Journal: Scientific Reports

Article Title: IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

doi: 10.1038/s41598-019-46605-x

Figure Lengend Snippet: IFT88 is recruited at k-fibers minus-ends after laser ablation and contributes to their re-anchoring into spindle. ( a ) Images from time-lapse microscopy of monopolar Emerald-IFT88 LLC-PK1 cells labelled for tubulin (SiR-Tubulin) before and after k-fiber laser ablation (upper panel). Hoechst live was used to identify k-fibers attached to chromosomes. Time post-ablation (s). α-tubulin, IFT88 and α-tubulin/IFT88 stainings (maximal intensity projection of 2 planes) show IFT88 accumulation at minus-end after laser ablation (white arrow). ( b ) Line scans representing α-tubulin and IFT88 fluorescence intensities measured from a to b along the yellow line shown in ( a ). ( c ) Western-blots showing the amount of IFT88 in GFP-α-tubulin LLC-PK1 cells transfected with control (CT) or IFT88 siRNA. α-tubulin: loading control. ( d ) Images from time-lapse microscopy of monopolar GFP-α-tubulin LLC-PK1 labelled for DNA (Hoechst live, red), to allow for k-fibers detection, in control (CT) and IFT88-depleted cells (left panels). Inverted contrast images of α-tubulin before and after k-fiber ablation (ablation site, red arrowhead) show a delay in k-fiber re-anchoring into spindle upon IFT88 depletion. Images were acquired every 3 s for 2 min. Time post-ablation (s). Single planes are shown. Insets: magnification of the ablated k-fibers, dashed boxes regions. Red boxes indicate k-fibers re-anchoring. ( e ) Quantification of the time (s) required for k-fiber re-anchoring into spindle after laser ablation in CT and IFT88-depleted cells. n ≥ 30 ablated k-fibers (1 ablated k-fiber per cell), 3 experiments. Mean +/− s.e.m ** P < 0.01 compared to control ( t test). Scale bars: 5 µm.

Article Snippet: LLC-PK1 (CLS Cell Lines Services GmbH, Germany), GFP-αtubulin LLC-PK1 (Gift from P. Wadsworth) , GFP-αtubulin/mCherry-H2B LLC-PK1 previously generated and YFP-NuMA LLC-PK1 cells were grown in a 1:1 mixture of Opti-MEM/HAM’s F10 media supplemented with 10% fetal bovine serum (FBS).

Techniques: Time-lapse Microscopy, Fluorescence, Western Blot, Transfection, Control

Journal: Scientific Reports

Article Title: IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

doi: 10.1038/s41598-019-46605-x

Figure Lengend Snippet: IFT88 interacts with NuMA and contributes to its enrichment at k-fibers minus-ends after laser ablation. ( a ) Inverted contrast and merged images from time-lapse microscopy of monopolar YFP-NuMA LLC-PK1 combined with MT labelling (SiR-Tubulin) in control (CT) and IFT88-depleted cells before and after k-fiber laser ablation. The 3 s time-point after ablation is shown. Single planes are shown. Insets: magnification of the ablated k-fibers, dashed boxes regions. Hoechst live was used to identify k-fibers attached to chromosomes. Line scans (right) representing NuMA and α-tubulin fluorescence intensities, measured from a to b (control) or from c to d (siRNA IFT88) along the yellow line (left inset on the image), show an accumulation of NuMA at minus-ends of k-fibers after laser ablation in CT cells but not in IFT88-depleted cells. Scale bars: 5 μm. ( b ) Percentage of cells with ablated k-fibers associated with NuMA enrichment. n ≥ 29 ablated k-fibers (1 ablated k-fiber per cell), 3 experiments. Mean +/− s.d. ** P < 0.01 compared to control ( t test). ( c ) Quantification of NuMA fluorescence intensity at minus-ends of MT after laser ablation. n ≥ 22 cells, 2 experiments. Mean +/− s.e.m. * P < 0.05 compared to control ( t test). ( d ) Line scans of IFT88 and NuMA fluorescence intensities overtime at the minus-end of k-fibers after laser ablation. x: pre-ablation; black arrow indicates the time of ablation. ( e ) Endogenous immunoprecipitation of IFT88 performed on LLC-PK1 cells (nocodazole + 3 min washout) shows an interaction with NuMA. Scale bars: 5 μm.

Article Snippet: LLC-PK1 (CLS Cell Lines Services GmbH, Germany), GFP-αtubulin LLC-PK1 (Gift from P. Wadsworth) , GFP-αtubulin/mCherry-H2B LLC-PK1 previously generated and YFP-NuMA LLC-PK1 cells were grown in a 1:1 mixture of Opti-MEM/HAM’s F10 media supplemented with 10% fetal bovine serum (FBS).

Techniques: Time-lapse Microscopy, Control, Fluorescence, Immunoprecipitation

Journal: Scientific Reports

Article Title: IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

doi: 10.1038/s41598-019-46605-x

Figure Lengend Snippet: IFT88 contributes to k-fibers reincorporation into spindle after nocodazole washout and is required for proper chromosomes alignment. ( a ) Immunofluorescence images of GFP-α-tubulin LLC-PK1 upon nocodazole treatment followed by 5 min washout showing defects in k-fibers reincorporation into the main spindle in IFT88-depleted cells compared to control. α-tubulin and α-tubulin/DNA stainings are shown (left panel). Percentage of mitotic cells with disorganized spindles or misaligned chromosomes upon nocodazole washout (right panel). n > 300 mitotic cells. 3 experiments. Mean +/− s.d. *** P < 0.001 compared to control ( t test). ( b ) Immunofluorescence images of GFP-α-tubulin LLC-PK1 cells upon nocodazole treatment followed by 2 min washout showing defects in NuMA minus-ends localization in IFT88-depleted cells. NuMA and α-tubulin/NuMA stainings are shown (left panel). Insets: magnified dashed boxes regions. Quantification of NuMA fluorescence intensity at the minus-ends of acentrosomal microtubule asters upon nocodazole washout in control and IFT88-depleted cells (right panel). n ≥ 15 cells per condition and n ≥ 73 acentrosomal microtubule asters per condition, 1 experiment shown, representative of 2 experiments. Mean +/− s.e.m. * P < 0.05 compared to control ( t test). ( c ) Images from time-lapse microscopy of LLC-PK1 GFP-α-tubulin/mCherry-H2B cells showing defects in spindle organization and chromosomes alignment in IFT88-depleted cells compared to control upon nocodazole washout. Time after washout (min). ( d ) Immunofluorescence images of GFP-α-tubulin LLC-PK1 cells showing defects in chromosomes alignment (without nocodazole challenge) upon IFT88 depletion. α-tubulin/DNA staining is shown (left). Quantification (right): percentage of mitotic cells with misaligned chromosomes (siRNA control, IFT88 and IFT88 #2 as indicated). n > 100 mitotic cells. 3 experiments. Mean +/− s.d. ** P < 0.01 compared to control ( t test). ( e ) Immunofluorescence images (left) showing α-tubulin and DNA stainings in HCT116-AID-IFT88 cells. Control (No auxin) and auxin (30 h)-induced AID-YFP-IFT88 degradation conditions are shown. Quantification (middle): percentage of mitotic cells with misaligned chromosomes upon auxin treatment (30 h). n > 50 mitotic cells. 3 experiments. Mean +/− s.e.m * P < 0.05 compared to control ( t test). Western-blots (right) showing AID-YFP-IFT88 depletion in HCT116 cells upon auxin treatment. α-tubulin: loading control. In all panels, maximum projections are shown, scale bars: 5 or 10 μm.

Article Snippet: LLC-PK1 (CLS Cell Lines Services GmbH, Germany), GFP-αtubulin LLC-PK1 (Gift from P. Wadsworth) , GFP-αtubulin/mCherry-H2B LLC-PK1 previously generated and YFP-NuMA LLC-PK1 cells were grown in a 1:1 mixture of Opti-MEM/HAM’s F10 media supplemented with 10% fetal bovine serum (FBS).

Techniques: Immunofluorescence, Control, Fluorescence, Time-lapse Microscopy, Staining, Western Blot