3834 Research Article

IntroductionNcd, a minus-end kinesin-14 motor, plays an essential role inassembly of the anastral Drosophila oocyte meiosis I spindle(Matthies et al., 1996; Endow and Komma, 1997; Endow andKomma, 1998), where it is thought to crosslink and bundlemicrotubules to form foci or asters that migrate towards thechromosomes and nucleate microtubules for spindle assembly(Sköld et al., 2005). The motor also acts during oocyte meiosis Ispindle assembly to form lateral interactions between microtubule-coated bivalent chromosomes and stabilizes the interactions bysliding microtubules against one another (Sköld et al., 2005). TheNcd motor binds to centrosomes and microtubules throughout themitotic spindle (Hatsumi and Endow, 1992; Endow et al., 1994;Endow and Komma, 1996), and is thought to function in the mitoticspindle by binding to microtubules and hydrolyzing ATP to produceforce to maintain spindle length or elongate the spindle (Saundersand Hoyt, 1992). In particular, Ncd has been proposed to act as abrake in the spindle by opposing outward-acting forces (Sharp etal., 1999; Sharp et al., 2000; Tao et al., 2006). The motor also hasan important role in attaching centrosomes to mitotic spindle polesand chromosomes to spindles in early embryos (Endow et al., 1994;Endow and Komma, 1996).

Binding by the kinesin microtubule motors to the spindle isassumed to be by the highly conserved motor domain or head, whichcontains an invariant microtubule-binding motif and can bind tomicrotubules in vitro when expressed by itself (Chandra et al.,1993b; Hirose et al., 1995; Lockhart et al., 1995). The Ncd tail hasalso been reported to contain regions that bind tightly tomicrotubules in vitro and has been speculated to mediate interactionsof the motor with microtubules in vivo (Karabay and Walker,1999a), although tests of this hypothesis have not been reported.Information regarding the way in which Ncd and other spindle

motors bind to the spindle is essential to understand how the motorsperform their functions and produce force in the spindle.

Here we analyze Ncd head and tail binding interactions with thespindle using tailless and headless motors expressed in Drosophilaoocytes and embryos as fusions to green fluorescent protein.Unexpectedly, we found that the tailless Ncd motor does not bindto oocyte meiosis I spindles and binds weakly to mitotic spindlesand centrosomes. By contrast, headless Ncd binds to both oocytemeiotic and mitotic spindles. Thus, rather than the conserved motordomain playing a dominant role, Ncd binding to the spindle andcentrosomes is mediated predominantly by the tail. Tight bindingby the motor to spindle microtubules results in motor transport fromthe poles to the equator during spindle assembly in early metaphase,as revealed by analysis of fluorescence flow in the spindle, whichwas opposite to the direction of movement of the minus-end Ncdmotor along spindle microtubules.

ResultsHLNcdVenus and TLNcdVenusTo determine the role of the Ncd head and tail in binding to thespindle, we recovered germline transformants that express headless(HL) or tailless (TL) Ncd, regulated by the native ncd promotorand fused to Venus, an enhanced green fluorescent protein (Nagaiet al., 2002) (Fig. 1A). HLncdVenus encodes the Ncd tail and coiled-coil stalk, and TLncdVenus, the stalk and conserved motor domain,both joined at the C terminus to Venus. FLncdVenus, expressingfull-length Ncd fused to Venus, was made as a control.

HLncdVenus and TLncdVenus fail to rescue an ncd-null mutantHLncdVenus and TLncdVenus flies homozygous for the transgeneand the ncd-null mutant cand were tested for rescue of cand embryoinviability caused by elevated levels of chromosome nondisjunction

The Ncd kinesin-14 motor is required for meiotic spindleassembly in Drosophila oocytes and produces force in mitoticspindles that opposes other motors. Despite extensive studies,the way the motor binds to the spindle to perform its functionsis not well understood. By analyzing Ncd deleted for theconserved head or the positively charged tail, we found that thetail is essential for binding to spindles and centrosomes, but boththe head and tail are needed for normal spindle assembly andfunction. Fluorescence photobleaching assays to analyze bindinginteractions with the spindle yielded data for headless and full-length Ncd that did not fit well to previous recovery models.We report a new model that accounts for Ncd transport towardsthe equator revealed by fluorescence flow analysis of earlymitotic spindles and gives rate constants that confirm the

dominant role the Ncd tail plays in binding to the spindle. Bycontrast, the head binds weakly to spindles based on analysisof the tailless fluorescence recovery data. Minus-end Ncd thusbinds tightly to spindles and is transported in early metaphasetowards microtubule plus-ends, the opposite direction to thatin which the motor moves, to produce force in the spindle laterin mitosis.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/22/3833/DC1

Key words: Kinesin-14 motor, Ncd, Mitotic spindle, FRAP, Bindingkinetics, Transport

Summary

Ncd motor binding and transport in the spindleMark A. Hallen, Zhang-Yi Liang and Sharyn A. Endow*Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA*Author for correspondence (e-mail: endow@duke.edu)

Accepted 27 August 2008Journal of Cell Science 121, 3834-3841 Published by The Company of Biologists 2008doi:10.1242/jcs.038497

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and loss. Embryo viability of HLncdVenus; cand

(0.114, n=19, total=167) and TLncdVenus; cand linesM23M4 (0.187, n=25, total=134) and F26F2 (0.101,n=17, total=168) was similar to cand (0.113, n=144,total=1277) (Endow and Komma, 1997). By contrast,FLncdVenus cand (0.698, n=113, total=162) and atransgenic line expressing four copies of ncd fusedto S65T gfp (Endow and Komma, 1997; Heim et al.,1995), ncdgfp* cand 4121 (0.868, n=79, total=91),showed much higher embryo viability. HLncdVenus(χ2=116.6, 1 d.f., P<0.0001) and TLncdVenus linesM23M4 (χ2=75.0, 1 d.f., P<0.0001) and F26F2(χ2=121.9, 1 d.f., P<0.0001) differ significantly fromFLncdVenus; they also differ significantly fromncdgfp* 4121. Thus, Ncd deleted for either the tailor head fails to rescue embryo inviability of an ncd-null mutant caused by frequent chromosome mis-segregation during meiosis in oocytes and mitosis inearly embryos.

HLNcdVenus and TLNcdVenus in the oocytemeiosis I spindleMature HLncdVenus; cand oocytes expressing Ncdwithout the head showed multi-polar or multiple smallmeiosis I spindles (n=11, total=11) (Fig. 1B),consistent with previous findings that Ncd is requiredfor spindle assembly in oocytes (Kimble and Church,1983; Matthies et al., 1996; Endow and Komma,1997; Sköld et al., 2005). The spindles did notresemble the mature metaphase-arrested FLncdVenusoocyte meiosis I spindle, but exhibited abnormalitiestypical of null or severe loss-of-function mutants, suchas ncd2, which is blocked in the initial stages ofspindle assembly (Sköld et al., 2005). The multi-polaror multiple small oocyte spindles demonstrate that theNcd tail does not rescue cand for spindle assembly.They show that the Ncd tail is sufficient to bind tomeiotic spindles and does not require the head, butthe head is needed for normal assembly of the oocytemeiosis I spindle. The multi-polar and multiple smallspindles indicate that the head is needed to form and stabilize lateralinteractions between microtubule-associated bivalent chromosomesduring assembly of the meiosis I spindle (Sköld et al., 2005).

Immature TLncdVenus; cand oocytes showed cytoplasmicfluorescence but the germinal vesicle was dark, indicating thatTLNcdVenus was expressed but was excluded from the nucleus.Low fluorescence was also observed in mature oocytes followinggerminal vesicle breakdown, but meiosis I spindles were notobserved. Oocytes expressing wild-type Ncd fused to a monomericred fluorescent protein (mRFP) (Campbell et al., 2002) togetherwith TLNcdVenus were examined to provide a marker for themeiosis I spindle. The spindles fluoresced red, but showed no greenfluorescence, indicating little or no TLNcdVenus binding to thespindle (n=8, total=8) (Fig. 1B). The lack of binding byTLNcdVenus to the meiosis I spindle is consistent with the highfrequency of TLNcdVenus; cand embryo lethality, similar to the cand-null mutant.

HLNcdVenus and TLNcdVenus in the mitotic spindleHLncdVenus embryos exhibited bright spindle and centrosomefluorescence throughout the early cleavage divisions, similar to

FLncdVenus and ncdgfp* embryos (Endow and Komma, 1996), butabnormal mitotic spindles were frequently observed in HLncdVenusembryos (n=29, total=29). The abnormalities included spindle spursor branches caused by mis-segregating chromosomes detaching fromthe spindle or moving off the metaphase plate (Endow and Komma,1996), bent anaphase spindles as a result of unequal forces in thespindle and poorly formed midzones in telophase (Fig. 1C;supplementary material Movie 1). Free centrosomes were presentin some embryos that arose from failure of spindles to form orcentrosome loss from a spindle pole, based on analysis of the time-lapse sequences. A few embryos (n=3, total=29) showed highlyaberrant divisions caused by abnormal oocyte meiotic divisions, butmost embryos (n=26, total=29) displayed some spindles thatappeared normal and others with abnormalities that increased infrequency in later divisions, similar to those in embryos of the severeloss-of-function mutant ncd2 (Endow and Komma, 1996). The timerequired for HLncdVenus; cand cycle 10 embryos to progress fromnuclear envelope breakdown (NEB) in prometaphase to midzoneformation in telophase was 248±5 seconds (n=15), not significantlydifferent from FLncdVenus; cand (263±9 seconds, n=9) or ncdgfp*cand 4121 (245±3 seconds, n=17).

Fig. 1. Ncd in the meiotic and mitotic spindle. (A) Ncd constructs. FLNcdVenus consists of anN-terminal basic, proline-rich tail, α-helical coiled-coil stalk (grey) and conserved motordomain or head, with the Venus fluorescent protein (green) at the C terminus. HLNcdVenus isdeleted for the head and TLNcdVenus, for the tail. (B) FLncdVenus; cand oocyte meiosis Ispindle (top), abnormal multi-polar and multiple small HLncdVenus; cand meiosis I spindles(middle) and absence of TLNcdVenus fluorescence in a TLncdVenus; ncdmRFP cand meiosisI spindle (bottom). Scale bars: 5 μm. (C) Prometaphase to telophase in a HLncdVenus cycle 10(left) and TLncdVenus cycle 9 (right) mitotic division. Abnormal fused, spurred, bridged orbent spindles, or failure of midzone to form (arrows). Time, minutes:seconds. Scale bar:10 μm.

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TLncdVenus; cand embryos showed dark interphase nuclei priorto mitosis and uniform cytoplasmic fluorescence, indicating thatTLNcdVenus was expressed but was excluded from the nucleus,as in oocytes. The nuclei became fluorescent immediately followingNEB and faintly fluorescent spindles were apparent soon after (Fig.1C). TLncdVenus mitotic spindles showed a gap in fluorescence atthe metaphase plate, which was similar to the gap at thechromosomes observed in rhodamine-tubulin-labeled spindles(Endow and Komma, 1996); centrosomes showed faint fluorescence(Fig. 1C; supplementary material Fig. S1), differing from those inHLncdVenus and FLncdVenus. The pole-to-pole Ncd fluorescencein mitotic spindles at metaphase is thought to be caused by Ncdfibers or filaments that traverse the chromosomes (Endow andKomma, 1996). The gap at the chromosomes was not observed inHLncdVenus spindles; its presence in TLncdVenus spindles ispresumably caused by dependence of fiber or filament formationon the Ncd tail. The gap disappeared in early anaphase as the spindleelongated (Fig. 1C; supplementary material Movie 2). TLncdVenus;cand embryos frequently displayed abnormally spurred or bridgedspindles, nuclei that failed to form spindles or different-sizedspindles or nuclei, an indication of chromosome loss or mis-segregation (n=12, total=12). The time required for cycle 10embryos to progress from NEB to midzone formation (357±7seconds, n=14) was significantly longer than HLncdVenus,FLncdVenus or ncdgfp*. The TLncdVenus; cand embryosdemonstrate that the Ncd head without the tail binds weakly to themitotic spindle and frequently causes abnormal spindles to form.

HLncdVenus spindles are longer than those in the wild typeHLncdVenus; cand mitotic spindles were longer by 1-2 μm thanFLncdVenus; cand spindles in prometaphase and metaphase; thedifference in length increased to 3-4 μm in anaphase and telophase,reaching ~4 μm in mid-telophase (HLncdVenus, 23.7±0.3 μm, n=32;FLncdVenus, 19.9±0.2 μm, n=38) (Fig. 2; Table 1). The longer thannormal HLncdVenus spindles imply that Ncd regulates spindlelength during anaphase and telophase, presumably by opposing othermotors in the spindle. These results parallel previous reports thatcand spindles lacking Ncd are longer than those in the wild type inlate cleavage divisions (Sharp et al., 1999; Sharp et al., 2000).TLncdVenus spindles showed little or no centrosome fluorescence,making it difficult to measure pole-to-pole spindle lengths duringmitosis. Measurements made in mid-telophase, the time at whichthe greatest difference was observed between HLncdVenus andFLncdVenus spindles, and where the centrosomes were faintlyvisible against the dark telophase nuclei, gave a length of 18.8±0.3μm (n=26), close to the value for FLncdVenus spindles of 19.9±0.2μm, indicating that the TLNcdVenus motor can function to regulatespindle length.

NcdVenus fluorescence recovery in the mitotic spindlePhotobleaching assays were performed to estimate kinetic constantsfor Ncd binding interactions with the mitotic spindle. Two different-sized regions of interest (ROIs) at the equator (radius, w=2.66 μmand 1.3 μm) were photobleached at high laser power in spindles ofcycle 9-11 embryos and images were recorded rapidly (~165mseconds/frame) at low laser power to monitor recovery. Thefluorescence intensity in the bleach spot was normalized, correctedfor loss during imaging, averaged with data from replicate assaysand plotted against time. Assays of FLNcdVenus showed slowerfluorescence recovery in the large ROI (n=11) than the small ROI(n=5), as predicted if recovery is dependent on diffusion (Fig. 3A,B;

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supplementary material Movies 3 and 4). However, the large ROIshowed a greater delay than expected because of effects of diffusionalone. HLNcdVenus assays showed an even greater difference

Fig. 2. Spindle length in FLncdVenus and HLncdVenus embryos.(A) Centrosome-to-centrosome spindle length in cycle 10 embryos fromprometaphase to telophase. Data are the mean ± s.e.m. (B) Spindle imagesfrom time-lapse sequences. Note the difference in scale between theFLncdVenus and HLncdVenus images. ProM, prometaphase; M, metaphase;A, anaphase; T, telophase. Scale bars: 5 μm.

Table 1. Spindle length during mitosis

Length FLncdVenus (n) HLncdVenus (n) difference

Prometaphase 10.2±0.2 (38) 11.3±0.1 (37) 1.1Metaphase 11.8±0.2 (30) 13.5±0.1 (37) 1.7Late anaphase 16.8±0.3 (38) 19.6±0.2 (37) 2.8Early telophase 18.9±0.3 (39) 22.6±0.2 (32) 3.7Mid-telophase 19.9±0.2 (38) 23.7±0.3 (32) 3.8Late telophase 21.0±0.4 (25) 23.1±0.4 (28) 2.1Later telophase 20.8±0.5 (14) 23.4±0.4 (28) 2.6

Centrosome-to-centrosome spindle length (μm) and length differences(μm) between HLncdVenus spindles and FLncdVenus spindles. Late anaphaseto later telophase stages differ by 30-second intervals.

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between the large (n=10) and small ROI (n=8), and similarly shapedrecovery curves (Fig. 3C,D), indicating that the delayed recoveryof the large ROI could be attributed to Ncd tail effects. TLNcdVenusassays showed a much smaller difference between the large (n=11)and small ROI (n=8) recovery curves and more rapid recovery thanFLNcdVenus or HLNcdVenus for both ROIs (Fig. 3E,F), providingfurther evidence that the delayed recovery of the FLNcdVenus largeROI is caused by the Ncd tail, rather than the head.

Attempts to fit models for FRAP recovery to the FLNcdVenusand HLNcdVenus data based on binding or both diffusion andbinding (Sprague et al., 2004) gave poor fits (supplementarymaterial Fig. S2). By contrast, the TLNcdVenus FRAP recoverydata fit well to the diffusion-binding model reported previously,which accounts for recovery by both diffusion and bindinginteractions (Sprague et al., 2004). Concurrent fits of theTLNcdVenus data for the large and small ROI to this model (Hallenet al., 2008) yielded values for k*on, the pseudo-first-order bindingrate constant; koff, the dissociation rate constant; Ceq, the fraction

of the motor bound at equilibrium; Feq, the fraction free atequilibrium; and Deff, the effective diffusion coefficient in the spindle(Table 2). The TLNcdVenus dissociation and binding rate constantswere koff=0.06±0.01 second–1 and k*on=0.005±0.001 second–1. Thelow koff and k*on values and koff �k*on indicate weak binding, bothkinetically and thermodynamically. The low Ceq=0.07±0.02 andcorrespondingly high Feq=0.93 explain the low level of fluorescenceof TLncdVenus mitotic spindles.

Of the previous models, the FLNcdVenus and HLNcdVenusFRAP recovery data fit best to a two-state binding model (r2=0.98)and the diffusion-binding model (r2=0.98), but the data stillexhibited strong deviations from the models in several phases ofthe curve, including the beginning, which is strongly influenced byrapid diffusional effects, a quasi-linear middle region nonexistentin the model, and the end, which is dominated by bindinginteractions (supplementary material Fig. S2). Consequently, wesought to modify the diffusion-binding model to reduce thesedeviations and account for other factors that contribute to the

recovery. The marked difference in shape ofthe large and small ROI recovery curves forFLNcdVenus and HLNcdVenus indicate apronounced delay in recovery of the large ROI,suggesting depletion throughout recovery ofthe motor that is free to diffuse, possiblybecause of tight binding by FLNcd or HLNcdto spindle microtubules. This raised thepossibility that recovery occurs by growth ortransport of microtubules with bound Ncd intothe bleach spot.

As current FRAP models do not account forrecovery by transport, we derived a model thatassumes rapid diffusional equilibration ofunbleached free Ncd in the bleach spot andrecovery by Ncd binding to microtubules,followed by growth or transport ofmicrotubules with bound motor into the bleachspot. The new model fit the data well for bothFLNcdVenus and HLNcdVenus (r2=0.99),exhibiting a quasi-linear middle region, like thedata, that can be attributed to the effects of

Fig. 3. NcdVenus fluorescence recovery in the mitoticspindle. (A) FLncdVenus spindle FRAP assays at theequator in a small (radius, w=1.3 μm) (top) and large(w=2.66 μm) (bottom) ROI. (B) Mean fluorescencerecovery curves (magenta; small ROI, n=5; large ROI,n=11) after normalization to the first prebleach imageand correction for fluorescence loss during recoveryimaging. Inset, curve fits to the new diffusion-binding-transport model (dark green).(C) HLncdVenus spindle FRAP assays at the equatorin a small (w=1.3 μm) (top) and large (w=2.66 μm)(bottom) ROI. (D) Mean fluorescence recovery curves(purple; small ROI, n=8; large ROI, n=10) afternormalization and correction for loss during imaging.Inset, curve fits to the new diffusion-binding-transportmodel (dark green). (E) TLncdVenus spindle FRAPassays at the equator in a small (w=1.3 μm) (top) andlarge (w=2.66 μm) (bottom) ROI. (F) Meanfluorescence recovery curves (violet; small ROI, n=8;large ROI, n=11) after normalization and correctionfor loss during imaging. Inset, curve fits to thediffusion-binding model (Sprague et al., 2004) (darkgreen). Scale bars: 3 μm.

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transport. The model assumes that microtubules in the ROI aregrowing or sliding towards the spindle equator with constantvelocity, although each microtubule moves with a different velocity.The distribution of velocities is approximated as a half-Gaussianwith a peak at zero and width determined by fitting to the recoverydata. This is a tractable approximation to a Poisson distribution,which applies if microtubule growth rates form a Poissondistribution and microtubule velocity is proportional to the numberof motors propelling them, because the number of bound motorsis expected to follow a Poisson distribution. Net growth of spindlemicrotubules towards the equator occurs in early- to mid-metaphaseas spindles assemble, when the FRAP assays were performed.Sliding of microtubules towards the chromosomes is thought to bemediated by plus-end motors, e.g. the KLP61F kinesin-5 motoracting on parallel microtubules, or minus-end motors, such as Ncdor cytoplasmic dynein, sliding anti-parallel microtubules (Tao etal., 2006).

The new diffusion-binding-transport model was fitsimultaneously to the mean data sets for the large and small bleachspots using MATLAB. The curve fits yielded estimated values forkinetic parameters that included v, the mean velocity of Ncd-boundmicrotubule growth or transport into the bleach spot, koff, k*on andDeff (Table 2). The FLNcdVenus dissociation rate constant from thespindle and binding rate constant were koff=0.028±0.004 second–1

and k*on=0.019±0.003 second–1. The low koff and k*on � koff indicatetight binding to the spindle by the Ncd motor. HLNcdVenus alsoshowed a low koff=0.008±0.004 second–1 and k*on=0.006±0.003second–1, both three to four times lower than FLNcdVenus, withk*on�koff, indicating that the tight binding by the FLNcd motor couldbe attributed to the Ncd tail. FLNcdVenus and HLNcdVenus Deff

was two times lower than TLNcdVenus. The mean velocity of Ncd-bound microtubule growth or transport into the bleach spot wasv=0.026±0.002 μm/second for FLNcdVenus and v=0.042±0.002μm/second for HLNcdVenus.

The velocities correspond to the net microtubule growth,including depolymerization, poleward motion and flux, andtransport towards the spindle equator. The slower velocity ofFLNcdVenus movement towards the equator, compared withHLNcdVenus, could be due to poleward motor activity. Thevelocities are close and within a two- to threefold range of themicrotubule growth rate reported for interphase Drosophila S2 cellsof 0.063 μm/second (Rogers et al., 2002); they are also close tothe velocity of the plus-end spindle motor KLP61F (Tao et al.,2006) of 0.04 μm/second. The velocities of Ncd (0.1-0.2μm/second) (Chandra et al., 1993b) and yeast cytoplasmic dynein(0.085±0.030 μm/second) (Reck-Peterson et al., 2006) are higherin vitro, however, motor-mediated microtubule sliding in thespindle is thought to be slowed by plus-end motors (Tao et al.,2006).

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Ncd transport towards the equator in early metaphaseThe net growth or transport of microtubules bound to Ncd in earlymitotic spindles was analyzed by determining the median positionof FLNcdVenus fluorescence in each spindle half, the point alongthe spindle axis where half of the fluorescence is closer to theequator and half is further away. Image sequences of cycle 10spindles (n=7) recorded at 2.32 seconds/frame, were examinedfrom early metaphase to anaphase. Spindles were rotated andaligned manually, then cropped to eliminate centrosomes, astralmicrotubules and cytoplasm. The image sequences from spindlesin different embryos (n=4) were aligned in time by phase of mitosisand the median position of fluorescence in each spindle half overtime was determined relative to a stationary point outside thespindle using a MATLAB routine (fluorescence_medians). Themedian position values were normalized so that each data setstarted at zero and peaked at one to offset differences in spindlesize and length, then the normalized values were averaged (Fig.4A).

The median fluorescence position changed rapidly over timeduring the initial ~60 seconds, moving an average of 1.5±0.2 μmtowards the equator. This initial phase corresponds to rapidmicrotubule growth towards the equator during spindle assemblyand was followed by a plateau, roughly correlating with mid-

Table 2. FRAP kinetic parameters

Deff k*on koff v Recovery (μm2/second) (second–1) (second–1) Feq Ceq (μm/second) (%)

FLNcdVenus 2.9±0.3 0.019±0.003 0.028±0.004 0.59 0.41±0.01 0.026±0.002 99.7HLNcdVenus 3.6±0.4 0.006±0.003 0.008±0.004 0.554 0.446±0.009 0.042±0.002 106TLNcdVenus 5.9±0.4 0.005±0.001 0.06±0.01 0.93 0.07±0.02 – 99.0

Values for FLNcdVenus and HLNcdVenus from concurrent curve fits of the new diffusion-binding-transport model; Feq (the fraction of protein free atequilibrium) was calculated by Feq=1–Ceq, where Ceq is the fraction of protein bound at equilibrium, and k*on (the pseudo first-order binding constant) byk*on=(koff Ceq)/Feq. Values for TLNcdVenus from a diffusion-binding model concurrent curve fit; Ceq=k*on/(k*on+koff) and Feq=1–Ceq (Sprague et al., 2004).Recovery values are the average of the values for the large and small ROIs. Value ± 95% confidence interval from the curve fit.

Fig. 4. Ncd transport in the mitotic spindle. (A) Median fluorescence positionin FLncdVenus cycle 10 mitotic spindles versus time from early metaphasethrough anaphase. Distance from a point outside the pole in each half-spindle(n=14), normalized from zero to one, and averaged. Data are the mean ± s.e.m.The approximate time at which FRAP assays were performed is indicated.(B) Images from time-lapse sequences showing the equator (red line) andmedian fluorescence position in each half-spindle (blue lines) at the start,maximal position towards the equator, and end. Arrows (blue) in A indicate thetimes at which the images were acquired. Bar, 3 μm.

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metaphase (Fig. 4B; supplementary material Movie 5). The medianposition then retreated from the metaphase plate as the spindleelongated. The time at which the FRAP assays were performedcorresponds to the phase of rapid fluorescence flow towards theequator (Fig. 4A,B).

The velocity during the initial ~60 seconds was 0.027±0.004μm/second, not significantly different from the velocity of growthor transport towards the equator estimated from curve fits to theFLNcdVenus FRAP data of v=0.026±0.002 μm/second. The analysisshowed that net flow of FLNcdVenus fluorescence occurs in theearly metaphase spindle and is directed towards the spindle equator.The fluorescence flow is presumed to result from growth ortransport of microtubules with bound Ncd towards the equator,opposite to the direction of movement of the minus-end motor alongspindle microtubules.

DiscussionA large number of kinesin motor proteins have now been shownto function in meiosis and mitosis, performing essential roles inspindle assembly and length regulation (Manning et al., 2007;Saunders et al., 2007; Zou et al., 2008), kinetochore to spindleattachment (Grishchuk et al., 2007), and chromosome oscillationand congression (Mayr et al., 2007; Stumpff et al., 2008). Thesemotors include minus-end Ncd, a kinesin-14 motor required forspindle assembly in Drosophila oocytes (Kimble and Church,1983; Matthies et al., 1996; Endow and Komma, 1997; Sköld etal., 2005) and chromosome segregation in early embryos (Endowet al., 1994; Sharp et al., 2000). Although several studies of thebiochemical and motile properties of partial or truncated Ncd head(Chandra et al., 1993a; Chandra et al., 1993b; Furuta andToyoshima, 2008) and tail proteins (Chandra et al., 1993b;Karabay and Walker, 1999a; Karabay and Walker, 1999b) havebeen reported, the roles of the Ncd head and tail in the spindleare not well understood. By analyzing proteins that contain thecoiled-coil stalk but lack either the conserved head, which isneeded for ATP hydrolysis and force generation, or the basicproline-rich tail, including the microtubule-binding sites identifiedby Karabay and Walker (Karabay and Walker, 1999a), we findthat the tail is essential for motor binding to the spindle, but boththe head and tail are needed for normal spindle assembly and motorfunction in the spindle.

HLNcdVenus and TLNcdVenus in meiosisThe Ncd motor protein deleted for either the head or the tail wasnot sufficient to rescue embryo inviability of the ncd-null mutantcand, which is caused by high levels of chromosome mis-segregationin oocytes and early embryos. TLncdVenus females produced a lowfrequency of viable embryos, comparable to the cand-null mutant,and TLNcdVenus did not bind to the oocyte meiosis I spindle. Thus,the Ncd tail is necessary for spindle binding and motor function inthe meiosis I spindle. HLNcdVenus binds to spindles, but thespindles are abnormal. Mature oocytes that express HLNcdVenusexhibit abnormal multi-polar meiosis I spindles or multiple smallspindles in which lateral interactions have failed to form (Sköld etal., 2005), typical of cand or severe loss-of-function ncd mutants,indicating that the Ncd head is required for normal meiosis I spindleassembly. The multi-polar and multiple small spindles suggest thatthe head is needed to form lateral interactions between spindle-associated bivalent chromosomes for bipolar spindle assembly(Sköld et al., 2005), presumably by sliding and crosslinkingmicrotubules.

HLNcdVenus and TLNcdVenus in mitosisHLNcdVenus binds to mitotic spindles, but the spindles are frequentlybent, indicating unequal forces in the spindle, or spurred or bridgedto other spindles, evidence of mis-segregating chromosomes (Endowand Komma, 1996), and the midzones frequently fail to form. Thelonger than normal HLncdVenus spindles in late anaphase to telophaseindicates that the Ncd head plays a role in regulating spindle lengthby acting as a brake, opposing forces that cause spindle elongation(Sharp et al., 1999; Sharp et al., 2000; Saunders et al., 2007).HLNcdVenus shows that the tail alone can bind to the spindle, butis not sufficient to prevent chromosomes from detaching from themitotic spindle, resulting in spurs and bridges. The bent anaphaseand telophase spindles in HLncdVenus embyros indicate that HLNcdis defective in counteracting other spindle forces, resulting in longerthan normal spindles in late anaphase and telophase. TLncdVenusembryos showed only faint mitotic spindle fluorescence and little orno centrosome staining, and mitotic spindles were frequentlyabnormal. TLncdVenus cell cycle progression was also markedlydelayed and was 90-100+ seconds longer than FLncdVenus orHLncdVenus embryos, indicating that the Ncd head without the tailis not sufficient for motor function in the spindle.

Ncd binds to the spindle by its tailTight binding to spindle microtubules by the Ncd tail was confirmedby estimating binding constants for FLNcdVenus and HLNcdVenusin the mitotic spindle. Data from FRAP assays were fit to a newmathematical model that accounts for fluorescence recovery bydiffusion into the bleach spot of unbleached free Ncd and bindingto spindle microtubules, together with growth or transport ofmicrotubules with bound Ncd into the ROI. Analysis ofFLNcdVenus gave a low koff that was comparable to k*on, consistentwith tight binding to the mitotic spindle. The HLNcdVenus koff andk*on were both lower than those for FLNcdVenus, but did not differsignificantly from one another. By contrast, the TLNcdVenus koff

was much higher than FLNcdVenus or HLNcdVenus, whereask*on was the same or lower, giving koff �k*on, explaining the muchfaster recovery and weaker binding by TLNcdVenus. Deff forTLNcdVenus was also larger than FLNcdVenus or HLNcdVenus,indicating greater diffusional movement in the mitotic spindle.Tight binding by Ncd to the spindle can thus be attributed to thetail. A tailless Ncd has been reported previously to be a weak-binding diffusional motor in vitro (Chandra et al., 1993a); the weakfluorescence of TLncdVenus mitotic spindles and absence offluorescence in meiosis I spindles, together with FRAP kineticconstants showing koff �k*on and Deff greater than FLNcdVenus orHLNcdVenus, indicate that TLNcdVenus binds in a weakdiffusional manner to the spindle in vivo. Weak binding by theNcd motor domain to microtubules in vitro has been reportedpreviously (Kd,ATP=5.8±1.1 μM) (Song and Endow, 1998),compared with the tight binding by Ncd tail regions (<1 μM)(Karabay and Walker, 1999a). Ncd binding to the mitotic spindleand centrosomes thus requires the tail; the head binds little, if atall, to the oocyte meiosis I spindle and shows only weak, diffusionalbinding interactions with early embryo mitotic spindles.

Ncd is transported towards the equator in early metaphaseAnalysis of FLNcdVenus in the mitotic spindle demonstrates a netflow of fluorescence from the pole towards the equator in earlymetaphase with a rate of movement that decreases in mid-metaphase. The velocity is the same as the microtubule growth ortransport rate towards the equator estimated from curve fits of the

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new diffusion-binding-transport model to our photobleachingrecovery data. The data indicate that transport of FLNcdVenusoccurs during early metaphase as a result of tight binding by themotor to growing or sliding microtubules that move into the bleachspot. The net flow towards the spindle equator is opposite to thedirection of movement of the minus-end Ncd motor. Tight bindingby Ncd to spindle microtubules during early metaphase thus resultsin transport of the motor towards the equator, positioning it in thespindle for function later in mitosis.

Ncd force production in the mitotic spindleTransport of Ncd towards the spindle equator in early metaphaseto position the motor in the spindle for function later in mitosiscorrelates with evidence that Ncd is required in late anaphase tobalance forces in the spindle and regulate spindle length. Mitoticspindles of HLncdVenus cycle 9-11 embryos are abnormally bentand longer than those in wild-type embryos in late anaphase totelophase, consistent with the proposal that the Ncd motor producesforce in the spindle in the later stages of mitosis to regulate lengthby opposing other spindle forces. By contrast, mid-telophaseTLncdVenus spindles are comparable in length to those ofFLncdVenus embryos, indicating that the Ncd motor without thetail can function to regulate spindle length. Previous studies haveshown that Ncd produces force in the mitotic spindle in cycle 12-13 cleavage divisions that suppresses spindle collapse and regulatescentrosome spacing during prometaphase and metaphase, but haslittle effect in anaphase or later stages of mitosis (Sharp et al., 1999;Sharp et al., 2000). Ncd is a maternally expressed protein, thus thesedifferences in observations could be due to reduced or alteredactivity in the later cleavage divisions as the number of spindlesincreases, compared with the earlier stages examined in the presentwork. Further studies to clarify how Ncd function is regulated inthe spindle and the mechanism by which motor deficiency causesmitotic chromosome loss should be highly informative.

Materials and MethodsTransgenic fliespCaSpeR (Thummel et al., 1988) plasmids encoding full-length, headless and taillessNcd fused to the Venus fluorescent protein (Nagai et al., 2002), regulated by thenative ncd promotor, were constructed using conventional methods. FLncdVenusencodes the Ncd tail, coiled-coil stalk and motor domain (M1-K700) with a changeof D699L at the C terminus to create an AflII site to fuse Venus. HLncdVenus codesfor the Ncd tail and coiled-coil stalk (M1-G347+N348) with an added A349 tocreate an NcoI site to join Venus, and TLncdVenus encodes the stalk and conservedmotor domain (MES-L209-K700) with a change of D699L to fuse Venus. Plasmidswere injected into w; Δ2–3 Sb/+ embryos and w+ transformants were selected andmade homozygous for the ncd-null mutant cand, or heterozygous for cand and theTM3 balancer chromosome. Homozygous flies from different lines were analyzedby PCR using primers that give different length products for the cDNA transgeneand native ncd to confirm the absence of ncd +. FLncdVenus F28M1; cand,HLncdVenus M6M1; cand, TLncdVenus M23M4; cand and TLncdVenus F26F2; cand

were tested for viability by arraying embryos on grape juice agar plates andmonitoring hatching over 3-4 days. Transgenic flies expressing full-length Ncd fusedat the C-terminus to a monomeric red fluorescent protein (mRFP) (Campbell et al.,2002) were similarly recovered, transferred into a cand background and tested byPCR.

Live imagingLive oocytes or embryos expressing Venus or mRFP fusion proteins were preparedas described (Sciambi et al., 2005; Sköld et al., 2005; Zou et al., 2008) and imageswere collected at 20°C using a Bio-Rad Radiance2100 confocal scanhead mountedon an Axioskop2 microscope (Carl Zeiss Inc.) with a �40/1.3 NA Plan-NeoFluaroil-immersion objective and recorded with Bio-Rad LaserSharp 2000 software. Imageanalysis, including centrosome-to-centrosome spindle length measurements, wereperformed in ImageJ v. 1.39t (NIH). Images were cropped and assembled intomontages in ImageJ, brightness and contrast were adjusted in Adobe Photoshop v.8.0, and final changes in size were made in Adobe Illustrator v. 11.0.0. Adjustmentsto image contrast and brightness were linear.

Journal of Cell Science 121 (22)

Photobleaching assaysFRAP assays at the equator of cycle 9-11 mitotic spindles were performed at 22-25°C using an LSM510 confocal microscope and LSM510 software (Carl Zeiss, Inc.),a �40/1.3 NA Plan-NeoFluar oil-immersion objective and the 488 nm line of a 30mW Ar laser operating at 75% power. Briefly, six prebleach images were recorded,three or four photobleaching scans were performed at 100% laser power in an ROIof radius w=2.66 μm or 1.3 μm, followed by 494 rapid recovery images at ~165mseconds/image (recovery time=82.5 seconds) and low laser power (1-3%) tominimize photobleaching. Data were normalized and corrected as described (Zou etal., 2008; Hallen et al., 2008), then fit concurrently in MATLAB (The Mathworks)(Hallen et al., 2008) to kinetic models that account for FRAP recovery by binding,or both diffusion and binding (Sprague et al., 2004). None of the available modelsfor fluorescence recovery gave good fits to both the large and small ROI recoverydata. Because of the unusually slow recovery of the large ROI, we hypothesized theexistence of a binding state that depletes the free, diffusing Ncd, markedly affectingrecovery of the large ROI compared with the small ROI. Tight binding by Ncd tothe spindle would result in fluorescence recovery by microtubule growth or transportwith bound Ncd into the bleached ROI. We derived a model that accounts for this,together with recovery by diffusion and binding to microtubules in the ROI. Thefluorescence recovery as a function of time, frap(t), normalized to converge to 1 atlarge t, is given by:

where Feq is the fraction of Ncd that is free at equilibrium, Ceq is the fraction boundto microtubules, Feq+Ceq=1, w is the bleach spot radius, D is the diffusion cofficient,I0 and I1 are modified Bessel functions, koff is the dissociation rate constant, and σis the width of the velocity distribution. The mean velocity is given by σ . Fitsof the FLNcdVenus and HLNcdVenus FRAP data at the spindle equator to the newmodel, denoted the diffusion-binding-transport model, yielded the kinetic parametersshown in Table 2. The coefficient of multiple determination r2 was calculated usingthe MATLAB routine leasqr.m (Shrager et al., 1994; http://octave.sourceforge.net/doc/f/leasqr.html) as a measure of the goodness of fit by different recovery modelsto the data.

Similar to the diffusion-binding model it expands upon, the diffusion-binding-transport model requires that k*onw2/D�1. It reduces to the diffusion-binding modelfor small v or large koff, and to the Soumpasis pure-diffusion model (Soumpasis, 1983)for small Ceq. In these domains, the effect of transport on recovery is small anddetermination of velocity may be difficult. The data for TLNcdVenus, which exhibita large koff, are an example of this reduction; fits of the tailless data to the diffusion-binding-transport model gave values that were not significantly different from thediffusion-binding model, and v�0.

Ncd transport in the spindleFluorescence in FLncdVenus mitotic spindles was analyzed in time-lapse sequencesacquired at 2.32 second intervals using the Radiance2100 confocal system describedabove. Early metaphase through anaphase spindles from cycle 10 embryos were rotatedand aligned as necessary in ImageJ and cropped to eliminate centrosomes, astralmicrotubules, and cytoplasm. Image sequences were visually aligned in time by phaseof mitosis, based on the microtubule density and bundling in the spindle. Alignmentsbased on the ratio of pole to equator fluorescence or the image with maximalfluorescence displacement from the poles gave a higher overall s.e.m. after averagingand normalization. The equator was defined as the midpoint between poles in thefirst and last image of the sequence, and the median fluorescence position in eachspindle half was calculated using the MATLAB routine fluorescence_medians. Datafor the half-spindles from different embryos were averaged, the median fluorescencepositions were normalized from zero to one, and the mean normalized data wereplotted versus time in Kaleidagraph v. 3.6.4 (Synergy Software).

We thank Vann Bennett for the use of a Zeiss LSM510 confocalmicroscope, Roger Tsien for an mRFP plasmid, and Kevin Su forperforming HLncdVenus embryo viability tests. This work wassupported by grants to S.A.E. from the National Institutes of Healthand March of Dimes Foundation. M.A.H. is a 2008 Goldwater Scholar.

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σ2

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vt

w) +

vt w2 − v2 t2

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dv0

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⎤

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⎥⎥

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