Fig. 8 | Nature Communications

Fig. 8

From: The binding of Borealin to microtubules underlies a tension independent kinetochore-microtubule error correction pathway

Fig. 8

Borealin-mediated non-centromeric CPC-microtubule interaction is required for robust phosphorylation of the kinetochore substrates by the CPC. a Schematic of experimental procedure, for c and d, Borealin and Bub1 siRNA-mediated knockdown of endogenous Borealin and Bub1 in cells expressing chimeric CenpBDBD-INCENP747-918 transgene. b Cartoon showing domain structure of INCENPWT and chimeric LAP-CenpB-INCENP747-918 protein. c Cells expressing LAP-CenpB- INCENP747-918, for targeting Aurora-B to the centromeres, were treated with control siRNA (siLuc) or siBorealin to deplete endogenous Borealin as shown in a. Bub1 siRNA and Haspin inhibitor, 2 μM 5-ITU, were added before fixation in order to remove the inner-centromeric CPC localization signal. Cells were immunostained with antibodies against Hec1 pS44 and Aurora-B and representative images are shown. d Box and whisker graph of normalized Hec1 pS44 intensity and normalized Aurora-B intensity (n = 150 (from 13 cells) for cells treated with siLuc and n = 117 (from 12 cells) for cells treated with siBub1 and siBor). e Schematic of experimental procedure for fi. f Cells were treated as in e, LAP-CenpB- INCENP747-918 and LAP-BorealinWT or LAP-BorealinMTBM expressing cells were treated with siBub1 and haspin inhibitor (2 μM 5-ITU) to delocalize the endogenous CPC from the inner-centromere. Endogenous Borealin was depleted with siBorealin treatment and cells were immunostained with Hec1 pS44 (f) and Aurora-B (h) antibodies, representative images are shown. g Box and whisker graph of normalized Hec1 pS44 and normalized GFP intensity from f (n = 186 (from 12 cells) for WT and n = 171 (from 8 cells) for MTBM). i Box and whisker graph of normalized Aurora-B and GFP intensity from H (n = 135 for WT and n = 147 for MTBM; data from 11 cells per condition). Statistical analysis performed using Mann Whitney Test, ***P < 0.0001 and ns P > 0.05. All representative immunofluorescence images and quantitation is from one of two independent replicates. All Box and whisker plots represent the median (central line), 25th–75th percentile (bounds of the box) and 5th–95th percentile (whiskers). Scale bar 5 μm

Back to article page