Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles - PubMed (original) (raw)

Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles

Jedidiah Gaetz et al. J Cell Biol. 2004.

Abstract

During cell division metaphase spindles maintain constant length, whereas spindle microtubules continuously flux polewards, requiring addition of tubulin subunits at microtubule plus-ends, polewards translocation of the microtubule lattice, and removal of tubulin subunits from microtubule minus-ends near spindle poles. How these processes are coordinated is unknown. Here, we show that dynein/dynactin, a multi-subunit microtubule minus-end-directed motor complex, and NuMA, a microtubule cross-linker, regulate spindle length. Fluorescent speckle microscopy reveals that dynactin or NuMA inhibition suppresses microtubule disassembly at spindle poles without affecting polewards microtubule sliding. The observed uncoupling of these two components of flux indicates that microtubule depolymerization is not required for the microtubule transport associated with polewards flux. Inhibition of Kif2a, a KinI kinesin known to depolymerize microtubules in vitro, results in increased spindle microtubule length. We find that dynein/dynactin contribute to the targeting of Kif2a to spindle poles, suggesting a model in which dynein/dynactin regulate spindle length and coordinate flux by maintaining microtubule depolymerizing activities at spindle poles.

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Figures

Figure 1.

Figure 1.

Dynein/dynactin inhibition increases the length of spindle microtubules in the presence or absence of centrosomes. (A–C) Tubulin distribution in untreated spindles during live recordings. (D–G) p150-CC1 addition (2 μM, ∼3 min before image at t = 0) caused spindles to increase in length. (H and I) Higher magnified spindle pole regions indicated in F (Videos 1 and 2). (J) p150-CC1 was added to assembled spindles, samples were fixed after 8 or 15 min, spindle lengths were measured (mean ± SD, n = 15, two independent experiments), and normalized to the length of untreated spindles (40 μm). (K–M) Spindles fixed 8 min after addition of control buffer (K), 2 μM p150-CC1 (L), or 1 mg/ml 70.1 (M) (tubulin, red; DNA, blue). (N–P) Higher magnified, contrast-adjusted regions indicated in K–M, respectively. (Q and R) Spindles assembled in 18 μm p50 dynamitin were treated with control buffer (Q) or 2 μm p150-CC1 (R) and fixed after 15 min (tubulin, red; DNA, blue). (S and T) Spindles assembled in the absence of centrosomes, around DNA-beads (tubulin, red; DNA, blue). (S) Buffer control. (T) p150-CC1–treated (2 μM, 8 min). (U and V) Higher magnified, contrast-adjusted regions indicated in R. Times are in min:s. Bars, 10 μm.

Figure 2.

Figure 2.

Dynactin inhibition with p150-CC1 suppresses microtubule depolymerization at spindle poles. Spindle microtubule dynamics were analyzed using fluorescent speckle microscopy. (A) Tubulin speckles in a control spindle. (B) Polewards velocities of tubulin speckles in control (white bars; 2.1 ± 0.3 μm/min, mean ± SD), or p150-CC1–treated (2 μM p150-CC1, black bars; 2.1± 0.2 μm/min, mean ± SD) spindles (n = 12 for each condition, 120 speckles). Velocities were binned in 0.5 μm/min increments. (C–E) Images from a time-lapse video of a p150-CC1–treated spindle (2 μM p150-CC1 added ∼3 min before image at t = 0) showing tubulin speckles (C and D) and tubulin distribution (E). The black lines in A, C, and D indicate the regions used to generate the kymographs shown in F and G, respectively (Videos 3 and 4). Times are in min:s. Bars, 10 μm.

Figure 3.

Figure 3.

NuMA inhibition with LGN-N increases the length of spindle microtubules, in the presence or absence of centrosomes. (A–D) Tubulin distribution in an LGN-N–treated spindle (0.7 μM, added ∼3 min before image at t = 0) during live recordings (Video 5). (E) LGN-N was added to assembled spindles, samples were fixed after 8 or 15 min, spindle lengths were measured (mean ± SD, n = 15, two independent experiments), and normalized to the length of untreated spindles (40 μm). (F–H) Spindles assembled in the absence of centrosomes, around DNA beads (tubulin, red; DNA, blue). (F) Buffer control. (G) LGN-N–treated (2 μM, 8 min). (H) Higher magnified image of the region indicated in G. Times are in min:s. Bars, 10 μm.

Figure 4.

Figure 4.

Kif2a is required for bipolar spindle assembly and the regulation of spindle microtubule length. (A) Western blot of Xenopus egg extracts stained with anti-Kif2a. Molecular weight standards are shown. (B–E) Anti-Kif2a inhibits bipolar spindle assembly. Anti-Kif2a (0.7 mg/ml; B and C) or control buffer (D and E) were added at the start of assembly reactions. (B and D) Tubulin alone. (C and E) Overlay (tubulin, red; DNA, blue). (F–K) Anti-Kif2a (0.7 mg/ml) was added to assembled spindles. (F and G) 8 min after antibody addition. Long microtubule bundles extended beyond (white arrowheads), and buckled (green arrowheads) within the spindle. (F) Tubulin alone. (G) Overlay (tubulin, red; DNA, blue). (H–K) Real-time analysis of a spindle treated with anti-Kif2a (added ∼3 min before image at t = 0; Video 6). Times are in min:s. Bars, 10 μm.

Figure 5.

Figure 5.

Treatment with p150-CC1 or 70.1 displaces Kif2a, but not MCAK from spindle poles. Assembled spindles, after addition of buffer, p150-CC1, or 70.1, were processed for immunofluorescence. MCAK staining in control (A and D), p150-CC1–treated (2 μM, 15 min; B and E), and 70.1-treated (1 mg/ml, 15 min; C and F) spindles. (A–C) Overlays (tubulin, red; DNA, blue; MCAK, green). (D–F) MCAK alone. (G–I) Line scans of fluorescence intensity (MCAK, green; tubulin, red; arbitrary units) across the pole to pole axis of the spindles shown in A–C. Kif2a staining in untreated (J and M), p150-CC1–treated (2 μM, 15 min; K and N), and 70.1-treated (1 mg/ml, 15 min; L and O) spindles. (J–L) Overlays (tubulin, red; DNA, blue; Kif2a, green). (M–O) Kif2a alone. (P–R) Line scans of fluorescence intensity (Kif2a, green; tubulin, red; arbitrary units) across the pole to pole axis of the spindles shown in (J–L). (S and T) Spindles were treated for 15 min with p150-CC1 (4 μM) or control buffer and the relative amount of Kif2a (S), or MCAK (T), and tubulin associated with partially purified spindle pellets was analyzed by immunoblotting. (U) Quantitation of spindle-associated Kif2a and MCAK relative to tubulin from measurements of immunoblot band intensities (mean ± SD, two independent experiments), normalized to intensities from untreated spindles. Bars, 10 μm.

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