Jones LA , Villemant C , Starborg T , Salter A , Goddard G , Ruane P , Woodman PG , Papalopulu N , Woolner S , Allan VJ .

098390 Wellcome Trust , WT086077MA Wellcome Trust , Biotechnology and Biological Sciences Research Council , Medical Research Council , BB/G012652/1 Biotechnology and Biological Sciences Research Council , WT098390 Wellcome Trust , BB_BB/G012652/1 Biotechnology and Biological Sciences Research Council , Wellcome Trust , G0900930 Medical Research Council , G0600253 Medical Research Council

	Figure 1. Loss of LICs does not affect dynein complex stability. (A) LIC KnD in HeLaM cells was assessed by immunoblotting with isoform-specific LIC antibodies (asterisk, a cross-reacting band; arrow, LIC2) with lamin A/C depletion (LA) as a control. Black line indicates that intervening lanes have been spliced out. Tub, tubulin. (B) Extracts made from untreated (wild type [WT]) or HeLaM cells depleted of lamin A/C, both LICs (LIC), or DHC (HC), were immunoblotted for IC and DHC, with tubulin as a loading control. (C) KI-treated extracts from control (cont.; top) or LIC1- and 2-depleted (bottom) HeLaM cells were run on sucrose gradients containing 0, 100, or 150 mM KI and immunoblotted for IC. (D) Silver-stained SDS-PAGE of dynein isolated from control and LIC1- and 2-depleted cells. Molecular mass markers (in kilodaltons) are shown.
	Figure 3. Mitosis is prolonged when both LICs are depleted. (A) Mitotic index of HeLaMs after KnDs (>731 cells in total per condition, three independent experiments; means ± SEM. ***, P < 0.001, Student’s t test). (B) The duration of mitosis after depleting LICs or DHC or lamin A/C (LA) was determined from phase contrast time-lapse videos. The plot shows the percentage of cells completing mitosis in a given time (158–1,133 cells per treatment combined from three independent experiments; see Results for survival analysis p-values). (C) Mitotic index of epithelial cells in Xenopus embryos injected with a control MO (Ctrl) or MOs targeting LIC 1, 2, or both LICs (*, P < 0.05; ***, P < 0.001; one-way ANOVA, n = 3 independent experiments; means ± SEM). (D) Mitotic progression in Xenopus embryos injected with control or LIC1 and LIC2 MOs was assessed by time-lapse confocal imaging and plotted as a cumulative frequency (n = 43 and 51 spindles analyzed in four and five independent experiments for control MO and LIC1 plus LIC2 MOs, respectively).
	Figure 4. Depletion of LICs leads to multipolar spindles in Xenopus embryos. (A) Confocal images of spindles in embryos injected with control (Ctrl MO) or LIC 1 and 2 MOs (LIC1&2 MO) labeled with anti–α-tubulin and DAPI. Bars, 10 µm. (B) Quantification of spindle morphology in Xenopus embryos after depletion of LIC1, LIC2, or both LICs. Spindles categories: two poles = bipolar; three to four poles = minor multipolar; more than four poles = major multipolar. (n = 4 independent experiments, ≥27 embryos scored in total, 240–400 spindles categorized for each condition, means ± SEM). (C) Embryos injected with control or LIC1 and 2 MOs were rescued with a GFP plasmid or a mixture of plasmids encoding LICs 1 and 2 and scored as in B (a total of 98–159 mitotic cells from 13–18 embryos scored per condition, means ± SEM). Two-way ANOVA analysis versus controls: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For other comparisons, see Results. C, control.
	Figure 5. Multipolar spindles form by two mechanisms in LIC1 and 2 morphant embryos. Mitotic spindles were imaged live in Xenopus embryos using GFP–α-tubulin (green) and mCherry-H2B (magenta). (A) Bipolar spindles assembled and proceeded normally through mitosis and cytokinesis in controls (Ctrl). (B) An LIC morphant that formed a multipolar spindle at the onset of mitosis and subsequently failed cytokinesis (arrows). (C) An LIC morphant that assembled a bipolar spindle, which became multipolar when spindle poles fragmented (arrows). (D) Pole fragmentation was also seen in multipolar LIC MO spindles (arrows). Bars, 10 µm; time stamps display minutes and seconds.
	Figure 6. Multipolar LIC1 and 2 morphant spindle poles contain single centrioles. (A–C) Spindle pole composition was analyzed in control (Ctrl; A) and LIC1 and 2 morphant (B and C) embryos using 3View EM. Chromosomes (blue) and centrioles (red) were reconstructed using Imaris software. Sequential z slices of spindle poles showing a pair of centrioles in control (Ai and Aii) and single centrioles in a multipolar LIC morphant spindle (Bi–Bv). (Ci and Cii) An LIC morphant cell with a bipolar spindle has an increased distance (white double-headed arrows) between centrioles compared with control. Bars: (A, B, and C) 5 µm; (Ai, Aii, Bi–Biv, and Cii) 1 µm. Black arrows mark centrioles.
	Figure 7. Premature centriole disengagement and PCM fragmentation occurs after LIC depletion in HeLa cells. (A, B, and D) Centrin-1–GFP HeLa cells treated with scrambled (control) or LIC 1 and 2 siRNAs were labeled with anti–α-tubulin, anti-PC, and DAPI. (A) Minor multipolar spindles in centrin-1–GFP HeLa cells. White arrowheads, mother centrioles; red arrowheads, daughter centrioles. PC spots without associated centrioles are common at minor poles (cyan arrows) and rare at major poles (yellow arrow). Bars, 5 µm. (B) Centriole number in interphase centrin-1–GFP HeLa cells was scored (>200 cells scored per condition in each of three to four independent experiments, means ± SEM). (C) HeLaM KnD cells were stained with anti-CETN3 to detect centrioles. Centriole number was counted per pole in 100 mitotic cells, in three independent experiments (means ± SEM). (D) Centriole and PC distribution at each major and minor spindle pole in LIC1- and 2-depleted centrin-1–GFP HeLa cells was scored (69 cells, from two independent experiments). ***, P < 0.001, Student’s t test.
	Figure 8. LIC depletion does not affect the distribution of motor-associated components or γ-tubulin. HeLaM cells treated with scrambled or LIC1 and 2 siRNAs were fixed and labeled with the indicated antibodies, and cells with minor multipolar spindles were imaged using the same exposure for control and LIC-depleted cells. Maximum projections of deconvolved z series are shown. Bar, 5 µm.
	Figure 9. Inhibition of Eg5, but not loss of K fibers, prevents the formation of minor multipolar spindles. (A, B, and F) HeLaM cells were treated as indicated, labeled, and scored for mitotic phenotype (>100 cells in each of four independent experiments, means ± SEM). (A) Scrambled (SC) or LIC siRNA-treated HeLaM cells were incubated for 48 h and then transfected with scrambled or Nuf2 siRNAs and fixed after a further 24-h incubation. (B) Depleted cells were treated with DMSO or 100 nM CENP-E inhibitor (GSK-923295; CI) for 90 min before fixation. (C) Confocal images of mitotic spindles in control (Ctrl) or LIC1 and 2 Xenopus morphants treated with DMSO or STLC to inhibit Eg5. Spindles were stained with anti–α-tubulin, DAPI, and anti–γ-tubulin. Insets show zoom in of a monopolar spindle: two adjacent γ-tubulin foci indicate a single pole. Bar, 20 µm. (D) Quantitation of spindle morphology in control and LIC1 and 2 morphants treated with DMSO or STLC (means ± SEM, six independent experiments). (E) The mitotic index of control (C) and LIC morphants was determined ± STLC treatment (ns = not significant, Student’s t test, means ± SEM, n = 6 independent experiments). (F) Scrambled or LIC siRNA-treated HeLaM cells were treated with DMSO or 2 µM STLC for 2 h. See Tables S1 and S2 for two-way ANOVA results for A, B, D, and E.

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