Journal: bioRxiv

Article Title: Microtubule occupancy at kinetochores links checkpoint silencing with mitotic memory

doi: 10.64898/2026.01.26.701783

Figure Lengend Snippet: (A) Schematic summary of the kinetochore response to partial laser microsurgery ablation. (B) Representative live-recording images of Indian muntjac cells expressing 2x-GFP-CENP-A and GFP-CENTRIN-1 (both in blue) and mScarlet-MAD1 (orange/ grey scale) upon partial kinetochore surgery or acute Nocodazole treatment. Insets: kinetochore pair that was perturbed. Arrows point to the ablated kinetochore region, pre- and post-surgery (solid and hollow arrow, respectively). Arrowheads show MAD1 recruitment. Scale bar: 2µm. (B’) Max normalized line intensity profile plot of MAD1/CENP-A signal ratio along the longitudinal axis of the kinetochores highlighted in A (surgery cell in orange; nocodazole cell in grey). (C) Dot plot showing the time from surgery to observable deformation (bending; n= 12 kinetochores) and MAD1 signal re-appearance (n = 14 kinetochores) at the sister kinetochore in cells where SAC recruitment was observed. Each dot represents an individual kinetochore from a different cell, recorded during 13 independent experiments. P <0.0015, Wilcoxon matched-pairs signed rank test. (D) Max normalized MAD1/CENP-A ratio intensity profile along the longitudinal axis of kinetochores after partial surgery of their sisters (orange) or upon acute Nocodazole treatment (NOC; grey). Thin lines show individual kinetochores and thick lines show average curves (surgery n = 11 kinetochores/cells, 10 independent experiments; Nocodazole n = 19 kinetochores/cells, 2 independent experiments). The graph depicts a cohort of cells that recruit MAD1 to the proximal tip of the kinetochore, relative to the surgery region (n = 11/16 MAD1 recruiting kinetochores). (E) Coefficient of variation of MAD1/CENP-A signal ratio along kinetochores after partial surgery of their sisters. Dots represent individual kinetochores pooled from all cells where MAD1 recruitment was observed (surgery n = 16 cells, 14 independent experiments; Nocodazole n = 19 cells, 2 independent experiments). P <0.0001, Mann-Whitney test. (F), (F’) Representative images from correlative live cell spinning disk (F) and fixed CH-STED microscopy (F’). Indian muntjac cells expressing 2x-GFP-CENP-A and GFP-CENTRIN-1 (both in blue) and mScarlet-MAD1 (orange/grey scale) were fixed after partial kinetochore surgery and stained to visualize microtubules (grey). Insets: kinetochore pair that was perturbed. Arrows point to the ablated kinetochore region, pre- and post-surgery (solid and hollow arrow, respectively). Arrowheads show MAD1 recruitment. Scale bar: 2µm. (F’’) Max normalized line intensity profile plot of MAD1/CENP-A (live cell data just before fixation, orange) and microtubules/CENP-A (fixed cell data, grey) signal ratio along the longitudinal axis of the kinetochore highlighted in F and F’.

Article Snippet: Cells were imaged using Nikon-Ti spinning-disk confocal microscope with a 100x 1.4 NA oil objective as detailed in the “Monitoring kinetochore MAD1 levels in live recordings” section.

Techniques: Expressing, MANN-WHITNEY, Microscopy, Staining