The cilia protein IFT88 is required for spindle orientation in mitosis

Journal name:
Nature Cell Biology
Volume:
13,
Pages:
461–468
Year published:
DOI:
doi:10.1038/ncb2202
Received
Accepted
Published online

Cilia dysfunction has long been associated with cyst formation and ciliopathies1. More recently, misoriented cell division has been observed in cystic kidneys2, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells3, 4. Several IFT proteins also localize to spindle poles in mitosis5, 6, 7, 8, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737orpk and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.

At a glance

Figures

  1. IFT88 depletion leads to mitotic defects in HeLa cells, kidney cells from the Tg737orpk mouse mutant and zebrafish.
    Figure 1: IFT88 depletion leads to mitotic defects in HeLa cells, kidney cells from the Tg737orpk mouse mutant and zebrafish.

    (a) Immunofluorescence microscopy images of control (GFP) and IFT88-siRNA-treated mitotic HeLa cells. α-tubulin (microtubules) and γ -tubulin (spindle poles, arrow) staining show spindle pole defects. CREST (kinetochores) or DAPI (DNA) staining shows misaligned chromosomes. Scale bars, 5μm. (b) Quantification of mitotic defects following IFT88- or control (GFP)-siRNA treatment in HeLa cells. Defects include disrupted poles (α - and γ -tubulin), misaligned chromosomes (DAPI staining) and spindle misorientation (spindle tilt, spindle poles in different focal planes). n=70 mitotic cells per experiment. (c) Side views of 3D reconstructed immunofluoresence images showing misoriented mitotic spindles in IFT88- versus control-siRNA-treated HeLa cells. Spindle (EB1), centrosomes (5051) and DNA (Phos-H3). (d) Histogram showing metaphase spindle-angle distribution in control- and IFT88-siRNA-treated cells. n=30 mitotic spindles. Schematic (top) showing spindle angle (α ) measurement. H, hypotenuse. O, opposite. (e,f) Quantification (e) and time-lapse microscopy images (f) showing uneven timing of daughter-cell flattening onto the substrate after mitosis (misoriented cell division) in IFT88-siRNA-treated HeLa cells, compared with control. n=50 mitotic cells per experiment. Arrows, time when the first daughter cell begins flattening. Time, min. Scale bar, 10μm. (g) Immunofluorescence microscopy images (left) showing a disrupted spindle pole (α -tubulin, arrow) in kidney cells derived from the IFT88 mouse mutant Tg737orpk (Tg737−/−), compared with wild type (Tg737+/+). Scale bars, 2μm. Graph (right), quantification of mitotic defects in wild-type and Tg737orpk mutant cells. (h) Immunofluorescence microscopy images of mitotic spindles from the pronephric ducts of whole-mount zebrafish embryos. Control embryo, cell with aligned chromosomes and mitotic spindle oriented in the longitudinal plane of the duct. IFT88-depleted embryo, cell with non-aligned chromosomes and misoriented spindle. Lines, pronephric duct border. Dashed lines, spindle orientation. MO, morpholino. Right, enlargements of spindles outlined by dashed rectangles. Scale bar, 5μm.

  2. IFT88 depletion disrupts astral microtubules and the spindle pole localization of proteins involved in microtubule nucleation in HeLa cells.
    Figure 2: IFT88 depletion disrupts astral microtubules and the spindle pole localization of proteins involved in microtubule nucleation in HeLa cells.

    (a) Immunofluoresence microscopy images (left) of mitotic spindles showing disrupted astral microtubules (α -tubulin) at spindle poles of IFT88-depleted cells, compared with control. Pixel intensity range increased to visualize astral microtubules (arrow). Enlargements, spindle pole region. Graph (right), quantification of cells with long astral microtubules (>3μm). n=70 mitotic spindles per experiment. (b) Side view of 3D reconstructed images (left) showing astral microtubules (EB1 staining) contacting the cortex in control cells (arrow, upper panel) and astral microtubules that fail to contact the cell cortex in IFT88-depleted cells (arrow, lower panel). Dotted lines, cell cortex. Graph (right), quantification of cells with both poles showing astral microtubules contacting cortex. n=50 mitotic spindles per experiment. (c,d) Immunofluorescence microscopy images (c) and quantification (d) of mitotic spindles showing loss of EB1 and γ -tubulin from spindle poles (arrow) in IFT88-depleted cells, compared with control. Graph (d), percentage of cells with disrupted pole localization of EB1 or γ -tubulin. n=50 mitotic spindles per experiment. Scale bar, 5μm. (e) Immunoblots (WB) showing that IFT88 co-immunoprecipitates with EB1 (left) and that γ -tubulin co-immunoprecipitates with IFT88 (right) from lysates of mitotic HeLa cells, demonstrating a mitotic interaction between the proteins, either direct or indirect. Ig, rabbit antibody, negative immunoprecipitation (IP) control. Input, 5% of total lysate used for immunoprecipitation. For full scan of immunoblots, see Supplementary Fig. S8. (f) Quantification of γ -tubulin intensity at spindle poles of mitotic cells showing γ -tubulin recruitment to poles in a microtubule regrowth experiment. t, time after nocodazole washout (min). Bar, median. Experiment shown is representative of three independent experiments. a.u., arbitrary unit. (g) Immunofluoresence microscopy images showing microtubule regrowth (α -tubulin) at mitotic spindle poles 0, 1 and 2min after nocodazole washout in IFT88- or GFP-depleted mitotic cells. t=0min shows no nucleation in GFP- and IFT88-depleted cells, and t=1min and 2min show decreased nucleation in IFT88-depleted cells, compared with control cells. Scale bar, 2μm. (h) Percentage of cells showing detectable nucleation (aster size ≥1μm) 0, 1 and 2min after nocodazole washout. n=50 mitotic cells per experiment; error bars, mean of at least three experiments ± s.d.

  3. IFT88 is required for the movement of peripheral microtubule clusters containing microtubule-nucleating components towards spindle poles in LLC-PK1 cells stably expressing GFP-
[alpha]
-tubulin.
    Figure 3: IFT88 is required for the movement of peripheral microtubule clusters containing microtubule-nucleating components towards spindle poles in LLC-PK1 cells stably expressing GFP– α -tubulin.

    (a) Immunofluoresence microscopy images showing IFT88 and dynein intermediate chain (Dyn) localizing to a peripheral microtubule cluster (GFP– α -tubulin) in a prometaphase cell. Pixel intensity range increased to visualize peripheral microtubule cluster. Scale bar, 5μm. Inset, peripheral microtubule cluster. See Supplementary Fig. S4a for negative controls. (b) Quantification of GFP– α -tubulin LLC-PK1 metaphase cells with ectopic microtubule clusters following IFT88- or control- (lamin) siRNA treatment. n=50 mitotic cells per experiment. (c,d) Immunofluoresence microscopy images of GFP– α -tubulin LLC-PK1 control or IFT88-depleted metaphase cells. γ -tubulin (c), EB1 (d, left) and dynein (d, right) localize to ectopic microtubule clusters. Insets, ectopic microtubule clusters. Scale bar, 5μm. (e) Selected still images from time-lapse microscopy movies of GFP– α -tubulin LLC-PK1 cells. Control prometaphase, minus-end-directed motion of peripheral microtubule clusters towards spindle pole. In IFT88-depleted cells, peripheral clusters formed, but showed no movement towards spindle poles. Full cell (left); enlargement of spindle pole and microtubule cluster (right). Time (min); arrowhead, microtubule cluster; arrow, spindle pole. (f) Immunofluorescence microscopy images (left) and quantification (right) of the relocalization of microtubule clusters to spindle poles in a spindle reassembly assay (α -tubulin, microtubule regrowth following nocodazole washout). The decrease in cells with ectopic microtubule clusters over time correlates with their movement towards the poles. IFT88 depletion delays relocalization of microtubule clusters to poles. Arrows, spindle poles (localization confirmed with centrosome protein staining). Arrowheads, ectopic microtubule clusters. n=40 mitotic cells per experiment per time point. t, time after nocodazole washout (min).

  4. IFT88 moves towards spindle poles and requires microtubules for its spindle pole localization.
    Figure 4: IFT88 moves towards spindle poles and requires microtubules for its spindle pole localization.

    (a) Microtubule pulldown assay shows that IFT88 co-pelleted with taxol-stabilized microtubules in mitotic HeLa cell lysates. Nocodazole (Noc), inhibition of microtubule polymerization used as negative control. α -tubulin, microtubules. (b) Immunofluoresence microscopy images showing IFT88 foci formation (lower panel) after nocodazole washout (α -tubulin, microtubule regrowth; upper panel) in HeLa cells. t, time after nocodazole washout (min). Control without nocodazole (no Noc). Scale bar, 5μm. (c,d) Immunofluorescence microscopy images showing the molecular composition of IFT88 foci in HeLa cells. Maximum projection of a cell with IFT88 foci 5min after nocodazole washout (c) showing that IFT88 foci co-stain for α -tubulin (α -tub) and dynein intermediate chain (Dyn). Enlargements, single plane of the outlined foci. Enlargements of IFT88 foci (d) showing that microtubule clusters (α -tubulin; α -tub) can be observed extending from some foci, and that IFT88 foci co-stain with microtubule-nucleating components (5051, centrosome protein marker; γ -tubulin; EB1). Pixel intensity range increased to visualize foci. Scale bar, 1μm. (e) Quantification of IFT88 intensity at spindle poles of mitotic HeLa cells showing IFT88 recruitment to poles following nocodazole washout. t, time after nocodazole washout (min). Experiment shown is representative of three independent experiments. Bar, median. a.u., arbitrary unit. No nocodazole (No Noc), untreated cells. (f) Still images from time-lapse microscopy imaging of a GFP–IFT88 LLC-PK1 cell line (left) showing one of the GFP–IFT88 foci (arrowhead) moving towards the GFP–IFT88-labelled spindle pole (arrow). Time elapsed is shown in seconds. Scale bar, 1μm. Schematic representation (right) of several GFP–IFT88 foci moving towards (red arrow) or away from (black arrowhead) the spindle pole (grey dot). Time between points, 1s. Arrows indicate the direction of the movement.

  5. IFT88 is part of a dynein1-driven transport complex in mitosis.
    Figure 5: IFT88 is part of a dynein1-driven transport complex in mitosis.

    (a) Immunoblots (left) showing fractions of mitotic HeLa cell lysates obtained after gel-filtration fractionation and probed for IFT88, dynein intermediate chain (Dyn IC), dynactin p150/glued, p50 dynactin, IFT52 and IFT20. Input, total lysate before gel filtration chromatography. Arrowheads, peak elution fraction for calibration proteins: bovine serum albumin (Mr66K), β -amylase (Mr200K), thyroglobulin (Mr669K). V, Void volume. Immunoprecipitation experiment (right) carried out on fractions 16–22 from gel filtration containing dynein. Immunoblots show that IFT88 co-immunoprecipitates with dynein (IC, intermediate chain) after gel filtration chromatography. For full scan of immunoblots, see Supplementary Fig. S8. (b) Immunofluorescence microscopy images of HeLa cells showing IFT88 redistribution from mitotic spindle poles to a more diffuse region surrounding the poles following dynein1 (D1) depletion, compared with control (GFP). α -tubulin; α -tub. Intensity profiles, lower left panels; spindle pole enlargement, lower right panels. Scale bar, 5μm. (c) Percentage of cells with focused IFT88 localization at poles following dynein1- (D1) or dynein2- (D2) siRNA treatment. n=70 mitotic spindles per experiment. (d) Schematic representation of IFT88 (green) redistribution in cilia when dynein2 (D2) is depleted, and around mitotic spindle poles when dynein1 (D1) is depleted. (e) Immunofluorescence microscopy images showing that D1 depletion in HeLa cells delays IFT88 (red) relocalization to spindle poles in a spindle reassembly assay (α -tubulin, green). The decrease of cytoplasmic foci over time, observed in control (GFP) cells correlates with the relocalization of IFT88 from foci to spindle poles. Despite the formation of microtubule clusters in D1-depleted cells, several IFT88 foci remain in the cytoplasm 30min after nocodazole washout. Arrows, spindle poles; arrowheads, IFT88 foci. (f) Percentage of cells with more than ten cytoplasmic foci. n=40 cells per experiment per time point. t, time after nocodazole washout (min). (g) Molecular model for IFT88 function in mitosis. IFT88 is depicted as a component of a minus-end-directed dynein1-driven transport complex. This complex is required for transport of microtubule clusters and their associated nucleating components (EB1 and γ -tubulin) to spindle poles. IFT88 thus contributes to the formation of astral microtubule arrays and consequently spindle orientation. Adapted from ref. 20.

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Affiliations

  1. Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Suite 210, Worcester, Massachusetts 01605, USA

    • Benedicte Delaval,
    • Alison Bright &
    • Stephen Doxsey
  2. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

    • Nathan D. Lawson

Contributions

B.D. and S.D. wrote the manuscript. B.D. conceived and planned the experimental work. B.D. and A.B. carried out the experimental work and analysed the data. N.D.L. provided the zebrafish facility and helped plan and guide the zebrafish experimental work.

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The authors declare no competing financial interests.

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