Journal of Cell Science 113, 597-609 (2000) 597 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0866 Evidence that actin and myosin are involved in the poleward flux of tubulin in metaphase kinetochore microtubules of crane-fly spermatocytes R. V. Silverman-Gavrila and A. Forer* Biology Department, York University, Toronto, Ontario M3J 1P3, Canada *Author for correspondence (e-mail: [email protected]) Accepted 1 December 1999; published on WWW 31 January 2000 SUMMARY We studied the effects of various drugs on the poleward flux We studied whether these drugs altered spindle actin. We of tubulin in kinetochore microtubules in metaphase-I used fluorescent phalloidin to visualize spermatocyte F- crane-fly spermatocytes. We used as a measure of tubulin actin, which was associated with kinetochore spindle fibers flux a ‘gap’ in acetylation of kinetochore microtubules as well as the cell cortex, the contractile ring and finger-like immediately poleward from the kinetochore; the ‘gap’ is protrusions at the poles. Spindle F-actin was no longer seen caused by a time lag between incorporation of new tubulin after cells were treated with cytochalasin D, swinholide A subunits at the kinetochore and subsequent acetylation of or a high concentration of latrunculin B, whereas a low those subunits as they flux to the pole. We confirmed concentration of latrunculin B, which did not completely that the ‘gap’ is due to flux by showing that the ‘gap’ remove the ‘gap’, caused reduced staining of spindle actin. disappeared when cells were treated briefly with the anti- Neither 2,3-butanedione 2-monoxime nor jasplakinolide tubulin drug nocodazole, which decreases microtubule altered spindle actin. These data suggest that an dynamics. The ‘gap’ disappeared when cells were treated actomyosin mechanism drives the metaphase poleward for 10 minutes with anti-actin drugs (cytochalasin D, tubulin flux. latrunculin B, swinholide A), or with the anti-myosin drug 2,3-butanedione 2-monoxime. The ‘gap’ did not disappear when cells were treated with the actin stabilizing drug Key words: Tubulin flux, Actin, Actomyosin, Kinetochore jasplakinolide. microtubule, Spindle, Spindle matrix, Metaphase, Acetylated tubulin INTRODUCTION kinetochore microtubule stubs moved poleward, often accompanied by their associated metaphase chromosomes Poleward flux of tubulin molecules in kinetochore (Spurck et al., 1997). microtubules of metaphase cells was demonstrated as poleward What might generate the forces that act on kinetochore movement of locally uncaged fluorescent tubulin in vivo microtubules? Mitchison and Sawin (1990) suggested that (Mitchison, 1989; Mitchison and Salmon, 1992; Zhai et al., forces on kinetochore microtubules might be generated within 1995) and in reconstituted spindles in vitro (Sawin and the spindle from motor molecules (kinesins) acting on Mitchison, 1991; Desai et al., 1998). Thus, tubulin kinetochore microtubules. Alternatively, Wilson and Forer polymerization occurs at the kinetochore (plus) ends of (1994) and Forer and Pickett-Heaps (1998a) proposed that kinetochore microtubules and depolymerization occurs at the actin and associated motor proteins apply force on kinetochore pole (minus) ends (Mitchison et al., 1986). microtubules; their arguments are based on the effects of Poleward flux in kinetochore microtubules might be due to ultraviolet microbeam irradiations on chromosome movement ‘treadmilling’, a natural occurrence for microtubules in (Forer and Wilson, 1994) and on evidence that anti-actin equilibrium in vitro, where the flux is driven by a higher drugs interfere with anaphase chromosome motion (Forer subunit-lattice affinity at the microtubule plus end relative to and Pickett-Heaps, 1998a). Consistent with the latter the minus end (Margolis and Wilson, 1981). But it also is possibility, Waterman-Storer and Salmon (1997) have shown possible that the flux is caused by forces acting on kinetochore experimentally that microtubule translocation in lamellipodia microtubules, as suggested by Mitchison and Sawin (1990), is inhibited by drugs that alter actin and myosin. Moreover, similar to other cell systems where lengthwise forces on recent work indicates that microtubule dynamics are modulated microtubules can promote microtubule polymerization (e.g. by actin-microtubule interactions (Hely and Willshaw, 1998) Putnam et al., 1998; Heidemann, 1990; Zheng et al., 1993). and that actin filaments bind to astral microtubules in extracts Direct evidence that forces act on kinetochore microtubules of Xenopus eggs (Sider et al., 1999). was obtained by experiments in which ultraviolet microbeam We have tested the idea that forces on kinetochore irradiations severed kinetochore microtubules: the remnant microtubules applied by actomyosin promote tubulin flux of 598 R. V. Silverman-Gavrila and A. Forer kinetochore microtubules in metaphase; we measured Optiphot microscope using Nikon or Zeiss 100× oil-immersion kinetochore microtubule flux in the presence of inhibitors of objective lenses (NA=1.3), were recorded in real time on videotape. actin and of myosin. We used as a measure of flux the acetylation of kinetochore microtubules. Kinetochore Cell treatments microtubules are not acetylated at their kinetochore end, but For every treated cell we first followed control cells from the same are acetylated closer to the pole (Wilson and Forer, 1989). preparations or from the contralateral testis, perfused only with IR or with the maximal DMSO concentration (0.3%) used in different drug Since acetylation of newly polymerized tubulin in solutions, to ensure that cells proceeded normally from metaphase to microtubules occurs with a 5-15 minute time lag after tubulin telophase. All experiments were performed at room temperature. incorporation (Wilson et al., 1994), this suggests that the ‘gap’ The dose and duration of treatments were chosen from the literature in acetylation at the kinetochore end is due to microtubule flux (Forer and Pickett-Heaps, 1998a) for latrunculin B (LATB) and from kinetochore to pole (Wilson and Forer, 1989; Wilson et cytochalasin D (CD), from our own work (unpublished) for al., 1994). Further experiments confirmed that the ‘gap’ can be nocodazole (NOC), and from analysis of effects on anaphase chromo- used to visualise tubulin flux since taxol, known to stabilize some movement for swinholide A (SWA), jasplakinolide (JAS) and microtubule dynamics (Schiff and Horowitz, 1980), caused the 2,3-butanedione 2-monoxime (BDM). We chose the minimum dose ‘gap’ in acetylation to disappear (Wilson and Forer, 1997); i.e. that affected anaphase chromosome movement. in the presence of nanomolar concentrations of taxol the Experimental cells were treated for 10 minutes with CD, LATB, SWA, JAS or BDM, or for 4 minutes with NOC. In some experiments kinetochore microtubules were acetylated at the kinetochore. cells treated with CD for 10 minutes were allowed to recover for 10 Thus the ‘gap’ is due to flux of tubulin from kinetochore to minutes by washing out the drug with fresh IR; some cells treated pole. with NOC for 4 minutes were allowed to recover for 20 minutes. At We studied effects of inhibitors of actin and of myosin on the end of the treatments (or of the recovery period) the cells were the ‘gap’ in acetylation at the kinetochore end of microtubules, processed for fluorescence microscopy, and either lysed directly as using the presence of the ‘gap’ to indicate that microtubule flux described by Wilson et al. (1994), for studying spindle microtubules, continues and its absence to indicate the absence of flux. Our or for treatment with one of a variety of methods described below, for results, that actin and myosin inhibitors cause the ‘gap’ to studying spindle actin. disappear, suggest that actomyosin is involved in driving the Fluorescence staining flux of tubulin in kinetochore microtubules in metaphase. For every session of staining we stained at least one control coverslip as a positive control for the procedures. MATERIALS AND METHODS Double staining for acetylated and tyrosinated α-tubulin We stained for acetylated and tyrosinated α-tubulin as described by Solutions Wilson et al. (1994). Briefly, the cells were lysed for 10 minutes in a The following were used for cell preparations: halocarbon oil lysis buffer (100 mM Pipes, final pH 6.9, 10 mM EGTA, 5 mM (Halocarbon Products Corp., River Edge, NJ, USA); insect Ringer’s MgSO4, 5% DMSO, 1% Nonidet P-40), fixed for 9 minutes in 0.25% solution (IR; 0.13 M NaCl, 0.005 M KCl, 0.001 M CaCl2, 6 mM glutaraldehyde in PBS (phosphate-buffered saline: 0.13 M NaCl, Sørensen’s phosphate buffer (3 mM KH2PO4, 3 mM Na2HPO4), final 6 mM Sørensen’s phosphate buffer, final pH 7), and then rinsed with pH 6.9); fibrinogen (Calbiochem, La Jolla, CA, USA), 10 mg/ml in PBS and incubated for 20 minutes in 1 mg/ml sodium borohydride IR; thrombin (Sigma Chemical Co., St Louis, MO, USA), 50 units/ml (NaBH4) in PBS. Then the coverslips were rinsed with PBS and stored IR; and valap (vaseline: lanolin: paraffin, 1:1:1). in 1:1 PBS/glycerol at 4°C until they were processed further. The Various drugs were dissolved in dimethylsulfoxide (DMSO); PBS/glycerol was removed by rinsing the coverslips with PBS prior measured portions were stored as stock solutions at −80°C until use, to staining. The cells were dual stained for acetylated α-tubulin by when they were thawed and diluted with IR. The concentrations after incubation with 6-11B-1, a mouse monoclonal antibody specific for dilution were 20 µM for Cytochalasin D and 0.75-1.5 µM for acetylated α-tubulin (Sigma-Aldrich) diluted 1:300, followed by rat- latrunculin B (Calbiochem, La Jolla, CA, USA), 50 nM for Swinholide adsorbed fluorescein-isothiocyanate (FITC)-conjugated goat anti- A (Kamiya Biomedical, Seattle, WA, USA), 0.3 µM for Jasplakinolide mouse IgG (Caltag Laboratories, San Francisco, CA, USA), diluted (Molecular Probes, Eugene, OR, USA) and 10 µM for Nocodazole 1:50, and for tyrosinated α-tubulin by incubation with YL1/2 rat (Sigma, St Louis, MO, USA). The final concentrations of DMSO in monoclonal antibody specific for tyrosinated α-tubulin (a gift from Dr the diluted drug solutions ranged from 0.06% to 0.3%.