dynein through a similar mechanism at representation in Fig. 3e). The probability of microtubule-sliding kinetochores, spindle poles and cortical attachment sites. events correlating with lateral pole movement in the same direction To test whether this mechanism is mediated by a physical interaction was determined for different values of σ . At σ < 30◦ we observed we precipitated complexes from whole-cell extracts using antibodies no pole movements. The probability of pole movement increased against MAP4 or p150Glued (a subunit of the dynein–dynactin complex), Glued to 40% when this angle was between 30◦ and 40◦ and rose to 73% and revealed an interaction between MAP4 and p150 (Fig. 4e,f). when it reached 100–110◦ (Fig. 3h). To confirm the correlation To test whether MAP4 directly affects the capacity of dynein–dynactin between microtubule-sliding events and pole movements we used complexes to generate force, we expressed and purified recombinant kymograph analyses to compare the trajectory of the pole (red in full-length mouse MAP4 from Escherichia coli (Fig. 4g), which was able Fig. 3a,i,j) with the direction of moving astral microtubules (green to bind directly to microtubules in vitro (data not shown). We then in Fig. 3a,i,j). In most cases (81.5 12.5%, n 10 cells), the spindle carried out microtubule-gliding assays using dynein–dynactin purified ± = pole followed one or two moving microtubules (Fig. 3i,j). These data from mouse brains. Decoration of microtubules with MAP4 did not pre- are consistent with a model in which the observed cortical microtubule vent dynein binding to microtubules. However, dynein-mediated mi- 1 1 movements are directly responsible for spindle mispositioning in crotubule sliding was reduced from 120 nm s− in control to 68 nm s− MAP4-depleted cells. in the presence of 50 nM MAP4 (Fig. 4h,i). Higher concentrations of

a b EB3–tdTomato 0 s 1 s 2 s 3 s 4 s 5 s 6 s

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Astral-MT growth and static dwell to side-on Astral-MT movement Dwelling (end-on) Movement (parallel to cortex) Catastrophe Spindle-pole movement

c Kymograph analyses of astral-MT de growth and static dwell Control siRNA 40 MAP4 siRNA P = 1.9 × 10–6 σ = cos–1((p2 + h2 – l2)/2ph) 30

Control siRNA Control l Spindle pole 20 p σ 10 s h 1 µ m 10 siRNA Percentage frequency

0 MAP4 0.5 1.0 1.5 2.0 2.5 Spindle pole Movement speed (µm s–1) f gh

Control siRNA 10 Control siRNA 80 10 MAP4 siRNA MAP4 siRNA 9 MAP4 siRNA 9 70 8 8 60 7 per pole) per pole) 7 50 –1 –1 6 6 5 5 40 4 4 30

3 movements 3 20 2 2 10 Frequency of lateral pole 1

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123456 123456 Percentage of lateral spindle pole 0 20 40 60 80 100 120 p (µm) p (µm) σ (°) 10 s i j 10 s

1 µm 1 µm siRNA MAP4 Control siRNA

Astral MT Astral MT Pole Pole

Figure 3 MAP4 depletion increases cortical astral-microtubule and lateral siRNA. Triangles in histograms represent mean values. (e) Parameters spindle-pole movements. (a) Image showing lines used for kymograph used in the analyses of astral-microtubule and lateral pole movements. analyses of astral microtubule (MT) number and dynamics. To assess (f,g) Relationship between pole-to-cortex distance (p) and frequency of astral microtubule growth and static dwell, a line (blue) was added to astral-microtubule (f) or lateral pole (g) movement in cells treated with images from the pole to the cortex (dashed yellow line). To assess cortical control and MAP4 siRNA. All movement frequencies and pole-to-cortex movement of astral microtubules, a line was drawn across half the cell distances were measured from 42–56 cells and seven experiments. perimeter (green), and for analyses of pole movement a line was aligned (h) Probability of lateral pole movement in cells treated with MAP4 siRNA with the pole (red). (b) Two possible outcomes, catastrophe or movement for different values of angle σ measured as illustrated in e. 218 values after transition from end-on to side-on attachment, of an EB3 comet of σ were measured for 34 astral-microtubule movement events from arriving at the cell cortex. (c) Kymographs of pole (bottom)-to-cortex (top) seven cells and two experiments. (i,j) Kymographs of astral-microtubule line scans of control and MAP4-depleted cells. The maximum projection and lateral pole movements in cells treated with control (i) and MAP4 (j) of a 20-pixel-wide blue line (illustrated in a) was plotted at 500 ms time siRNA where the poles are near the cortex. Kymographs were generated by intervals from left to right. Microtubule growth results in diagonal lines and the maximum projection of ﬁve-pixel-wide green and red lines (illustrated static dwells (white stars) at the cortex in horizontal lines. (d) Distribution in a) at 500 ms time intervals. Schematic illustrations of movements were of cortical-movement speeds in cells treated with control and MAP4 constructed by overlaying kymographs with red (poles) and green (astral siRNA where poles are near the cortex. Movements of all run lengths microtubules) lines. Only astral-microtubule movements greater than 1 µm were analysed from six cells from three independent experiments for each in length are shown.

absiRNA c d Control 60 35 siRNA 35 30 30 Control siRNA MAP4 DHC siRNA Mr (K) ControlControl Control MAP4 25 25 d + MAP4 + DHC + DHC siRNA 150 20 20 40 15 15 X ) °

( 10 10

Anti-MAP4 α α 5 5 0 0 Control siRNA 250 20 cells of Percentage 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Anti-DHC 35 35 MAP4 30 Control siRNA 30 siRNA 0 DHC 150 25 25 DHC d siRNA 20 + MAP4 siRNA 20 + MAP4 X Glued siRNA Anti-p150 MAP4 15 15 siRNA ControlControl Control+ DHC + DHC + MAP4 10 10

50 5 5 DHC 0 0 Percentage of cells of Percentage 0.5 1.0 1.5 2.0 2.5 3.0 Anti-α-tubulin 0.5 1.0 1.5 2.0 2.5 3.0 Displacement (µm) Displacement (µm) α-tubulin γ-tubulin CREST e IP Input g hi Glued AP4 eads -NTA M (K) B M p150 5% 1% r 2+ Ni

Short Anti-uMAP4 M (K) Ion exchange 400 150 exposure r Mr (K) Long 250 250 150 300 exposure 150 Anti-p150Glued 150 ) 0 nM MAP4 –1 200 f Input IP 100 100

(nm s 3 µm Glued 100 eads AP4 Mr (K) 5% 1% B M p150 Short 1 min 150 exposure 0

Long 0 50 100 200 50 nM MAP4 150 exposure Dynein-mediated microtubule sliding Anti-MAP4 [MAP4] (nM)

Figure 4 MAP4 suppresses dynein-dependent force generation. MAP4. Whole-cell lysates were immunoprecipitated with anti-p150Glued (a) Validation of DHC siRNA and efﬁciency of double siRNA of DHC and anti-MAP4 antibodies. Two different exposures of a western blot are and MAP4 by immunoblotting. The dynactin subunit p150Glued and shown. (f) Immunoprecipitation of MAP4 with dynactin subunit p150Glued. α-tubulin were used as loading controls. (b) Statistical box diagrams Whole-cell lysates were immunoprecipitated with anti-p150Glued and (presented as in Fig. 2g) of mitotic-spindle angle α in control, MAP4, anti-MAP4 antibodies. Two different exposures of a western blot are DHC and MAP4 DHC depleted cells. Measurements were made as shown. n 2 experiments. (g) SDS–polyacrylamide gel electrophoresis + = described in Fig. 2f. (c) Distribution of spindle position relative to cell and western blotting analyses of mouse His–MAP4 following protein centre d in HeLa cells after control, DHC, MAP4 and MAP4 DHC puriﬁcation on Ni-NTA (nitrilotriacetic acid) and ion-exchange columns. + siRNA treatment. Displacement was calculated as described in Fig. 2h. (h) Statistical box diagrams (presented as in Fig. 2g) of average speeds Triangles in histograms represent mean values. (d) Representative images measured in dynein gliding assays with microtubules in the presence of metaphase cells treated with control and DHC siRNA with no or little of varying amounts of His –MAP4. n 33–76 microtubules from two 6 = spindle displacement. Note the pole-focusing defect in DHC-depleted independent experiments. (i) Kymograph of a typical dynein-mediated cells. b–d, n 90 cells from three independent experiments. Scale bar, microtubule gliding event without or in the presence of 50 nM His –MAP4. = 6 10 µm. (e) Immunoprecipitation (IP) of dynactin subunit p150Glued with Uncropped images of blots are shown in Supplementary Fig. S5.

1 1 MAP4 further reduced the speed to 48 nm s− (100 nM) and 42 nm s− out siRNA complementation assays in cells treated with CLASP1 siRNA (200 nM) (Fig. 4h). These data confirm a direct role of MAP4 in and found that RNA interference-resistant GFP–CLASP1α (ref. 30), controlling force generation by dynein–dynactin motors. but not GFP–CLASP2α, or the empty vector, was able to rescue the spindle-displacement phenotype observed in cells treated with CLASP1 CLASP1 controls spindle positioning siRNA (Fig. 6). Thus, CLASP1, but not CLASP2, is required for spindle CLIP-associated proteins CLASP1 and CLASP2 are important positioning in human cells. Depletion of CLASP1 did not result in regulators of microtubule–cortex interactions during interphase30. any defects in z-axis spindle orientation (Fig. 5d,e), indicating that it They localize to the plus ends of astral microtubules31,32 and have genetically separates the mechanisms that control xy and z position of been implicated in dynein function33. To investigate the functional the mitotic spindle. Moreover, CLASP1 (but not CLASP2) depletion interaction between CLASP1, CLASP2, MAP4 and dynein we first was able to rescue the spindle misorientation in the z axis, and partially confirmed that MAP4, CLASP1 and CLASP2 are depleted to the rescued the positioning defects in the xy plane in MAP4-depleted cells. same extent in single- and double-siRNA experiments (Fig. 5a). The phenotype in cells treated with CLASP1 siRNA was dominant Depletion of CLASP1 caused spindles to be mispositioned in the over MAP4 depletion, indicating that CLASP1 functions upstream of 1 xy plane (Fig. 5b,c), although the rate of displacements (0.1 min− ) and MAP4, presumably by promoting the correct end-on capture of astral 1 rotations (0.08 min− ) was much lower than in MAP4-depleted cells microtubules at the cell cortex. By following EB3 dynamics, we observed (Fig. 5e–h and Supplementary Movies S11–S13). In contrast, depletion that astral microtubules underwent relatively frequent transitions to of CLASP2 had no effect on spindle positioning and co-depletion the side-on moving state in CLASP1-depleted cells, consistent with of CLASP1 and CLASP2 caused the same phenotype as CLASP1 CLASP1 functioning to stabilize end-on cortical attachments (Table 1, depletion alone (Fig. 5b,d). To confirm these observations, we carried Supplementary Fig. S2d and Movies S14, S15).

ab siRNA 35 35 35 P2 30 Control siRNA 30 Control siRNA 30 Control siRNA 25 25 MAP4 siRNA 25 CLASP1 siRNA CLASP2 siRNA 20 20 20 + MAP4 + CLASP1 + CLASP2 15 15 15 10 10 10 5 5 5 Mr (K) ControlControlControlMAP4 Control+ CLASP1MAP4 CLASP1+ CLASP2 + CLAS 0

Percentage of cells 0 0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 150 Anti-MAP4 35 35 35 30 Control siRNA 30 Control siRNA 30 Control siRNA 170 Anti-CLASP1 25 CLASP1 25 CLASP1 25 CLASP2 20 + CLASP2 20 + MAP4 siRNA 20 + MAP4 siRNA 170 Anti-CLASP2 15 siRNA 15 15 10 10 10 50 Anti-α-tubulin 5 5 5

Percentage of cells 0 0 0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Displacement (µm) Displacement (µm) Displacement (µm) c d Control siRNA CLASP1 siRNA CLASP2 siRNA 60

40 ) ° X d X d X ( α d 20

α-tubulin γ-tubulin CREST siRNA MAP4 MAP4 ControlControl Control Control + MAP4 CLASP1 + CLASP1+ CLASP2+ CLASP2+ CLASP1+ CLASP2 e 0′ 3′ 6′ 12′ 18′ 24′ 30′ 39′

0′ 3′ 6′ 12′ 18′ 24′ 30′ 33′ siRNA Control siRNA CLASP1 0′ 3′ 18′ 21′ 36′ 51′ 66′ 117′ siRNA MAP4 + CLASP1 fg h Control siRNA Control siRNA Control siRNA

MAP4 siRNA MAP4 siRNA MAP4 siRNA

Control Control Control + CLASP1 siRNA + CLASP1 siRNA + CLASP1 siRNA CLASP1 CLASP1 + MAP4 siRNA CLASP1 + MAP4 siRNA + MAP4 siRNA 0 0 0 0.1 0.2 0.3 0.05 0.10 0.15 0.20 0.25 0.05 0.10 0.15 0.20 0.25 z rotation (min–1) xy rotation (min–1) xy displacement (min–1)

Figure 5 Depletion of CLASP1 leads to spindle-positioning defects in the as in Fig. 2g) of mitotic-spindle angle α in cells treated with xy plane, but does not affect spindle orientation in z .(a) Immunoblot of control, MAP4, CLASP1, CLASP2, MAP4 CLASP1, MAP4 CLASP2 + + whole-cell extracts from cells transfected with control, MAP4, CLASP1, and CLASP1 CLASP2 siRNAs. Measurements were carried out as + CLASP2, MAP4 CLASP1, MAP4 CLASP2 and CLASP1 CLASP2 described in Fig. 2f. n 90 cells from three independent experiments. + + + = siRNAs and probed with antibodies as indicated. α-tubulin was used (e) Successive frames from live-cell movies of HeLa cells expressing as a loading control. (b) Distribution of spindle position d relative to H2B–eGFP/mRFP–α-tubulin in control siRNA cells (top row), CLASP1 the cell centre in HeLa cells after control, MAP4, CLASP1, CLASP2, siRNA cells (middle row) and MAP4 CLASP1 siRNA cells (bottom + MAP4 CLASP1, MAP4 CLASP2 and CLASP1 CLASP2 siRNA row). (f–h) Frequency of xy rotation (f), xy displacement (g) and + + + treatment. The displacement was calculated as described in Fig. 2h. z rotation (h) in cells transfected with control, MAP4, CLASP1 and Triangles in histograms represent mean values. n 90 cells from MAP4 CLASP1 siRNAs. Control and MAP4 siRNA values were taken = + three independent experiments. (c) Representative images of control from Fig. 2. Quantiﬁcation was derived from live-cell movies (n 100 cells = and CLASP1- and CLASP2-depleted metaphase cells indicating the per condition). Error bars represent s.d. Scale bars, 10 µm. Uncropped extent of spindle displacement. (d) Statistical box diagrams (presented images of blots are shown in Supplementary Fig. S5.

a Control siRNA + Control siRNA + with only 0.06% in cells treated with control siRNA; Supplementary Control siRNA + eGFP GFP–CLASP1α GFP–CLASP2α Fig. S1h). These results demonstrate that MAP4-mediated control of dynein activity is crucial for maintaining a stable spindle position and d d d correct cell division. X X X DISCUSSION Our model proposes that CLASP1 and MAP4 are components of the eGFP γ-tubulin GFP-CLASP1α γ-tubulin GFP-CLASP2α γ-tubulin CLASP1 siRNA + CLASP1 siRNA + control system for cortical force generation (Fig. 7d,e). Theoretical CLASP1 siRNA + eGFP GFP–CLASP1α GFP–CLASP2α models specify that force generators within the equatorial region (e- FGs; Fig. 7d) of a rounded-up mammalian mitotic cell are in an active state, as a consequence of cortical cues13, whereas those on the hemi- d d d X X spheres (h-FGs; Fig. 7d) are less active. This maintains the spindle in a X stable horizontal position, as e-FGs exert greater pulling forces on astral microtubules than h-FGs. Loss of MAP4 would cause excessive engage- eGFP γ-tubulin GFP-CLASP1α γ-tubulin GFP-CLASP2α γ-tubulin ment of force generators independently of their location. Deregulated b 35 35 pulling events by h-FGs would move the spindle out of its horizontal Control siRNA + 30 Control siRNA + 30 eGFP GFP–CLASP1α orientation, whereas deregulated e-FG-mediated microtubule-sliding 25 CLASP1 siRNA + 25 CLASP1 siRNA + events would cause the observed spindle displacement/rotation in the 20 eGFP 20 GFP–CLASP1 α xy plane. MAP4 and CLASP1 thus operate as a ‘safety mechanism’ that 15 15 10 10 prevents microtubules at the cortex escaping from ‘end-on’ attachment 5 5 that drives pushing and depolymerization-coupled pulling events that Percentage of cells Percentage of cells 0 0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 stabilize spindle position (Fig. 7e). The switch to ‘side-on’ observed in Displacement (µm) Displacement (µm) MAP4 or CLASP1 depletion leads to a depolymerization-uncoupled

50 state, enabling dynein-dependent microtubule sliding and the gen- Control siRNA + eration of torque that mispositions the spindle. Physical interaction 40 GFP–CLASP2α Glued CLASP1 siRNA + between endogenous MAP4 and p150 and reconstitution of the 30 GFP–CLASP2α microtubule–MAP4–dynein–dynactin system in vitro directly supports 20 this model. Our in vitro experiments also indicate that MAP4 limits 10 dynein activity, rather than access to the microtubule lattice, as Percentage of cells 0 0.5 1.0 1.5 2.0 2.5 3.0 decoration of microtubules with MAP4 did not prevent binding to Displacement (µm) dynein-coated coverslips. We note, however, that astral-microtubule sliding along the cell cortex, although infrequent, can be observed Figure 6 CLASP1, but not CLASP2, rescues the spindle mispositioning in CLASP1-depleted cells. (a) Representative images of cells treated with in wild-type cells. Motorized sliding of microtubules could therefore control or CLASP1 siRNA, transfected with either empty vector, RNA contribute to the force-generating mechanisms in mammalian cells. interference-resistant GFP–CLASP1α or GFP–CLASP2α and stained with We further propose that CLASP1 is crucial for the initial microtubule anti-γ-tubulin (red). (b) Quantiﬁcation of spindle displacement d in CLASP capture at the cell cortex (Fig. 7e), as it seems to be required for rescue experiments as outlined in a. n 60–100 cells from two independent = experiments. Triangles in histograms represent mean values. exertion of force on astral microtubules by h-FGs. Thus, MAP4 Scale bar, 10 µm. depletion, which enables excess engagement of force generators globally, does not lead to vertical spindle rotations when CLASP1 is MAP4 and CLASP1 ensure cell division along the correct axis depleted. Why does the spindle become displaced in the xy plane when Spindle position is strongly linked to the cell-division axis1,13,34. To CLASP1 is depleted? We predict that additional pathways of cortical directly test whether the absence of MAP4 or CLASP1 results in division- microtubule capture must be active at the cell equator. NuMA (nuclear plane errors, we observed dividing immortalized primary epithelial mitotic apparatus protein) and LGN (leucine–glycine–asparagine hTERT-RPE1 cells on adhesive substrate patterns that promote a single repeat-enriched protein) are excellent candidates because they localize division plane12. In control cells, more than 50% of cell divisions to the equatorial region during mitosis and are required for occurred in the direction guided by the extracellular pattern with a spindle positioning16,17,35. Imbalanced cortical interactions of such precision of 6◦ (Fig. 7a–c). Consistent with the spindle-orientation proteins with astral microtubules would explain the observed spindle- ± defects reported here, MAP4 or CLASP1 depletion led to wider angular displacement phenotype in the absence of CLASP1. Unravelling the distributions of cell-division axes (Fig. 7a–c). This distribution was spatiotemporal regulation and crosstalk between the different cortical not random, indicating that the mechanisms of establishing and microtubule-capturing complexes in relation to extracellular cues is interpreting environmental cues for spindle positioning34 are intact. a future challenge. Correct spindle positioning in the horizontal and However, the force imbalances described above do not permit a stable vertical axes is clearly of wide biological importance, and controlling spindle position, and therefore the correct division plane, in the absence force generators by distinct mechanisms would be a good way to of MAP4 or CLASP1. Live-cell imaging experiments using HeLa regulate three-dimensional movements. cells expressing H2B–eGFP also showed that 37% of MAP4-depleted Loss of MAP4 function also caused defects in chromosome cells underwent anaphase vertical to the substrate (in comparison alignment and led to hyper-focusing of the spindle poles (Fig. 1d

a –10 min 0 min 10 min 25 min 30 min 140 min θ

20 µm

b Control siRNA MAP4 siRNA CLASP1 siRNA 90 90 0.6 90 120 60 0.3 120 60 0.3 120 60 0.5 0.4 0.2 0.2 0.3 150 30 150 30 150 30 0.2 0.1 0.1 0.1 0 180 0 0 180 0 0 180 0 0.1 Frequency Frequency 0.2 0.1 Frequency 0.1 0.3 330 210 210 330 210 330 0.4 0.2 0.2 0.5 240 300 0.6 0.3 240 300 0.3 240 300 270 270 270 Division axis θ n = 100 Division axis θ n = 100 Division axis θ n = 30

c 90 90 90 0.3 120 60 0.3 120 60 0.3 120 60

0.2 0.2 0.2 150 30 150 30 150 30 0.1 0.1 0.1

0 180 0 0 180 0 0 180 0 Frequency Frequency 0.1 Frequency 0.1 0.1 210 330 210 330 210 330 0.2 0.2 0.2

0.3 240 300 0.3 240 300 0.3 240 300 270 270 270 Division axis θ n = 40 Division axis θ n = 40 Division axis θ n = 30

d e End-on Side-on Cortex FG Limit FGs low Capture Dynein MAP4 CLASP1

MAP4

CLASP1 FGhigh Erzin Astral microtubule Astral NuMA Dynactin Fpull LGN mDia Fpush Fpull Surface Spindle pole

Figure 7 MAP4- and CLASP1-mediated control of cortical force generators CLASP function in the context of extracellular cues. FG, force generator; ensures an accurate cell-division axis. (a) Example of a MAP4-depleted RPE1 mDia, mammalian diaphanous-related formin. (e) Schematic representation H2B–mRFP cell grown on an H pattern. Cells adopt a pseudo-square shape of proposed functions of MAP4 (green) in limiting accessibility of growing in interphase (cell outline in interphase highlighted with dashed lines) and astral microtubules (red arrows) to dynein motors (grey). CLASP1 (brown) preferentially align the spindle along the vertical axis (θ 90◦). (b,c) Angular functions to capture microtubules and stabilize the end-on state. In the = distribution of division axes θ on H patterns (b) and disc patterns (c) in absence of MAP4 or CLASP1, excess motors engage with the microtubule the siRNA conditions indicated. Note that distributions for control cells on and slide it side-on along the cell cortex. This generates lateral pulling H patterns are plotted on a different scale. (d) Model of proposed MAP4 and forces, some of which will result in torque on the spindle pole. and Supplementary Fig. S1a,h,i). MAP4’s function to reduce dynein overactive dynein, leading to ‘hyper-focusing’ of microtubule minus engagement and force generation would explain these phenotypes. ends (Fig. 1d). At the kinetochore, increased dynein activity could Dynein is involved in focusing microtubules at the spindle pole36 increase poleward transport of chromosomes out of the metaphase (Fig. 4d and Supplementary Fig. S3c). Loss of MAP4 would result in plate. Our observation that chromosomes ‘pop’ out of the metaphase

10 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLES plate is consistent with this model (Supplementary Fig. S1a,i,j). 9. Gonczy, P., Pichler, S., Kirkham, M. & Hyman, A. A. Cytoplasmic dynein is required As unaligned chromosomes activate the spindle checkpoint37, this for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J. Cell Biol. 147, 135–150 (1999). model also explains the mitotic delay following MAP4 depletion 10. Kozlowski, C., Srayko, M. & Nedelec, F. Cortical microtubule contacts position the (Supplementary Fig. S1b,c), which is partially rescued after co-depletion spindle in C. elegans embryos. Cell 129, 499–510 (2007). 11. Grill, S. W., Howard, J., Schaffer, E., Stelzer, E. H. & Hyman, A. A. The of dynein (Supplementary Fig. S3f). We speculate that MAP4 operates distribution of active force generators controls mitotic spindle position. Science 301, as a universal mechanism to control dynein motor activity within 518–521 (2003). 12. Théry, M. et al. The extracellular matrix guides the orientation of the cell division axis. the cell. This motor-control function is consistent with our finding Nat. Cell Biol. 7, 947–953 (2005). that MAP4 depletion does not affect astral- and spindle-microtubule 13. Théry, M., Jiménez-Dalmaroni, A., Racine, V., Bornens, M. & Jülicher, F. Experimental and theoretical study of mitotic spindle orientation. Nature 447, dynamics (Table 1; data not shown). Moreover, Tau limits the 493–496 (2007). engagement of dynein with the microtubule lattice, and MAP2 inhibits 14. Woodard, G. E. et al. Ric-8A and Gi α recruit LGN, NuMA, and dynein to the cell the ATPase of dynein38,39, indicating that MAP–dynein motor-control cortex to help orient the mitotic spindle. Mol. Cell Biol. 30, 3519–3530 (2010). 15. Gonczy, P. 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Cell Biol. 158, 873–884 (2002). ACKNOWLEDGEMENTS 20. Green, R. A., Wollman, R. & Kaplan, K. B. APC and EB1 function together in We thank A. Garrod for selecting the double stable EB3–tdTomato eGFP–CENP-A mitosis to regulate spindle dynamics and chromosome alignment. Mol. Biol. Cell cell line, K. Kaseda for selecting the mCherry–α-tubulin cell line and D. Roth for 16, 4609–4622 (2005). assistance with cloning. We are grateful to I. Titley at the Institute of Cancer Research 21. Wang, X. M., Peloquin, J. G., Zhai, Y., Bulinski, J. C. & Borisy, G. G. Removal of in Sutton, UK, for help with cell sorting, N. Galjart for the gift of CLASP1 antiserum MAP4 from microtubules in vivo produces no observable phenotype at the cellular and A. Akhmanova for providing CLASP1 and CLASP2 rescue constructs. This level. J. Cell Biol. 132, 345–357 (1996). work was supported by programme grants to A.D.M. and A.S. from Marie Curie 22. Shiina, N. & Tsukita, S. 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METHODS containing 10% FCS (Invitrogen). Images were acquired every 3 min for 10 h using a Cell culture, siRNA transfection and drug treatments. HeLa-E1 and HeLa-K 40 oil NA 1.3 objective on an Olympus Deltavision Personal microscope (Applied × cells were grown at 37 ◦C in DMEM-GlutaMAX medium containing 10% FCS, Precision, LLC) equipped with a DAPI–GFP–tetramethyl rhodamine isothiocyanate 1 1 100 U ml− penicillin and 100 µg ml− streptomycin with 5% CO2 in a humidified (TRITC) filter set (Chroma). The MC049 cell line (described above) was imaged incubator. Human RPE1 cells immortalized with hTERT (Clontech) were grown at at 37 ◦C using a 100 oil NA 1.4 objective on an Olympus Deltavision Personal 1 × 37 ◦C in DMEM/F-12 medium containing 10% FCS, 2.3gl− sodium bicarbonate, microscope (Applied Precision, LLC). Fluorescence microscopy 1,024 1,024 pixel 1 1 × 100 U ml− penicillin and 100 µg ml− streptomycin with 5% CO2 in a humidified images were acquired at 50% neutral density with 300 ms exposure at a temporal incubator. To establish the stable HeLa-K cell line expressing centromere protein resolution of 500 ms for 120 s using a TRITC filter set (Chroma). Images were A (CENP-A)–eGFP and EB3–tdTomato, a CENP-A–eGFP stable cell line40 was deconvolved with a medium noise filtering method for ten iterations using SoftWorx transfected with pKan–CMV–Mapre3–tdTomato and positive cells were selected (Applied Precision, LLC). 1 with 200 µg ml− Geneticin (Sigma). Clonal lines were then FACS sorted to generate cell line MC049. This cell line was maintained in DMEM-GlutaMAX Measurement of astral-microtubule dynamics. For analyses of astral- 1 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U ml− peni- microtubule dynamics, cells were considered only where both spindle poles, each 1 1 1 cillin, 100 µg ml− streptomycin, 200 µg ml− Geneticin (Sigma) and 0.5 µg ml− at least 2 µm away from the cell cortex, were positioned in one focal plane and puromycin. The RPE1 HR7 cell line was generated by transfecting hTERT-RPE1 exhibited no major movements during the observation time. Astral-microtubule 1 cells (Clontech) with H2B–mRFP followed by selection with 500 mg ml− Geneticin nucleation frequencies were quantified by counting the number of EB3 comets in (Sigma). A stable HeLa-K FRT mCherry–α-tubulin cell line (MC042) was the focal plane that originated from each spindle pole during the observation time. constructed by transfecting pCherry–tubulin–IRES–puro2 (pMC206) and selecting Average cytoplasmic growth speeds for individual comets were measured as the 1 positive clones with 0.4 µg ml− puromycin. Short RNA-mediated interference total distance travelled from the spindle pole towards the cell cortex divided by oligonucleotides (60 nM); control siRNA (5-GGACCUGGAGGUCUGCUGU- the time. Cortical contact frequencies were determined by counting how many EB3 3), MAP4-1 siRNA (5-GAAACAGAAGUAGUUCUCAUCAAGA-3), MAP4-4 comets nucleated by each spindle pole touched the cell cortex during the observation siRNA (5-CCCACCACCAUUGGUGGGUUGAAUA-3), MAP4-3 siRNA (5- time. For each of these, static dwell time was measured as the period that an EB3 CGAUACUACAGGGUCUCCAACUGAA-3) (Invitrogen), DHC siRNA (ref. 41), comet spent at the cortex before undergoing catastrophe or starting to move. Only CLASP1 siRNA (ref. 30) and CLASP2 siRNA (ref. 30) were transfected using cortical movements of at least 1 µm in length were considered. For the analysis Oligofectamine (Invitrogen) as per the manufacturer’s instructions and analysed of astral-microtubule movements in cells where poles were adjacent to the cell 48 h (MAP4, CLASP1, CLASP2 and DHC), 72 h (DHC) or 96 h (DHC) after trans- cortex, all cortical movements independent of run length were taken into account. fection. For experiments in Figs 2, 3, 5 and 7 Table 1 the MAP4-3 oligonucleotide For each of these, instantaneous cortical movement speed was measured between was used. All siRNA treatments were validated by immunofluorescence or successive frames. Kymographs of astral-microtubule and spindle-pole movements immunoblotting (Figs 1a–c, 4a, 5a). For drug treatments, HeLa-E1 cells were treated were obtained using MetaMorph image-analysis software (Molecular Devices). with 1 µM MG132 or 0.1–1 µM sodium orthovanadate for 1 h before fixation for immunofluorescence. Protein purification and dynein gliding assays. 6 His-tagged MAP4 was × expressed in E. coli strain BL21-CodonPlus-(DE3) and expression was induced Whole-cell lysates were prepared using a liquid-nitrogen Immunoblotting. with 0.5 mM isopropyl-β-d-thiogalactoside at 37 ◦C. Bacteria were lysed in binding grinding method as described previously42. Immunoblotting was carried out as buffer (50 mM NaPO4 buffer, pH 8.0; 300 mM NaCl; 2 mM β-mercaptoethanol; 43 described using rabbit anti-MAP4 (1:1,000 dilution; H-300, Santa Cruz), mouse 15% glycerol) by sonication. MAP4 was bound to Ni-NTA resin (Qiagen) and Glued anti-α-tubulin (1:10,000; DM1A, Sigma-Aldrich), mouse anti-p150 (1:1,000, eluted with 250 mM imidazole in binding buffer. After twofold dilution with BD Biosciences), mouse anti-DHC (1:200; Sigma-Aldrich), rabbit anti-CLASP1 low-salt buffer (20 mM MES, pH 6.8; 1 mM EGTA; 0.5 mM MgCl2) the MAP4- (1:3,000; Rb2292; ref. 30), rat anti-CLASP2 (1:2,000; KT68, Absea Biotechnology), containing fractions were loaded on SP Fast Flow Sepharose (GE Healthcare), anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies washed with low-salt buffer and eluted with a step gradient of high-salt buffer (Amersham) and anti-rat horseradish peroxidase-conjugated antibodies (Abcam). (20 mM MES, pH 6.8; 1 mM EGTA; 0.5 mM MgCl2; 1 M NaCl). All purification was carried out at room temperature to minimize protein aggregation. Proteins Plasmid construction and siRNA-rescue experiments. To generate were analysed by SDS–polyacrylamide gel electrophoresis and SimplyBlue staining pKan–CMV–Mapre3–tdTomato, complementary DNA encoding the long isoform (Invitrogen). Buffer exchange to BRB80 (80 mM PIPES, pH 6.8; 1 mM MgCl ; of mouse EB3 (ref. 44) was amplified by polymerase chain reaction to introduce 2 1 mM EGTA) was carried out using Vivaspin spin columns (Sartorius) according SacI and SacII restriction sites at 5 and 3 ends respectively and cloned to to the manufacturer’s protocol. Cytoplasmic dynein was purified from mouse pEGFP-N1 (Clontech). Subsequently, eGFP was replaced by tdTomato45 using brains as described previously47,48. Dynein gliding assays were carried out in a 10 µl a BamHI–BsrGI digest. The resulting frameshift was corrected by cutting with flow chamber made from a glass slide, coverslip and double-sided tape. Assays BamHI, treating with mung-bean nuclease and religating the blunt ends. To were done in motility assay buffer (MAB: 10 mM Na-PIPES, 50 mM potassium generate pCherry–tubulin–IRES–puro2 (pMC206), mCherry–αtubulin45 was acetate, 5 mM MgSO4, 1 mM EGTA, pH 7.0). The chamber was first incubated cloned as an NheI–BamHI fragment into pIRES–puro2 (Clontech). To generate 1 with 0.05 mg ml− anti-dynein antibody (mAB, 1618, Millipore) for 5 min. A BSA pKan–CMV–eGFP–uMAP4, the ubiquitous isoform of mouse MAP4 (uMAP4) 1 wash was then added and incubated for 5 min (1 mg ml− BSA, 10 µM Taxol open reading frame (GenBank accession M72414) was amplified from C2C12 in MAB). Next, three chamber-fulls of dynein, with 5 min incubations each, cDNA, thereby introducing SalI and SacII restriction sites at 5 and 3 ends enabled the dynein to coat the chamber surface. Rhodamine-labelled microtubules respectively and cloned into pEGFP-C1 (Clontech). To generate 6 His-tagged 1 × (0.05 mg ml− , in 20 µM Taxol and MAB), pre-incubated with either MAP4 or MAP4, the uMAP4 open reading frame from above was cloned into the MscI S 46 BRB80 in a 1:1 ratio for 20 min, were allowed to bind for 2 min. Finally, an ATP site of the pCAP vector , subsequently digested with SalI and MfeI and 1 1 mix (MAP4 or BRB80, 1 mM ATP, 10 µM Taxol, 15 mg ml− glucose, 0.5 mg ml− cloned into XhoI- and EcoRI-digested pRSET-A vector (Invitrogen). For rescue 1 glucose oxidase, 0.63 mg ml− catalase in MAB) was added to the chamber. Five experiments, cells were transiently transfected with pKan–CMV–eGFP–uMAP4, minute movies were taken using epifluorescence, imaging every 5 s with a 500 ms RNA interference-resistant GFP–CLASP1α (ref. 30) or GFP–CLASP2α (ref. 30) exposure time using a TiE epifluorescence microscope (Nikon) with a 60 24 h after siRNA transfection using FuGENE 6 reagent (Roche) according to the × 1.49 NA oil-coupled objective, a Cascade II EM–CCD (charge-coupled device) manufacturer’s protocol and analysed 48 h later. camera (Roper Scientific) and Nikon Elements software. Average microtubule Immunofluorescence microscopy. Cells were fixed for 6 min in cold methanol velocities were determined from stacks of 16 bit tiffs using the ImageJ plug-in, at 20 ◦C. Primary antibodies were used as follows: rabbit anti-MAP4 (1:500 MtrackJ (http://www.imagescience.org/meijering/software/mtrackj/). Kymographs − dilution; H-300, Santa Cruz), mouse anti-α-tubulin (1:1,000; Sigma-Aldrich), of 3-pixel-wide lines were constructed using the ImageJ kymograph plugin (http:// rabbit anti-γ-tubulin (1:200, Abcam) and CREST antisera (1:250; Antibodies www.embl.de/eamnet/html/body_kymograph.html). Incorporated). Cross-adsorbed secondary antibodies (Molecular Probes) were used. Three-dimensional image stacks were acquired in 0.2 µm steps using an 100 oil Cell division on patterned substrates. RPE1 HR7 cells were treated with × NA 1.4 objective on an Olympus Deltavision RT microscope (Applied Precision, siRNA for 48 h, trypsinized and seeded onto CYTOO Starter’s chips as per the LLC) equipped with a DAPI–fluorescein isothiocyanate–Rhod/TR–CY5 filter set manufacturer’s instructions 3 h before imaging. CYTOO chips were transferred to (Chroma) and a Coolsnap HQ camera. Deconvolution of three-dimensional image a CYTOO chamber and imaged at 37 ◦C, 5% CO2 in an ONICS stage incubator (Tokai Hit) on an Olympus Deltavision Personal microscope (API) using a 10 stacks and quantitative measurements were carried out with SoftWorx (Applied × Precision, LLC). objective. On each chip, 48 views covering 1,728 medium-sized H patterns and 32 views covering 1,152 medium-sized disc patterns were imaged every 5 min Live-cell imaging. HeLa cells expressing H2B–eGFP and mRFP–α-tubulin (ref. 43) using bright field and an mCherry filter set. The division angle was determined were imaged at 37 ◦C in LabTech II (NUNC) chambers in Leibovitz’s L-15 medium in the first image where furrow ingression was visible as shown in Fig. 7a

NATURE CELL BIOLOGY © 2011 Macmillan Publishers Limited. All rights reserved. DOI: 10.1038/ncb2297 METHODS using Softworx (Applied Precision, LLC). Data representation was done using 44. Straube, A. & Merdes, A. EB3 regulates microtubule dynamics at the cell cortex and Origin (OriginLab). is required for myoblast elongation and fusion. Curr. Biol. 17, 1318–1325 (2007). 45. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent 40. Jaqaman, K. et al. Kinetochore alignment within the metaphase plate is regulated proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, by centromere stiffness and microtubule depolymerases. J. Cell Biol. 188, 1567–1572 (2004). 665–679 (2010). 46. Schlieper, D., von Wilcken-Bergmann, B., Schmidt, M., Sobek, H. & Muller-Hill, B. 41. Draviam, V. M., Shapiro, I., Aldridge, B. & Sorger, P. K. Misorientation and reduced A positive selection vector for cloning of long polymerase chain reaction fragments stretching of aligned sister kinetochores promote chromosome missegregation in based on a lethal mutant of the crp gene of Escherichia coli. Anal. Biochem. 257, EB1- or APC-depleted cells. EMBO J. 25, 2814–2827 (2006). 203–209 (1998). 42. McClelland, S.E. & McAinsh, A.D. Hydrodynamic analysis of human kinetochore 47. Ross, J. L., Wallace, K., Shuman, H., Goldman, Y. E. & Holzbaur, E. L. Processive complexes during mitosis. Methods Mol. Biol. 545, 81–98 (2009). bidirectional motion of dynein–dynactin complexes in vitro. Nat. Cell Biol. 8, 43. McAinsh, A. D., Meraldi, P., Draviam, V. M., Toso, A. & Sorger, P. K. The human 562–570 (2006). kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome 48. Bingham, J. B., King, S. J. & Schroer, T. A. Purification of dynactin and dynein from segregation. EMBO J. 25, 4033–4049 (2006). brain tissue. Methods Enzymol. 298, 171–184 (1998).

DOI: 10.1038/ncb2297 0"12-$%34 $

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Figure S1 MAP4 depletion causes a delay in mitotic progression. (a) Frames MG132. Chromosomes in metaphase cells were counted as unaligned if from live-cell movies of HeLa cells expressing H2B-eGFP/α-tubulin-mRFP they were located outside the central 30% of the mitotic spindle. n=60 cells (to mark chromosomes and MTs) in siControl (upper panel) and siMAP4 cells from 2 independent experiments. (e) Representative images of siControl and (lower panel). Scale bar = 10 µm. (b) Cumulative frequency plots of anaphase siMAP4 treated cells arrested in metaphase after a 1 hour treatment with onset times as determined from live-cell movies in siControl (black line) and MG132 and stained with α -tubulin (red), γ-tubulin (green) and CREST (blue) siMAP4 (red line) treated cells. t = 0 min is defined as NEBD and anaphase antisera. Scale bar = 10 µm. (f) Examples from a live-cell movie showing a onset as the time point where the chromatids first separate. After 60 min lagging chromosome and a DNA bridge in anaphase. Scale bar = 10 µm. (g) 95% of control cells have initiated anaphase compared to only 35% in MAP4- Percentage of cells with chromosome segregation problems in siControl or depleted cells. Note that the CENP-A-GFP EB3-tdTomato cell line which is siMAP4 treated cells. Segregation problems were scored using live-cell movies. used in this study (see Fig. 3, Fig. S2b,c and Table 1) has a normal timing Error bars represent SD. n=150 cells from 3 independent experiments. (h) from NEBD to anaphase onset. n=150 cells from 3 independent experiments. Percentage of cells that underwent vertical anaphase in siControl and siMAP4 (c) Cumulative frequency plots of initial metaphase plate formation (all treated cells. Error bars represent SD. n=150 cells from 3 independent chromosomes aligned on spindle equator) times in siControl and siMAP4 experiments. (I) Zoom of the white box in (a) siMAP4 t = 141min, showing treated cells, with NEBD defined as t = 0 min, as determined from live-cell a de-aligned chromosome. Scale bar = 2 µm. (j) Percentage of cells with movies. By t = 42 min, 95% of siControl cells had formed a metaphase plate unstable metaphase plate in siControl or siMAP4 treated cells. We define compared to 70% in siMAP4 treated cells. n=150 cells from 3 independent “unstable metaphase plate” as a metaphase plate that has chromosomes experiments. (d) Percentage of cells with misaligned chromosomes in siControl leaving the plate after the initial alignment is complete. Error bars represent or siMAP4 treated cells after a 1 hour treatment with the proteasome inhibitor SD. n=150 cells from 3 independent experiments.

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Supplementary Movie Legends

Movie S1- MAP4 depleted cell with transient bent spindle after nuclear envelope breakdown. Movie S2-- MAP4 depleted cell with transient diamond spindle after nuclear envelope breakdown. Movie S3- H2B-EGFP/α-tubulin–mRFP Control-depleted cell undergoing mitosis Movie S4- H2B-EGFP/α-tubulin–mRFP MAP4-depleted cell undergoing mitosis Movie S5- Growth of spindle and astral MT plus ends in a control cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end. Movie S6- Growth of spindle and astral MT in a control cell stably expressing mCherry-α-Tubulin. Red arrow: microtubule catastrophe event. Yellow arrow: pre- warning of cortical microtubule sliding event or a catastrophe event. Green arrow: microtubule side-on attachment. Movie S7- Growth of spindle and astral MT plus ends in a MAP4 depleted cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end. Movie S8- Movement of astral MT plus ends and spindle poles in a MAP4 depleted cell stably expressing EB3-tdTomato where the pole to cortex distance is < 1 µm. Note frequent cortical movement events. Movie S9- Growth of spindle and astral MT plus ends in a dynein depleted cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end. Movie S10- Growth of spindle and astral MT plus ends in a MAP4 and dynein depleted cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end. Movie S11- H2B-EGFP/α-tubulin–mRFP Control-depleted cell undergoing mitosis Movie S12-. H2B-EGFP/α-tubulin–mRFP CLASP1-depleted cell undergoing mitosis Movie S13- H2B-EGFP/α-tubulin–mRFP CLASP1- and MAP4-depleted cell undergoing mitosis Movie S14- Growth of spindle and astral MT plus ends in a CLASP1 depleted cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end. Movie S15- Growth of spindle and astral MT plus ends in a MAP4 and CLASP1 depleted cell stably expressing EB3-tdTomato. Red arrow: pre-warning of cortical microtubule sliding event. Yellow arrow: arrival of microtubule plus end. Green arrow: moving microtubule plus end.