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. 2017 Jul 28;8:150. doi: 10.1038/s41467-017-00209-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Spatially distinct pools of Aurora-B impede distinct steps of the end-on conversion process. a Images of cells expressing CenpB-INCENP-GFP exposed to Monastrol and MG132 with either ZM447439 or DMSO (solvent control), as indicated, prior to immunostaining with antibodies against Tubulin, SKAP and GFP. Cropped images show lateral (upper row) and end-on (lower row) kinetochores. Scale: 5 μm in main and 2 μm in cropped images. Boxed areas correspond to cropped images. b Graph shows percentage of lateral, end-on and detached kinetochores in CenpB-INCENP-GFP expressing cells treated as in a. Each circle represents values from one cell. Black bar marks average values from two independent experimental repeats. * indicates statistically significant difference on the basis of P-values obtained using unpaired Student’s t-test. c Single-plane time-lapse images of Z-stacks show the fate of a lateral or end-on KT (red) bound to MT (green). Time-lapse images of HeLa cells expressing mKate2-Tubulin and CenpB-INCENP-GFP treated with Monastrol. White arrowheads mark KT tracked. Yellow arrows mark the tip of lateral K-fibre. Scale: 2 μm. d Graph shows percentage of lateral or end-on kinetochores that transitioned into other attachment states (D-detached, L-lateral and E-end-on) in time-lapse movies as in c. Error bars are SEM values across three experimental repeats e Cumulative frequency plots show time spent by lateral kinetochores on MT-walls, before changing into another attachment state, in cells expressing either Mis12-INCENP-GFP or CenpB-INCENP-GFP. Error bars are SEM values across three experimental repeats. Note: Mis12-INCENP-GFP curve values were obtained from data presented in Fig. 3d. T50 values are in seconds derived from the cumulative frequency plot. f Table contrasts the consequence of localizing Aurora-B at the outer kinetochore (KT) vs. centromere (cen), using Mis12-INCENP-GFP and CenpB-INCENP-GFP fusion proteins, respectively. Both fusion proteins disrupt end-on conversion but they differently control the fate of lateral attachments (within yellow box). Active Aurora-B status (Active AurB) was assessed using immuno-staining with antibodies against AuroraB-pThr232 as in Fig. 1a. KT-MT attachment status was obtained from fixed-cell studies. Fate of lateral kinetochore and instances of non-productive end-on attachments and productive end-on conversion are from live-cell movies