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First published online 17 June 2008
doi: 10.1242/jcs.024018
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Research Article |
1 Harvard Medical School, Department of Systems Biology, 200 Longwood Avenue, WAB536, Boston, MA 02115, USA
2 Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA
3 Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
* Author for correspondence (e-mail: aaron_groen{at}student.hms.harvard.edu)
Accepted 22 April 2008
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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-tubulin became redistributed to the spindle equator, suggesting that proper kinesin-5 function is required for pole assembly.
Key words: Kinesin-5, Mitosis, Small-molecule inhibitor, Xenopus-extract spindles
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Introduction |
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; McIntosh and Euteneuer, 1984; Salmon and Begg, 1980). In most spindles, a plus-end-directed tetrameric motor from the kinesin-5 family (called Eg5 in Xenopus) is essential for spindle bipolarity (Cole et al., 1994; Heck et al., 1993; Kapitein et al., 2005; Sawin et al., 1992; Walczak et al., 1998). Kinesin-5 is proposed to function by sliding anti-parallel microtubules apart (Sharp et al., 1999). In spindles from Xenopus egg extract, it is also proposed to drive continuous, poleward microtubule sliding during the metaphase steady state (Kapitein et al., 2005; Miyamoto et al., 2004).
An important sub-question for understanding spindle assembly is how spindle poles assemble, particularly in anastral spindles, which lack pre-existing microtubule-nucleating centers. Previous work has shown that kinesin-5 has a role in organizing anastral spindle poles, because poles assembled without kinesin-5 appear as asters with large holes (`holey asters') (Gaglio et al., 1996; Sawin et al., 1992; Sawin and Mitchison, 1994). However, analysis of such disorganized structures is complicated, making further conclusions about how kinesin-5 focuses microtubules into a spindle pole difficult.
Here, we used a novel ATP-competitive inhibitor of kinesin-5, compound-3, also known as 2-[1-(4-fluorophenyl)cyclopropyl]-4-(pyridin-4-yl)thiazole and which we will refer to hereafter as FCPT (Rickert et al., 2008). We found that FCPT induced a tight binding of kinesin-5 onto microtubules and induced loss of microtubules selectively at the poles of Xenopus-extract spindles without altering microtubule dynamics. We also found that FCPT blocked poleward microtubule sliding and redistributed the spindle-pole proteins TPX2 and -tubulin. Our data suggest that kinesin-5 might have a role in pole assembly in bipolar spindles by either directly or indirectly maintaining microtubule assembly.
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Results |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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; Maliga et al., 2002). Members of this class are called `non-competitive inhibitors' and induce the formation of monoaster spindles in dividing cells. In this study, we used a representative non-competitive inhibitor, S-trityl-L-cysteine (STLC, see Fig. 1A) (DeBonis et al., 2004), and a novel kinesin-5 inhibitor, FCPT, developed at Merck, that competes with the ATP substrate (Luo et al., 2007; Rickert et al., 2008) (Fig. 1A).
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). Inhibition of the microtubule-stimulated ATPase activity of the kinesin-5 motor domain by FCPT was competitive with the ATP substrate (Rickert et al., 2008), unlike non-competitive inhibitors (Maliga et al., 2002; Cochran et al., 2004). The inhibitor constant (Ki) for inhibition of ATPase activity was 110 nM (Rickert et al., 2008).
To test the effect of FCPT on the interaction between kinesin-5 and microtubules, we expressed monomeric motor domain and performed co-sedimentation assays with microtubules. AMP-PNP was used as a control known to promote tight microtubule binding. We found that both FCPT [half-maximal inhibition (EC50) 
65±10 µM] and AMP-PNP (EC50 116±17 µM) promoted a dose-dependent increase in the amount of motor domain co-sedimenting with microtubules, whereas very little motor domain co-sedimented in the presence of 1 mM ATP and no drug (Fig. 1B,C).
To calculate the apparent dissociation constant (Kd), we performed co-sedimentation assays with varying concentrations of microtubules (Fig. 1D). Whereas the non-competitive inhibitors reduced the affinity of kinesin-5 for microtubules in the absence of ATP (Kd without inhibitor = 0.7 µM compared with Kd with inhibitor = 2.3 µM), we found that FCPT only enhanced binding in the presence of ATP (data not shown), suggesting that an aspect of the ATPase cycle is important for the activity of FCPT (Cochran et al., 2005). Co-sedimentation of microtubules and kinesin-5 required either FCPT (apparent Kd = 1.7±0.437 µM) or AMP-PNP (apparent Kd = 6.3±1.96 µM); very little kinsin-5 co-sedimented without drug (or AMP-PNP). Unlike AMP-PNP, the activity of FCPT was reversible upon resuspension of the microtubule pellet without drug (data not shown). These experiments show that FCPT, similar to AMP-PNP, locks the motor into a conformation with an increased affinity for microtubules. We will refer to this as `tight binding'.
To test the efficacy and specificity of the tight-binding effect in cytoplasm, we added taxol to clarified Xenopus egg extract with or without FCPT, pelleted the assembled microtubule asters and immunoblotted the pellets for motors involved in spindle morphogenesis. FCPT enhanced recruitment of kinesin-5 (by approximately threefold), whereas recruitment of MCAK, XCTK2, and dynein-dynactin was not affected (Fig. 1E; data not shown).
To test whether FCPT promoted tight binding of kinesin-5 to microtubules in spindles, we added small amounts (approximately 1 µM) of full-length kinesin-5 labeled on random lysines with rhodamine [prepared as in Kapoor and Mitchison (Kapoor and Mitchison, 2001)] to pre-assembled Xenopus-extract spindles and imaged the resulting kinesin-5 speckles by spinning-disc confocal microscopy. Without drug, the motor became transiently immobilized, as evidenced by short lines at various angles in the kymograph (Fig. 1F, control). Addition of FCPT (at 200 µM) greatly increased the recruitment of X-rhodamine-labeled kinesin-5 to spindles as quickly as we could assay (less than 2 minutes), and also caused the speckles to become immobilized for many seconds, as evidenced by the bright, vertical lines in the kymograph (Fig. 1F, FCPT). Thus, the drug also promoted tight binding in the context of assembled spindles. These data also suggest that poleward microtubule sliding is blocked, a point we confirm below with tubulin imaging.
FCPT altered spindle morphology while maintaining bipolarity
FCPT and the non-competitive inhibitors of kinesin-5 each have dramatic but different effects on spindle morphology in meiotic Xenopus egg extracts. As previously studied, non-competitive kinesin-5 inhibitors, such as STLC, induced the formation of monoaster spindles in dividing cells (Brier et al., 2004; Mayer et al., 1999; Mitchison et al., 2005). Similar to other non-competitive kinesin-5 inhibitors, we found that STLC addition caused pre-formed Xenopus egg-extract spindles to collapse into monoasters over the course of 20 minutes, changing the average pole-pole length from 35 µm to approximately zero (using labeled anti-NuMA IgG to mark the poles; Fig. 2A,B) (Kapoor et al., 2000). The EC50 for STLC in this experiment was 2 µM (concentration of STLC required to assemble 50% monoasters), making it 20 times more potent than the inhibitor monastrol (Brier et al., 2004; DeBonis et al., 2004).
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5 minutes, until 50% of the initial amount remained (Fig. 2A,C), suggesting a decrease in microtubule density near spindle poles. Tubulin fluorescence near the equator remained approximately constant and, unlike with STLC addition, the pole-pole distance was not changed (Fig. 2B). Despite the decrease in microtubule density at the poles, some pole structure remained, as evidenced by largely unchanged levels of anti-NuMA (Fig. 2A). The EC50 of FCPT (concentration required to reduce the ratio of pole:equator fluorescence to approximately 0.8 or 50% of the maximum reduction) for promoting morphological change was 75 µM, and we used the compound at 200 µM in most experiments. This EC50 was higher than that for inducing tight binding with pure proteins, which is typical for hydrophobic drugs in extract, perhaps because much of the drug partitions into lipids. These data from fixed samples were confirmed by time-lapse imaging of drug-treated spindles and the morphological effects observed using labeled tubulin alone were identical in the absence of the anti-NuMA pole marker (data not shown). The addition of FCPT before spindle assembly induced different morphological changes. Whereas STLC addition, whether added before or after spindle assembly, induced the formation of monoaster spindles, FCPT inhibited spindle assembly and produced structures with disorganized, elongated microtubules (Fig. 2D; data not shown). Thus, consistent with its role in perturbing an essential meiotic kinesin, such as kinesin-5, FCPT affects spindle morphology whether added before or after spindle assembly.
FCPT altered spindle-pole composition of bipolar spindles
We found that FCPT lowered microtubule density at spindle poles of bipolar spindles (Fig. 2). To test whether FCPT also affects the protein composition of spindle poles, we measured the effect of FCPT on the localization of the spindle-pole markers TPX2 and -tubulin. Each is required for assembly of microtubules during spindle assembly in extract (Gruss et al., 2001; Zheng et al., 1995). To visualize TPX2 and -tubulin, we used a TPX2-GFP fusion protein and a non-perturbing, labeled anti--tubulin antibody (Fig. 3A,B). FCPT caused TPX2 and -tubulin to relocalize from the poles to the band of remaining microtubules at the equator. The redistributions occurred over the same time scale as the microtubule loss at the spindle poles (data not shown; Fig. 2). Additionally, the kinesins XCTK2 and MCAK also disappeared from the FCPT-treated spindle poles (at the same rate as tubulin) but, unlike TPX2 and -tubulin, neither were significantly enhanced in the spindle equator (Fig. 3C,D). We also found that FCPT did not enhance the binding of either TPX2, -tubulin, XCTK2 or MCAK to microtubules, as shown by western blot analysis of taxol-polymerized microtubule pellets isolated from extracts (Fig. 1E; supplementary material Fig. S1A). Thus, FCPT might perturb the protein composition of intact poles of bipolar spindles by either affecting microtubule dynamics (transport, polymerization or depolymerization) or directly changing the recruitment of spindle-pole factors.
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Aster poles did not undergo FCPT-dependent morphological changes
To better understand the effect that FCPT has on spindle poles, we added FCPT to microtubule asters. We induced the formation of microtubules in extracts with DMSO, which assembled into aster-like structures after 30 minutes (Fig. 3E). As shown previously, such structures localized NuMA, TPX2 and -tubulin (Groen et al., 2004; Wittmann et al., 1998; Wittmann et al., 2000). Similar to bipolar spindles, we found that the addition of FCPT to preformed asters induced the recruitment and immobilization of kinesin-5 to the aster (data not shown). FCPT addition before aster assembly abolished the assembly of all organized structures and mislocalized the pole markers TPX2 and NuMA (Fig. 3E; supplementary material Fig. S2A). However, surprisingly, unlike bipolar spindles, we found that the addition of FCPT after aster assembly did not affect the aster structure and pole (Fig. 3E). We found that the localization of pole markers such as NuMA, TPX2 and -tubulin were also not drastically changed (Fig. 3E; supplementary material Fig. S2A; data not shown). Similar effects were observed for RanGTP-induced asters or STLC-induced monoasters (supplementary material Fig. S2B; data not shown). Our data suggest that FCPT requires bipolarity (anti-parallel microtubules) to influence spindle-pole structure.
Effect of FCPT on microtubule dynamics
Microtubules in extract spindles undergo poleward sliding, driven at least in part by kinesin-5 (Miyamoto et al., 2004). Non-competitive kinesin-5 inhibitors inhibit poleward sliding, so we wanted to test whether FCPT had similar effects. FCPT caused a dose-dependent decrease of the poleward microtubule-sliding rate, measured by tubulin speckle imaging (Sawin and Mitchison, 1991; Waterman-Storer et al., 1998), with an EC50 of 30 µM (Fig. 4A,B). Similar to the recruitment of rhodamine-labeled kinesin-5 to spindles, the inhibition of poleward sliding occurred as fast as we could assay (less than 2 minutes), suggesting that the inhibition of poleward sliding is most probably due to perturbation of kinesin-5.
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; Tirnauer et al., 2004). On control spindles, as shown previously, EB1 localized throughout the spindle, tracking along growing microtubule tips, suggesting that polymerization occurs throughout the spindle (Tirnauer et al., 2002a). In FCPT-treated spindles, EB1 disappeared from the spindle pole over a similar time scale as the microtubule loss at the spindle poles (approximately 5 minutes) and remained localized to the band of microtubules at the equator with no significant change in overall tip-tracking dynamics in this region (Fig. 4C; supplementary material Movies 1, 2; data not shown). Thus, FCPT locally inhibits microtubule polymerization at the spindle pole of a bipole, but does not directly affect microtubule polymerization elsewhere.
Specificity of FCPT effects
We found that the EC50 for poleward-sliding inhibition (30 µM) was lower than that for the morphological changes at spindle poles (75 µM; Fig. 2; Fig. 4B), suggesting the possibility of two targets with different binding affinities for FCPT. Although we cannot rule out this possibility, there are also many other equally possible explanations. For example, microtubule assembly (and/or the transport of assembly factors) might depend on poleward microtubule sliding and therefore the loss of microtubule assembly at the spindle pole might only occur when sliding is inhibited above a particular threshold, leading to a higher EC50 for the morphological changes. Consistent with this model, we found that the FCPT-dependent inhibition of poleward sliding always occur (temporally) before the morphological changes at the spindle pole. Further experiments should address these models.
To further evaluate the specificity of the effects of FCPT on Xenopus-extract spindles, we tested a structurally dissimilar compound that might act by a similar mechanism. Biphenyl sulfamide 20 [referred to here as GSK246053 (Fig. 1A)] was developed as a specific ATPase-competitive kinesin-5 inhibitor by Cytokinetics and GlaxoSmithKline (Parrish et al., 2007). Mutations in the non-competitive site of kinesin-5, which block binding of non-competitive inhibitors, had no effect on ATPase inhibition by GSK246053, suggesting that it binds at a novel site (Parrish et al., 2007). GSK246053 had the same effect as FCPT in the co-sedimentation assay in Xenopus extract and in all of our spindle assays (supplementary material Fig. S1B-D). This similarity of effects between two structurally unrelated compounds, both developed as specific kinesin-5 inhibitors, provides confidence that our observed gain-of-function phenotypes result from successfully targeting the active site of kinesin-5.
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Discussion |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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; Kapoor et al., 2000; Mitchison et al., 2004), such experiments suggest that the observed FCPT phenotypes either require anti-parallel microtubules or that the poles of monoasters/asters and bipolar spindles rely on different assembly mechanisms. Further experiments are required to test this hypothesis.
From our data, it is not clear whether the reduction of EB1 at spindle poles reflects spatial changes in microtubule turnover (i.e. increase in catastrophes at spindle poles) or spatial changes in microtubule nucleation (i.e. less microtubule nucleation at spindle poles). The FCPT-dependent loss of microtubule density at spindle poles could directly result from the inhibition of the transport of microtubules to the pole. Additionally, it could result from the inhibition and/or mislocalization of polymerization factors (such as TPX2 and -tubulin) or the activation and/or mislocalization of depolymerization factors (such as Op18 and MCAK). Although we did not observe the FCPT-dependent recruitment of MCAK to the spindle pole (Fig. 3C), there could be other unidentified depolymerization factors recruited to the spindle pole after FCPT treatment. Further experiments need to address these issues.
TPX2, -tubulin and NuMA were previously argued to accumulate at poles owing to dynein-dynactin-mediated transport (Buendia et al., 1990; Merdes et al., 1996; Verde et al., 1991; Wittmann et al., 1998), a mechanism that should be insensitive to kinesin-5 perturbation with FCPT. Interestingly, the pole localizations of TPX2 and -tubulin were perturbed by FCPT treatment, whereas the localization of NuMA was unaffected, suggesting that TPX2 and -tubulin depend upon other mechanisms, such as kinesin-5-dependent poleward sliding, to concentrate on spindle poles. Although perturbation of the dynein-dynactin complex does not inhibit poleward sliding (Maddox et al., 2003), the dynein-dynactin complex has a role in organizing microtubules into poles as well as in transporting materials along them (Merdes et al., 1996; Wittmann and Hyman, 1999). However, distinguishing transport with dynein-dynactin from transport by attachment to sliding microtubules will be difficult, and we suspect that both mechanisms are important. Overall, FCPT is an interesting tool for mitosis research and a testament to the rich pharmacology of motor proteins.
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Materials and Methods |
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1-(4-fluorophenyl)cyclopropanecarbonitrile
See Fig. 5 for structure. 10.00 g (7.4 mmol) (4-fluorophenyl)-acetonitrile and 9.56 ml (11.1 mmol) 1,2-dibromoethane were dissolved in 60 ml Toluene followed by addition of 2.0 g (7.4 mmol) tetra-N-butylammonium chloride and 60 ml aqueous 12 M sodium hydroxide. The reaction mixture was stirred at room temperature. After 24 hours, 200 ml ethyl acetate was added, and the organic layer was separated and washed twice with water. The combined organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified on silica (Hexanes/ethyl acetate 5:1) to yield 5.80 g (49%) of the desired product as yellow oil.
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7.30 (m, 2H), 7.06 (t, 2H, J=8.6), 1.73 (q, 2H, J=5.1), 1.38 (q, 2H, J=5.3).
1-(4-fluorophenyl)cyclopropanecarbothioamide
See Fig. 6 for structure. 5.20 g (32.3 mmol) 1-(4-fluorophenyl)-cyclopropane - carbonitrile, 7.22 g (35.5 mmol) magnesium chloride hexahydrate and 10.8 g (19.4 mmol) sodium hydrogen sulfide were dissolved in 100 ml N,N-dimethylformamide (Manaka, 2005). The reaction mixture was heated to 50°C. After 3 hours the reaction mixture was poured into 600 ml water whereby a white precipitate forms. The aqueous layer was extracted three times with ether and the combined organic layers were washed with water. The combined organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure to yield an off-white fluffy solid that was triturated twice with hexanes and dried under reduced pressure to yield 4.80 g of the desired product as white powder.
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7.85 (s, 1H), 7.40 (m, 2H), 7.09 (t, 2H, J=8.6), 6.57 (s, 1H), 2.06 (q, 2H, J=3.7), 1.32 (q, 2H, J=3.7).
2-[1-(4-fluorophenyl)cyclopropyl]-4-(pyridin-4-yl)thiazole (FCPT)
See Fig. 1A for structure. 2.0 g (10.2 mmol) 1-(4-fluorophenyl)-cyclopropane - carbothioic acid amide and 2.88 g (10.2 mmol) 2-bromo-1-(pyridin-4-yl)ethanone hydrobromide were suspended in 100 ml ethanol and heated to reflux for 2 hours. Upon cooling, the product precipitated. The precipitate was filtered off, washed with cold ethanol and dried under reduced pressure to yield 2.44 g (63%) of the desired product as hydrobromide (yellow powder) without additional purification.
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8.85 (d, 2H, J=6.4), 8.43 (d, 2H, J=6.4), 8.11 (s, 1H), 7.47 (dd, 2H, J=5.5, 8.3), 7.09 (t, 2H, J=8.6), 1.86 (dd, 2H, J=3.7, 6.3), 1.51 (dd, 1H, J=3.8, 6.4). Synthesis of GSK246053 was performed with minor modifications according to the procedure reported in Coleman, P. J., Garbaccio, R. M. and Neilson, L. A. (2005). `Preparation of substituted thiazoles as mitotic kinesin inhibitors.' vol. US2005176776 (ed. U. S. P. A. Publ.). USA.
3-fluoro-4'-(trifluoromethyl)biphenyl-4-amine
See Fig. 7 for structure. A solution of 1.6 g tetrakis triphenylphosphane palladium in 10 ml DMF was added to a solution of 6.61 g (34.8 mmol) 4-(trifluoromethyl)phenyl boronic acid and 5.51 g (29 mmol) 4-bromo-2-fluoro-anilin in 40 ml DMF and 50 ml 2 M aqueous potassium carbonate. The reaction mixture was thoroughly purged with argon and stirred at 100°C for 16 hours. After cooling to room temperature, the reaction mixture was poured in 400 ml of half-saturated aqueous sodium bicarbonate solution and extracted three times with ether. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified on silica (20% ethyl acetate in hexanes) to yield 5.5 g (72%) of the desired product as a white powder.
1H NMR (500 MHz, CDCl3) 7.65 (d, 2H, J=8.4), 7.61 (d, 2H, J=8.4), 7.27 (dd, 1H, J=1.8, 12.2), 7.22 (dd, 1H, J=1.8, 8.2), 6.86 (t, 1H, J=8.7), 3.90 (s, 2H).
GSK246053
See Fig. 1A for structure. 149 µl of water was added to a solution of 718 µl (8.25 mmol) chlorosulfonyl isocyanate in 17.5 ml acetonitrile at 0°C. The reaction was stirred for 1 minute at 0°C and for 3 hours at 25°C. After cooling to 0°C, a solution of 1.91 g (7.5 mmol) 3-fluoro-4'-(trifluoromethyl)biphenyl-4-amine in 17.5 ml acetonitrile and 1.25 ml pyridine was added dropwise. The reaction mixture was then warmed to room temperature. After 15 hours, the reaction mixture was diluted with brine and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified on silica gel (35% ethyl acetate in hexanes) to yield 660 mg (26%) of the desired product as white solid.
1H NMR (500 MHz, CDCl3/CD3OD) 7.70 (t, 2H, J=7.2), 7.65 (t, 2H, J=8.3), 7.39 (dd, 2H, J=5.7, 17.3), 6.71 (s, 1H).
Xenopus egg-extract spindles
Cycled Xenopus egg-extract spindles were assembled from meiotic Xenopus egg extracts [cytostatic factor (CSF)] as described previously (Desai et al., 1999; Murray, 1991). Briefly, spindles were assembled from CSF extract containing demembranated sperm nuclei (500/µl) and cycled from interphase (with addition of 0.4 mM CaCl2) for 80 minutes back to meiosis (with addition CSF). After spindle assembly (1-2 hours after CSF addition), spindle reactions were supplemented with various amounts of either FCPT (diluted in DMSO to 200 mM), 200 µM STLC (diluted in DMSO to 100 mM) or 200 µM GSK246053. Tubulin was visualized by the addition of purified bovine tubulin (20 µg/ml) directly labeled with X-rhodamine (Invitrogen) or Alexa Fluor 647 (Invitrogen) as described previously (Hyman et al., 1991; Sawin and Mitchison, 1991). Generally, experiments were repeated three to six times using three different extracts.
Real-time imaging
Real-time images were obtained using a 100x/1.4 NA Plan Apochromat objective (Nikon) on a Nikon TE 300 inverted microscope fitted with a Yokogawa spinning-disc confocal head (Perkin Elmer) using an Andor EM-CCD camera (512 x 512 pixel sensor) driven by Andor software (Andor).
Real-time images and fixed images were obtained using either a 20x/0.75 NA Plan Apochromat or a 40x/0.95 NA Plan Apochromat objective (Nikon) on an upright Nikon Eclipse 90i using an Orca ER cooled CCD camera (Hamamatsu) driven by Metamorph software (Molecular Devices).
NuMA and -tubulin were imaged by addition of 10 µg/ml of Alexa-Fluor-488 (Invitrogen) directly labeled antibodies (Groen et al., 2004) after spindle assembly and FCPT treatment.
Recombinant EB1 was labeled with NHS-ester-Alexa-Fluor-488 (Invitrogen) and imaged by addition of 50 nM to extracts before spindle assembly.
TPX2 was imaged by addition of approximately 20 nM of purified human GFP-TPX2 to extracts before spindle assembly. Human TPX2-GFP was purified from HeLa S3 suspension cells. Briefly, 1 l of cells was transfected using 750 µl Lipofectamine (Invitrogen) with 500 µg of purified pcDNA3.1-GFP-hTPX2 (a kind gift from S. Garrett and T. Kapoor, Rockefeller University). Lysates containing GFP-TPX2 were created by sonicating frozen pellets (created by pelleting and washing suspension cells three times in PBS, 1000 g, 5 minutes each spin, 4°C) in 10x the volume of pellet in XB with no sucrose added (10 mM HEPES, pH 7.7, 100 mM KCl, 1 mM MgCl2, 100 µM CaCl2) supplemented with 1 mM DTT and protease inhibitors (Roche Applied Scientific). The lysates were clarified (50,000 g for 30 minutes) and GFP-TPX2 was purified first with a 5 ml HiTRAP S column (GE Healthcare) and eluted with XB (no sucrose added) supplemented with 250 mM KCl. The fraction containing GFP-TPX2 was applied to a Superose-12 10/300 gel-filtration column (GE Healthcare) equilibrated with XB + 10% sucrose, 1 mM DTT. GFP-TPX2-containing fractions were frozen in liquid nitrogen.
Microtubule pelleting assay
To determine the FCPT EC50 for binding of kinesin-5 to microtubules in vitro, Taxol (Sigma)-stabilized microtubules (10 µM) and 4 µM clarified (200,000 g for 20 minutes) gel-filtered (Superose-6 10/300 gel) recombinant HIS-kinesin-5 motor domain [amino acids 1-370; a kind gift from S. Kim and E. Wojcik, Louisana State University (Wojcik et al., 2004)] were incubated with varying concentrations of either AMP-PNP, FCPT or DMSO (control) for 5 minutes at room temperature. For the apparent Kd, varying concentrations of taxol-stabilized microtubules were incubated with 8 µM kinesin-5 and 10 µM of either FCPT or AMP-PNP. All samples were pelleted through a 20% glycerol cushion (150,000 g for 10 minutes). All buffers contained saturating amounts of ATP (1 mM ATP) in 1x BRB-80 plus 10 µM taxol and the appropriate concentration of FCPT. The supernatant and the pellet of each reaction were supplemented with sample buffer for Coomassie-stained SDS-PAGE. The fraction of kinesin-5 co-sedimenting with microtubules was determined using Image Pro (NIH). The best-fit curves (for the apparent Kd and EC50 calculations with a 95% confidence) were determined using Igor Pro (Wavemetrics) fitting to the equations: (kinesin-5 fraction bound) = (cxx)/(x+EC50) and (kinesin-5 fraction bound) = (cxx)/(x+Kd), where x is the concentration of drug for EC50 measurements or concentration of tubulin for apparent Kd measurements. A correction constant (c) was necessary to fit each curve.
Taxol (10 µM; Sigma)-induced microtubules from clarified Xenopus meiotic egg extracts (crude CSF spun 200,000 g, 2 hours) containing either 200 µM FCPT or DMSO (control) were pelleted (20,000 g, 10 minutes). The pellet was washed three times with 500 µl CSF-XB supplemented with 1 mM ATP, 1 mM GTP, 1 mM DTT, taxol (10 µM) and either 200 µM FCPT or GSK246053. The final pellet was resuspended in 20 µl of sample buffer for use in western blot analysis probing against Eg5, XCTK2, MCAK, TPX2, -tubulin and tubulin.
Image analysis
Kymographs were taken from real-time image sequences using lines of 3-µm-width over maximum intensity particles on Metamorph (Molecular Devices). Average flux rates were calculated from manually tracking 10-15 particles on kymographs for three to six spindles from three different extract experiments for each condition.
The images from EB1 movies were aligned with StackReg plugin (Thévenaz et al., 1998).
The ratio of spindle-pole:mid-spindle fluorescence was obtained by taking the average of the integrated intensity at each pole divided by the integrated intensity of the mid-spindle (within 1-3 µm of the chromatin) over a fixed rectangle for each spindle using ImageJ (NIH).
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Acknowledgments |
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Footnotes |
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References |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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Brier, S., Lemaire, D., Debonis, S., Forest, E. and Kozielski, F. (2004). Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5. Biochemistry 43, 13072-13082.[CrossRef][Medline]
Buendia, B., Antony, C., Verde, F., Bornens, M. and Karsenti, E. (1990). A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. J. Cell Sci. 97, 259-271.
Cochran, J. C. and Gilbert, S. P. (2005). ATPase mechanism of Eg5 in the absence of microtubules: insight into microtubule activation and allosteric inhibition by monastrol. Biochemistry 44, 16633-16648.[CrossRef][Medline]
Cochran, J. C., Sontag, C. A., Maliga, Z., Kapoor, T. M., Correia, J. J. and Gilbert, S. P. (2004). Mechanistic analysis of the mitotic kinesin Eg5. J. Biol. Chem. 279, 38861-38870.
Cochran, J. C., Gatial, J. E., 3rd, Kapoor, T. M. and Gilbert, S. P. (2005). Monastrol inhibition of the mitotic kinesin Eg5. J. Biol. Chem. 280, 12658-12667.
Cole, D. G., Saxton, W. M., Sheehan, K. B. and Scholey, J. M. (1994). A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos. J. Biol. Chem. 269, 22913-22916.
DeBonis, S., Skoufias, D. A., Lebeau, L., Lopez, R., Robin, G., Margolis, R. L., Wade, R. H. and Kozielski, F. (2004). In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol. Cancer Ther. 3, 1079-1090.
Desai, A., Murray, A., Mitchison, T. J. and Walczak, C. E. (1999). The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61, 385-412.[Medline]
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