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Plk1 protects kinetochore–centromere architecture against microtubule pulling forces

2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês

10.15252/embr.201948711

1469-3178

Robert F. Lera, Roshan X. Norman, Marie Dumont, Alexandra Dennee, Joanne Martin‐Koob, Daniele Fachinetti, Mark E. Burkard,

Ubiquitin and proteasome pathways

Article30 August 2019free access Transparent process Plk1 protects kinetochore–centromere architecture against microtubule pulling forces Robert F Lera Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Roshan X Norman Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Marie Dumont Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Alexandra Dennee Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Joanne Martin-Koob Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Daniele Fachinetti orcid.org/0000-0002-8795-6771 Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Mark E Burkard Corresponding Author [email protected] orcid.org/0000-0002-4215-7722 Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Robert F Lera Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Roshan X Norman Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Marie Dumont Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Alexandra Dennee Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Joanne Martin-Koob Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Daniele Fachinetti orcid.org/0000-0002-8795-6771 Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Mark E Burkard Corresponding Author [email protected] orcid.org/0000-0002-4215-7722 Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Search for more papers by this author Author Information Robert F Lera1,2, Roshan X Norman1,2,‡, Marie Dumont3,‡, Alexandra Dennee1,2, Joanne Martin-Koob1,2, Daniele Fachinetti3 and Mark E Burkard *,1,2 1Division of Hematology/Oncology, Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA 2UW Carbone Cancer Center, University of Wisconsin, Madison, WI, USA 3Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +1 608-262-2803; E-mail: [email protected] EMBO Rep (2019)20:e48711https://doi.org/10.15252/embr.201948711 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During mitosis, sister chromatids attach to microtubules which generate ~ 700 pN pulling force focused on the centromere. We report that chromatin-localized signals generated by Polo-like kinase 1 (Plk1) maintain the integrity of the kinetochore and centromere against this force. Without sufficient Plk1 activity, chromosomes become misaligned after normal condensation and congression. These chromosomes are silent to the mitotic checkpoint, and many lag and mis-segregate in anaphase. Their centromeres and kinetochores lack CENP-A, CENP-C, CENP-T, Hec1, Nuf2, and Knl1; however, CENP-B is retained. CENP-A loss occurs coincident with secondary misalignment and anaphase onset. This disruption occurs asymmetrically prior to anaphase and requires tension generated by microtubules. Mechanistically, centromeres highly recruit PICH DNA helicase and PICH depletion restores kinetochore disruption in pre-anaphase cells. Furthermore, anaphase defects are significantly reduced by tethering Plk1 to chromatin, including H2B, and INCENP, but not to CENP-A. Taken as a whole, this demonstrates that Plk1 signals are crucial for stabilizing centromeric architecture against tension. Synopsis Plk1 inhibition compromises kinetochore integrity during chromosome segregation without activating mitotic checkpoint, thereby leading to formation of micronuclei. Loss of Plk1 activity leads to major loss of kinetochore proteins including CENP-A from the chromosome in response to microtubule pulling forces. As cells progress into anaphase, the chromosomes lacking CENP-A lag behind the segregating masses. Depletion of DNA helicase PICH partially rescues the kinetochore disruption induced by Plk1 inhibition. Plk1 inhibition does not activate mitotic checkpoint, and therefore leads to formation of micronuclei. Introduction The human kinetochore is a multi-protein complex essential for accurate segregation of sister chromosomes during mitosis. Within this structure, microtubule-binding proteins of the KMN complex (Knl1, Mis12 complex, and Ndc80 complex) assemble on a constitutive layer of chromatin-embedded proteins, termed the constitutive centromere-associated network (CCAN). During each cell cycle, the CCAN is replenished and redistributed to the new sister chromosome generated by DNA replication. CENP-A, a histone H3 variant, epigenetically establishes and maintains the CCAN to ensure genomic integrity and cell viability 12345. Immediately following mitosis, new CENP-A is rapidly integrated into centromeric chromatin as a direct consequence of loss of Cdk1 activity 678, a process that requires HJURP, Mis18, and localized kinase signals from Plk1 9101112. In this manner, the CCAN and centromere specification is maintained in proliferating cells. Signals within the kinetochore are crucial to generate stable microtubule attachment and biorientation, in which replicated sister chromatids are linked to opposite spindle poles. Multiple protein kinases regulate kinetochore functions, including Mps1 to initiate the mitotic checkpoint 1314, BubR1 to control microtubule attachment and checkpoint signaling 1516, Aurora B to correct erroneous microtubule attachments 17181920, Haspin kinase to align chromosomes 21, and Plk1 to stabilize end-on microtubule attachments 222324 and promote CENP-A assembly following mitosis 12. Although these kinases all localize within the kinetochore, CCAN, or inner centromere, their distributions within these structures are distinct, reflecting their disparate roles. Plk1 phosphorylates substrates throughout the kinetochore–CCAN even though the ~ 5 nm size of a kinase domain is dwarfed by the ~ 100-nm scale of a kinetochore. Plk1 reaches substrates either by binding them directly or by binding adjacent proteins. Indeed, a number of binding partners within the kinetochore are known, including Bub1 25, BubR1 26, and CENP-U/PBIP 27. Multiple partners are important, as Plk1 tethered to distinct partners within the kinetochore can phosphorylate only regionally within this structure 28. Although most of these binding partners are at the outer kinetochore, Plk1 signals arising from the inner centromere and chromatin are crucial for proper chromosome alignment and accurate anaphase segregation 28. We previously found that partial loss of Plk1 activity leads to an anaphase segregation defect 29 that did not trigger the mitotic checkpoint. In that study, the ~ 15% of lagging chromosomes had a stretched appearance suggesting merotelic attachments, though these aberrant microtubule attachments were not directly observable. Here, we considered the alternative that partial Plk1 inhibition causes chromosome mis-segregation via a novel mechanism also silent to the mitotic checkpoint. Indeed, we find that Plk1 activity is required at the centromere to stabilize the CCAN and maintain genomic integrity against spindle tension. The mitotic spindle exerts ~ 700 pN force across the mitotic kinetochore 30, causing stretch between sister kinetochores and within each kinetochore 3132, which necessarily is transmitted across centromeric chromatin. In the absence of full Plk1 activity, chromosomes align on the metaphase plate initially, followed by loss of multiple kinetochore and CCAN components, including CENP-A. As cells progress into anaphase, the chromosomes lacking CENP-A lag behind the segregating masses, yielding cells with chromatin devoid of either ancestral or nascent CENP-A. Kinetochore disruption is restored, in part, by depletion of the DNA helicase PICH or enforcing Plk1 signals at chromatin or the inner centromere. Thus, "kinetochore rupture" is a new effect of Plk1 inhibition that is not detected by the mitotic checkpoint. Results To confirm previous findings and to identify the specific roles of Plk1 at the kinetochore, we performed time-lapse videomicroscopy with histone H2B-GFP- and mCherry-tubulin-labeled RPE1 cells (Fig 1A–C and Movies EV1, EV2 and EV3). As expected, untreated cells efficiently aligned and segregated chromosomes. Next, these cells were challenged with low nanomolar concentrations of BI-2536, a specific inhibitor of Plk1 23. With treatment, chromosomes aligned in metaphase followed by a secondary misalignment (yellow arrowheads). Cells progressing to anaphase commonly exhibited lagging chromosomes, which were later ensconced into micronuclei in daughter cells (white arrowheads). These findings confirm previous observations that high Plk1 activity is required to maintain metaphase chromosome alignment and to ensure accurate chromosome segregation in anaphase, but not required for mitotic progression. Figure 1. Plk1 activity is important for outer kinetochore integrity during mitosis in RPE1 cells A, B. Maximum-intensity projection frames from live-cell fluorescence microscopy of RPE1 cells undergoing mitosis untreated (A) and exhibiting lagging chromosomes after Plk1 inhibition with 40 nM BI-2536 (B). Arrowheads in insets highlight micronucleus formation. Time, in min:s from metaphase onset. Scale bars, 5 μm. C. Maximum-intensity projection frames from live-cell fluorescence microscopy of RPE1 cell exhibiting misaligned chromosomes after Plk1 inhibition with 40 nM BI-2536. Yellow arrowheads highlight misaligned chromosomes. Time, in min:s from metaphase onset. Scale bars, 5 μm. D. Graph shows average percentage (± SEM) of anaphase cells exhibiting lagging chromosomes after Plk1 inhibition (BI-2536) or nocodazole washout (n > 25 cells/experiment; three independent experiments). E. Representative maximum-intensity projection micrographs of kinetochore protein localization in anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight lagging kinetochores, marked by ACA. Scale bars, 5 μm. F. Graphs show relative volume intensities of indicated proteins from (E) at the kinetochores of segregated (blue) and lagging (red) chromosomes. Each circle represents a single segregated (n = 4/cell) or lagging (n = 1–7/cell) kinetochore from the same cell (10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by Kruskal–Wallis test with Dunn's correction for multiple comparisons. Download figure Download PowerPoint Lagging chromosomes can arise by distinct mechanisms. One common mechanism is merotelic attachments, the link of a single kinetochore to both mitotic poles 33. Such attachments of single kinetochores to both poles are generated by nocodazole washout and impair the ability of a lagging chromosome to segregate in either direction. To compare this common mechanism with Plk1 inhibition, we generated lagging chromosomes with BI-2536 treatment or nocodazole treatment/washout (Fig 1D–F) and evaluated two components of the heterotetrameric Ndc80 complex that directly bind microtubules at the outer kinetochore. Strikingly, both Hec1/Ndc80 and Nuf2 intensities are largely reduced on lagging chromosomes with Plk1 inhibition compared to nocodazole washout segregants. By contrast, these Ndc80 components are retained in properly segregated Plk1-inhibited chromosomes, and in merotelic lagging chromosomes generated with nocodazole washout. To ensure the observations are a direct effect of Plk1 inhibition, we evaluated lagging chromosomes in Plk1as RPE1 cells, which express a modified Plk1 allele sensitive to the bulky ATP analog, 3-MB-PP1 2934. Indeed, partial inhibition of Plk1 yielded lagging chromosomes with marked reduction of Hec1 (Fig EV1A–C), consistent with our findings in Plk1-wild-type RPE1 cells. These results demonstrate a novel mechanism by which Plk1 regulates kinetochore integrity and chromosome segregation during human mitosis. Click here to expand this figure. Figure EV1. Defects in outer kinetochore integrity are specific to loss of Plk1 activity (related to Fig 1) Graph shows average percentage (± SEM) of EGFP-Plk1as-expressing RPE1 cells exhibiting lagging chromosomes in anaphase after Plk1as inhibition (200 nM 3-MB-PP1) or nocodazole washout (n = 30 cells/experiment; four independent experiments). Representative maximum-intensity micrographs of anaphase cells from (A). Insets highlight presence/absence of Hec1 at lagging kinetochores, marked by ACA. Scale bars, 5 μm. Graph shows relative volume intensities of Hec1 (left) and ACA (right) at segregated (blue) and lagging (red) kinetochores from (A, B). Each circle represents a single kinetochore (n = 10 segregated kinetochores/cell, 1–8 lagging kinetochores/cell; eight cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by Kruskal–Wallis test with Dunn's correction for multiple comparisons. Download figure Download PowerPoint Mechanistically, the lack of the Ndc80 complex on lagging chromosome could be explained by failed recruitment or by secondary removal. To test if Plk1 is required for Hec1/Ndc80 recruitment, cells were arrested in mitosis using monastrol or nocodazole and exposed to low (40 nM) or high (200 nM) concentrations of BI-2536. Neither concentration of BI-2536 impaired kinetochore recruitment of Hec1 (Fig 2A and B), which is consistent with prior findings 35. To test for secondary removal, cells were challenged with MG-132 to prevent anaphase entry along with BI-2536 or microtubule poisons (nocodazole or paclitaxel) to generate misaligned chromosomes (Fig 2C). As expected, nocodazole- and paclitaxel-challenged cells exhibited equal Hec1 kinetochore intensity in the majority (> 90%) of misaligned chromosome pairs (Fig 2D and E). Strikingly, misaligned chromosome pairs in the Plk1-inhibited group exhibited unequal Hec1 distribution. The loss was restricted to the "pole-distal" kinetochore, consistent with a model where the chromosome pair misaligns through pulling toward the retained kinetochore. These findings demonstrate that Plk1 mediates Hec1 maintenance at, but not recruitment to, the kinetochore. Figure 2. Plk1 activity is not required for Hec1 kinetochore recruitment, but maintains it against microtubule pulling forces Representative maximum-intensity projection micrographs of Hec1 kinetochore localization in prometaphase cells arrested with monastrol (left) or nocodazole (right) with or without Plk1 inhibition (BI-2536). Scale bars, 5 μm. Graph shows average relative volume intensity (± SEM) of total Hec1 and ACA from (A) (n = 10 cells/experiment; three independent experiments). Graph shows average percentage (± SEM) of metaphase cells with misaligned chromosomes after 2-h Plk1 inhibition (BI-2536) or nocodazole, paclitaxel, or combination challenge. MG-132 used to prevent mitotic cells from entering anaphase (n = 100 cells/experiment; ≥ 4 independent experiments). Representative maximum-intensity projection micrographs of cells from (C). Insets and linescans highlight 3 types of Hec1 intensity distribution observed between misaligned chromosome pairs. Tubulin indicates approximate position of spindle poles. Scale bars, 5 μm. Graph shows average percentage (± SEM) of misaligned chromosome pairs exhibiting each of the distribution types from (D) (n = 1–3 chromosomes/cell; 10 cells/experiment; ≥ 4 independent experiments). "Decreased" intensity indicates ≤ 50% intensity of sister kinetochore. Download figure Download PowerPoint To determine when Plk1 activity is required to retain Hec1 at kinetochores, we performed time-lapse imaging of Plk1as cells challenged with 3-MB-PP1 at distinct time points following release from an S-phase thymidine block (Appendix Fig S1A). This demonstrated similar rates of lagging chromosomes whether inhibition was initiated at imaging onset, or for the entire 8.5 h prior to imaging. We conclude that Plk1 inhibition in late G2 and mitosis is sufficient to yield the Hec1 mislocalization. Because the observed defects occur following initial chromosome alignment and are limited to one kinetochore of a sister pair, we hypothesized that impaired Plk1 activity weakens the kinetochore assembly on centromeric chromatin against spindle microtubule pulling forces, leading to stochastic rupture. Once a kinetochore ruptures, the tension is relieved across the kinetochore of the remaining sister chromosome, leading to poleward migration of the pair. Finally, the sister chromosome lacking a kinetochore would be predicted to fail to segregate in anaphase. To test this model, we employed low-concentration nocodazole or paclitaxel to relieve tension. As expected, these chemicals can generate misaligned chromosomes individually or with BI-2536 (Fig 2C). Notably, the misaligned chromosomes co-challenged with either spindle poison or BI-2536 retained Hec1 (Fig 2E), indicating that a reduction in microtubule pulling forces at kinetochores is sufficient to prevent kinetochore rupture. In a second experiment, we challenged cells with two distinct inhibitors of Mps1 to abrogate the spindle checkpoint. Inhibiting Mps1 markedly shortens the duration of mitosis, reducing the time for correct microtubule attachments and generation of tension across the kinetochore 363738. Consistent with our hypothesis, the kinetochores of lagging chromosomes remained intact when both Plk1 and Mps1 were inhibited (Appendix Fig S1B–D). Taken together, these data support a model where microtubule attachment and tension drive kinetochore loss in the setting of reduced Plk1 activity. Plk1 activity is required for integrity of the entire kinetochore To further evaluate the kinetochore components affected by Plk1 inhibition, we probed for the scaffolding protein Knl1, which facilitates kinetochore recruitment of BubR1 and Mad1 (Fig EV2A and C). Like Hec1, Knl1 intensity is markedly reduced in lagging chromosomes (Fig EV2B) or in pole-distal misaligned chromosomes prior to anaphase (Fig EV2D). Concordant with the loss of Knl1, we observed diminished BubR1 and Mad1 in pole-distal misaligned chromosomes also lacking Hec1 (Fig EV2E–H). Next, we evaluated two CCAN components that directly link the kinetochore to centromeric chromatin: CENP-C and CENP-T (Fig EV2A) 39404142. Consistent with our other findings with Plk1 inhibition, both CENP-C and CENP-T intensities were significantly reduced from kinetochores of lagging chromosomes (Fig 3A–D and Appendix Fig S2A) and disproportionately lost from pole-distal kinetochores of misaligned chromosomes in metaphase cells (Appendix Fig S2B and C). Click here to expand this figure. Figure EV2. Kinetochore disruption after Plk1 inhibition extends to Knl1, impairing checkpoint protein recruitment Illustrative map of the kinetochore highlighting positions of KMN (Knl1, Mis12, Ndc80) and CCAN complexes relative to microtubules and centromeric chromatin. Representative maximum-intensity projection micrographs of kinetochore protein localization in anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight lagging kinetochores, marked by ACA. Scale bars, 5 μm. Illustrative diagram of Knl1 indicating method of BubR1 and Mad1 recruitment. Representative maximum-intensity projection micrographs of metaphase cell with misaligned chromosome pairs after 2-h Plk1 inhibition (BI-2536). Inset highlights decreased localization of Knl1 and Hec1 to the pole-distal kinetochores, indicated by ACA. MG-132 was used to prevent mitotic cells from entering anaphase. Scale bar, 5 μm. Representative maximum-intensity projection micrographs of metaphase cells with misaligned chromosome pair after 2-h Plk1 inhibition (BI-2536) or nocodazole challenge. Insets highlight distribution of BubR1 and Hec1 at misaligned kinetochore pairs, indicated by ACA. MG-132 was used to prevent mitotic cells from entering anaphase. Scale bars, 5 μm. Graphs show average percentage of misaligned pole-distal kinetochores with localized (green) or decreased (red) BubR1 and Hec1 after 2-h Plk1 inhibition (BI-2536) or nocodazole challenge (n = 1–10 kinetochore pairs/cell; 10 cells/experiment; two independent experiments). "Decreased" intensity indicates ≤ 50% intensity of sister kinetochore. Representative maximum-intensity projection micrographs of metaphase cells with misaligned chromosome pair after 2-h Plk1 inhibition (BI-2536) or nocodazole challenge. Insets highlight distribution of Mad1 and Hec1 at misaligned kinetochore pairs, indicated by ACA. MG-132 was used to prevent mitotic cells from entering anaphase. Scale bars, 5 μm. Graphs show average percentage of misaligned pole-distal kinetochores with localized (green) or decreased (red) Mad1 and Hec1 after 2-h Plk1 inhibition (BI-2536) or nocodazole challenge (n = 1–10 kinetochore pairs/cell; 10 cells/experiment; two independent experiments). "Decreased" intensity indicates ≤ 50% intensity of sister kinetochore. Download figure Download PowerPoint Figure 3. The integrity defect extends throughout the kinetochore, including CENP-A Representative maximum-intensity projection micrographs of kinetochore protein localization in anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight lagging kinetochores, marked by ACA. Scale bars, 5 μm. Graphs show relative volume intensities of CENP-C, Hec1, and ACA at lagging chromosomes. Each circle represents a single kinetochore (n = 1–7 kinetochores/cell; 10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by two-tailed Mann–Whitney test. Representative maximum-intensity projection micrographs of kinetochore protein localization in anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight lagging kinetochores, marked by ACA. Scale bars, 5 μm. Graph shows relative volume intensities of CENP-T, Hec1, and ACA at lagging chromosomes. Each circle represents a single kinetochore (n = 1–7 kinetochores/cell; 10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by two-tailed Mann–Whitney test. Representative maximum-intensity projection micrographs of kinetochore protein localization in anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight lagging kinetochores, marked by ACA. Scale bars, 5 μm. Graph shows relative volume intensity of CENP-A at segregated (blue) and lagging kinetochores (red) from (E). Each blue circle represents all segregated kinetochores/cell (n = 10 cells/experiment; three independent experiments), whereas each red circle represents a single kinetochore (n = 1–6 kinetochores/cell; 10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by two-tailed Mann–Whitney test. Download figure Download PowerPoint Finally, we probed for CENP-A, the centromere-specific histone variant that provides the epigenetic mark for the CCAN, and thereby the kinetochore assembly. Strikingly, CENP-A intensity is significantly diminished in lagging chromosomes with Plk1 inhibition (Figs 3E and F, and EV3A) and, prior to anaphase, CENP-A is disproportionately lost from the pole-distal kinetochore of misaligned chromosomes (Fig EV3B and C). Intriguingly, we found only modest loss of ACA signal with Plk1 inhibition. ACA is known to recognize multiple centromere epitopes, including CENP-B, which binds DNA independently of CENP-A 43. Consistent with our ACA findings, CENP-B removal after Plk1 inhibition is less severe than the other examined proteins (Fig EV3D and E). Taken together, these findings indicate that Plk1 activity is required to maintain integrity of the mitotic kinetochore and CCAN, but not for CENP-B. Click here to expand this figure. Figure EV3. Kinetochore disruption after Plk1 inhibition includes CENP-A, but not CENP-B (related to Fig 3) Graph shows relative volume intensities of CENP-A (gray), Hec1 (green), and ACA (red) at segregated (left) and lagging (right) kinetochores after Plk1 inhibition or nocodazole washout (Fig 3E and F). Note CENP-A data are identical to data presented in Fig 3F. Each symbol in the left graph represents all segregated kinetochores/cell (n = 10 cells/experiment; three independent experiments), whereas each symbol in the right graph represents a single kinetochore (n = 1–6 kinetochores/cell; 10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by two-tailed Mann–Whitney test. Representative maximum-intensity projection micrographs of metaphase cells with misaligned chromosomes. Insets highlight distribution of CENP-A and Hec1 at misaligned kinetochore pair, indicated by ACA. Scale bars, 5 μm. Graph shows average percentage (± SEM) of misaligned chromosome pairs exhibiting each of the three distribution types after 2-h Plk1 inhibition (BI-2536) or nocodazole challenge (n = 1–8 chromosomes/cell; 10 cells/experiment; two independent experiments). "Decreased" intensity indicates ≤ 50% intensity of sister kinetochore. Representative maximum-intensity micrographs of anaphase cells after Plk1 inhibition (BI-2536) or nocodazole washout. Insets highlight the presence of CENP-B and Hec1 at lagging kinetochores, marked by ACA. Scale bars, 5 μm. Graph shows relative volume intensities of CENP-B (gray), Hec1 (green), and ACA (red) at segregated (left) and lagging (right) kinetochores from (B). Each symbol represents a single kinetochore (n = 10 segregated kinetochores/cell, 1–8 lagging kinetochores/cell; 10 cells/experiment; three independent experiments). Bars indicate median kinetochore intensity and interquartile range. Significance determined by two-tailed Mann–Whitney test. Download figure Download PowerPoint We next evaluated whether the observed effects altered CENP-A turnover at the centromere or were specific for its nascent or ancestral pools. Centromere loading of nascent CENP-A is largely restricted to early G1 6, and turnover of loaded CENP-A is not known to occur in mitosis 44. Nevertheless, it is possible that Plk1 activity restrains the CENP-A turnover during late G2 or mitosis. To test this idea, we performed fluorescence recovery after photobleaching (FRAP) on CENP-A in mitotic cells with and without Plk1 inhibition (Fig EV4A). Consistent with earlier findings 44, there was no observed turnover of CENP-A during mitosis, and this was unaffected by inhibiting Plk1. Second, we employed quench-pulse-chase CENP-A-SNAP labeling to differentiate nascent vs. ancestral CENP-A 6 (Appendix Fig S3). As expected, there was no nascent loading of CENP-A at the centromere during G2 and this was not altered with Plk1 inhibition, as previously shown 12. Finally, we considered that nascent CENP-A, loaded during the prior G1, may be more readily extracted from centromeric chromatin than the ancestral pool. To evaluate this, we generated cell lines expressing CENP-A-SNAP to differentially label nascent and ancestral CENP-A nucleosomes (Fig EV4B). We found that both ancestral and nascent CENP-A pools are extracted with Plk1 inhibition (Fig EV4C and D). We conclude that Plk1 signaling does not modulate CENP-A loading and turnover, nor is it required for specific stabilization of nascent or ancestral CENP-A. Click here to expand this figure. Figure EV4. CENP-A loss is not the result of dysregulated turnover; both ancestral and nascent CENP-