Gaetz J , Kapoor TM .

GM65933 NIGMS NIH HHS , R01 GM065933-02 NIGMS NIH HHS , R01 GM065933 NIGMS NIH HHS

	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. 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. 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. 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. 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.

Cassimeris, Accessory protein regulation of microtubule dynamics throughout the cell cycle. 1999, Pubmed, Xenbase

Cassimeris, Accessory protein regulation of microtubule dynamics throughout the cell cycle. 1999, Pubmed , Xenbase
Desai, The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. 1999, Pubmed , Xenbase
Desai, Kin I kinesins are microtubule-destabilizing enzymes. 1999, Pubmed , Xenbase
Du, A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. 2001, Pubmed , Xenbase
Du, LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation. 2002, Pubmed
Echeverri, Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. 1996, Pubmed
Gaglio, NuMA is required for the organization of microtubules into aster-like mitotic arrays. 1995, Pubmed
Ganem, The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK. 2004, Pubmed
Goshima, The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. 2003, Pubmed
Heald, Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. 1996, Pubmed , Xenbase
Heald, Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. 1997, Pubmed , Xenbase
Howell, Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. 2001, Pubmed
Hyman, Preparation of modified tubulins. 1991, Pubmed
Karki, Cytoplasmic dynein and dynactin in cell division and intracellular transport. 1999, Pubmed
King, Analysis of the dynein-dynactin interaction in vitro and in vivo. 2003, Pubmed
Lombillo, Minus-end-directed motion of kinesin-coated microspheres driven by microtubule depolymerization. 1995, Pubmed
Maddox, Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos. 2002, Pubmed
Merdes, A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. 1996, Pubmed , Xenbase
Merdes, Formation of spindle poles by dynein/dynactin-dependent transport of NuMA. 2000, Pubmed
Mitchison, Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. 1989, Pubmed
Noda, KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B. 1995, Pubmed
Quintyne, Dynactin is required for microtubule anchoring at centrosomes. 1999, Pubmed
Rogers, Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. 2004, Pubmed
Walczak, The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly. 2002, Pubmed , Xenbase
Walczak, XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. 1996, Pubmed , Xenbase
Waterman-Storer, Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. 1998, Pubmed , Xenbase
Waters, The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work. 1996, Pubmed
Wittmann, The spindle: a dynamic assembly of microtubules and motors. 2001, Pubmed
Wittmann, Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes. 1999, Pubmed , Xenbase
Yu, Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization. 2000, Pubmed