Journal: Nature Communications

Article Title: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

doi: 10.1038/s41467-023-41091-2

Figure Lengend Snippet: a High temporal resolution spinning-disk confocal time-series of GFP-CENP-E T422A at KTs in U2OS cells. Time: min:sec. Scale bar: 10 μm. Red and white arrowheads indicate a CENP-E T422A comet. b Quantification of GFP-CENP-E T422A stripping velocity represented with mean ± SD. A total of 11 comets in 9 cells from 3 independent experiments were analyzed. c Representative time-series of U2OS GFP-CENP-E WT/T422A cells entering mitosis in the absence of MTs. Time: hour:min. Scale bar: 10 μm. d Time-lapse images of GFP-CENP-E T422A reloading at KTs after MT depolymerization. Time: hour:min Scale bars: 10 μm. e Quantification of GFP-CENP-E T422A at the KTs over time after the addition of nocodazole. Average KT intensities in cells are represented with mean ± SD. N (number of KTs, number of cells, number of independent experiments): t = 0 min (520, 26, 3), t = 10 min (525, 226, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). p values are indicated. f Representative maximum intensity-projected confocal images of expanded fibrous coronas in HeLa cells transiently transfected with GFP-CENP-E WT/T422A. Insets display single KT pairs with expanded coronas. Scale bar: 5 μm; scale bar in insets: 1 μm. g Quantification of corona volume and GFP intensity normalized to CENP-C intensity at KTs. Average KT intensities in cells are represented with mean ± SD. N (number of coronas, number of cells, number of independent experiments) - Corona volume: WT (1038, 29, 3), T422A (1063, 30, 3); KT intensity: WT (899, 40, 3), T422A (781, 38, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). h Structured illumination microscopy (SIM) images of phosphorylated CENP-E at expanded coronas of RPE cells, detected using the anti-phospho-T422 antibody. Scale bar: 5 μm; scale bar in insets: 1 μm. i 3D surface rendering model of the fibrous corona represented in h . j Intensity profile plot of 40 coronas from 10 different cells from 2 independent experiments as represented in h . Thick and dashed lines represent averages and SD, respectively.

Article Snippet: For the generation of stable U2OS GFP-CENP-E/CENP-A-mCherry and HeLa DHC-GFP/CENP-A-mCherry cell lines, we performed lentiviral infection of the U2OS GFP-CENP-E (this study) and HeLa DHC-GFP (gift from Iain Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, USA) cell lines using pLenti CENP-A-mCherry vector (Addgene #89767), followed by fluorescence activated cell-sorting (FACS) of mCherry-positive cells.

Techniques: Stripping Membranes, MANN-WHITNEY, Two Tailed Test, Transfection, Microscopy

Journal: Nature Communications

Article Title: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

doi: 10.1038/s41467-023-41091-2

Figure Lengend Snippet: a Spinning-disk confocal time-series of HeLa cells stably expressing DHC-GFP/CENP-A-mCherry and labeling of DNA with SiR-DNA undergoing the indicated treatments. The red arrow shows the presence of dynein in one KT in early metaphase. Relative time from NEB is represented. Time scale: hour:min. Scale bar: 10 μm. b DHC-GFP dynamics at KTs after NEB. Dots and dashed lines represent mean values and SD, respectively. The solid thick line represents the fitted curve. N (number of cells, number of independent experiments): siCTRL (13, 2), siCENP-E (14, 2). c Representative maximum intensity-projected point-scanning confocal images of U2OS WT/T422A monopoles immunostained against the indicated proteins. Scale bar: 10 μm. d Fluorescence intensity profile plots of the indicated proteins (representing the region depicted by a white dashed line in c ). e Quantification of spindle pole accumulation of indicated proteins in U2OS GFP-CENP-E WT and T422A cells represented in c . Values are plotted with mean ± SD. N (number of cells, number of independent experiments): GFP-CENP-E in WT (51, 3), GFP-CENP-E in T422A (55, 3), SPDLY in WT (54, 3), SPDLY in T422A (52, 3), ZW10 in WT (33, 3), ZW10 in T422A (34, 3), MAD1 in WT (43, 3), MAD1 in T422A (39, 3), BUBR1 in WT (50, 3), BUBR1 in T422A (49, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). p values are indicated.

Article Snippet: For the generation of stable U2OS GFP-CENP-E/CENP-A-mCherry and HeLa DHC-GFP/CENP-A-mCherry cell lines, we performed lentiviral infection of the U2OS GFP-CENP-E (this study) and HeLa DHC-GFP (gift from Iain Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, USA) cell lines using pLenti CENP-A-mCherry vector (Addgene #89767), followed by fluorescence activated cell-sorting (FACS) of mCherry-positive cells.

Techniques: Stable Transfection, Expressing, Labeling, Fluorescence, MANN-WHITNEY, Two Tailed Test