Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F - PubMed (original) (raw)

Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

Pascale Bomont et al. EMBO J. 2005.

Abstract

Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce 'twinkling', demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule-kinetochore interface.

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Figures

Figure 1

Figure 1

Transcription-mediated siRNA and RNA duplex siRNA both result in >90% depletion of CENP-F. (A–D) CENP-F repression was obtained by transcription-mediated siRNA and RNA duplex siRNA. HeLa cells were transfected with (A) the corresponding siRNA plasmids along with pCD20 that encodes the cell surface marker CD20 or (B) siRNA duplex RNAs and a luciferase–Cy3 RNA duplex. Accumulated CENP-F levels were visualized by (C) immunoblotting whole-cell lysates and (D) immunofluorescence 48 h after transfection.

Figure 2

Figure 2

Depletion of CENP-F does not perturb cell cycle timing in S or G2, but affects the duration of mitosis. (A) HeLa cells were transfected with the corresponding siRNA plasmids along with pCD20. After 24 h, transfected cells were affinity isolated, replated and incubated with 2 mM thymidine for 18 h. At 8 h after release from the drug, CENP-F depletion was verified by (B) immunofluorescence and (C) immunoblot. (B) Thymidine-induced synchronization in S phase or at the G1–S boundary was verified by the increase in late G2/mitotic cells 8 h after release (as revealed by immunopositivity for phosphorylated histone H3) and the presence of dividing cells after 10 h (revealed by DAPI staining). (D) Determination of mitotic entry following thymidine release in control RNAi cells (blue lines) and CENP-F RNAi cells (red lines). Mitosis entry is defined by the time each cell became rounded before dividing, as observed by time lapse brightfield microscopy. (E) Determination of the duration of prometaphase/metaphase, as defined by the time each cell spent between becoming round and the beginning of cytokinesis. The cumulative proportion of the cells that have divided is represented to show that all control cells spent <1 h in mitosis, while this is extended to >5 h in CENP-F RNAi cells. Scale bar, 10 μm.

Figure 3

Figure 3

Premature anaphase and mitotic delay in the absence of CENP-F. (A–C) Time lapse imaging was performed 48 h after transfection of (i) HeLa cells with psiRNAi-control/-CENP-F with pH2B-EYFP and (ii) an HeLa cell line stably expressing H2B-EYFP with control/CENP-F antisense RNA duplexes along with a Cy3-tagged RNA duplex to luciferase. (i) Bright EYFP positive cells or the (ii) Cy3 positive cells were recorded from prophase. (A) Time spent in different stages of mitosis for control RNAi cells (in blue) and CENP-F RNAi cells (in red). The individual time for each cell is represented (in circle), as the average time (black line). Cells that were still at the corresponding phase when the filming was stopped are boxed. All other cells proceeded through mitosis and divided. (B) Relative proportions of the different phenotypes in control RNAi cells (blue) and CENP-F RNAi cells (red). (C) Time-lapse sequences of mitosis in a control RNAi cell (Supplementary Movie 1) and a prometaphase delay (1, see Supplementary Movie 2), metaphase delay (2, Supplementary Movie 3), abnormal anaphase (3, Supplementary Movie 4) and division without metaphase (4, Supplementary Movie 5) observed in CENP-F RNAi cells.

Figure 4

Figure 4

Reduced capture and/or stability of microtubules at kinetochores in the absence of CENP-F. (A) Reduced tension between sister kinetochores in the absence of CENP-F. At 48 h after cotransfection of the corresponding psiRNA with pH2B-EYFP, high expressing H2B-EYFP cells in metaphase were imaged. (Blue) H2B-EYFP; (red) tubulin (green) ACA. In total, 120 kinetochore pairs in which both sister kinetochores were in the same focal plane were analyzed for both psiRNA-CENPF and psiRNA control cells with and without nocodazole treatment. Insets 1–3 show enlargements of deconvolved planes for the psiRNA-control (1) without and with (3) 20 μM nocodazole for 3 h and (2) for psiRNA-CENP-F. Scale bar, 5 μm. The averages of 1.39±0.09, 1.05±0.11, and 0.87±0.04 μm for kinetochores in cells from (1) (2) and (3) are significantly different (using ANOVA single factor test with α values <0.05 and _P_-values <0.001). (**B**) Reduced stability of kinetochore fibers in the absence of CENP-F. psiRNA-control and psiRNA-CENP-F cells were cooled on ice for 10 min prior to extraction and fixation. High expressing H2B-EYFP were stained for (red) tubulin and (green) ACA and processed to deconvolution. Attachment of kinetochores to microtubules was determined by following >100 individual kinetochores through all the focal planes. Kinetochores in regions where fibers were not easily visualized were not taken into account. The images and enlargements correspond to the merge of selected focal planes. Kinetochores attached to microtubules (white arrows), and unattached ones (green arrows).

Figure 5

Figure 5

CENP-F affects the recruitment of CENP-E and checkpoint regulators to the kinetochore. Kinetochore localization in prometaphase and metaphase of (A) CENP-E, (B) BubR1, (C) Bub1, (D) Mad2 48 h after transfection of psiRNA-control/-CENP-F with pH2B-EYFP. (Blue) H2B-EYFP; (red) ACA; (green) CENP-E, BubR1, Bub1 or Mad2. Quantification of the normalized integrated intensity of (A′) CENP-E, (B′) BubR1, (C′) Bub1, and (D′) Mad2 at kinetochores in (blue) psiRNA-control and (red) psiRNA-CENP-F cells. Mad2 quantification was performed on all kinetochores, including those for which no signal was detected. The error bars represent standard errors. Heteroscedastic T-tests revealed significant differences at prometaphase between psiRNA-control and psiRNA-CENP-F: _P_-values of 0.0002 (CENP-E), 0.0394 (BubR1) and 0.0290 (Bub1). The same test gives a _P_-value of 0.0861 for Mad2 at metaphase. Aproximately 1000 kinetochores were analyzed corresponding to about 10 cells for each bar. Scale bar, 5 μm.

Figure 6

Figure 6

Continued recruitment of Mad2 at kinetochores of aligned chromosomes and intact checkpoint activity in CENP-F depleted cells. (A) Number of kinetochores with detectable Mad2 on aligned chromosomes in control and CENP-F depleted cells 48 h after cotransfection of the psiRNA-CENP-F/-control with pH2B-EYFP. (B) Mad2 intensity at individual kinetochores of aligned chromosomes of metaphase cells 48 h after cotransfection with the psiRNA-control/-CENP-F along with pH2B-EYFP. (Open diamonds) The average recruitment of Mad2 at kinetochores in control and CENP-F free cells after disruption of microtubule attachment (by the addition of colcemid for 30 min). (C) Mitotic indices 48 h after transfection with control or CENP-F duplexes, followed by incubation with or without 0.1 μg/ml colcemid for another 18 h. Mitotic indices of control RNAi and CENP-F RNAi are not statistically different in absence or presence of colcemid (done using Heteroscedastic _T_-test). In total, 20 independent fields of 100 cells were included for each siRNA. Immunoblotting confirmed the degree of CENP-F depletion.

Figure 7

Figure 7

The mitotic delay of CENP-F RNAi cells is characterized by both the constitutive and transient recruitment of Mad1 at aligned chromosomes. Stably expressing EYFP-Mad1 HeLa cell lines were imaged 48 h after transfection with pH2B-mRFP along with psiRNA-control or psiRNA-CENP-F. (A) Aligned chromosomes in control cells rapidly and uniformly lose Mad1 binding coincident with alignment. In this example, one chromosome is trapped behind one spindle pole and one or both of the kinetochores of this chromatid pair chronically recruit Mad1. Selected frames of Supplementary Movie 7 of a psiRNA-control cell 48 h after transfection. (White or green) Mad1; (red) histone H2B. During the 64 min long pseudometaphase, no Mad1 is found at any aligned kinetochore. This shows that in these cells a single lagging chromosome is able to sustain mitotic checkpoint signaling to delay anaphase. (B) Images from a CENP-F depleted cell (Supplementary Movie 8). All kinetochores initially recruit Mad1 but after alignment Mad1 is released from most (see frame 0:35). However, one (yellow arrow) continued to bind Mad1 for 47 min (from frames 0:35 to 1:22). Other weaker Mad1 signals are intermittently detected (see white arrows 1–5). Finally, 14 min after Mad1 signals are lost from all kinetochores (from frames 1:22 to 1:36) anaphase initiates. (C, D). Mad1 twinkling. (C) Schematic representation of the Z-cross sections collected over time. A total of five Z-sections were collected, sampling ∼80% of the metaphase cell depth. (D) Distribution of Mad1 in every Z-section and during time in another CENP-F depleted cell. The constitutively bound Mad1 kinetochore in the lower panel moves slowly and progressively through the focal plane at 1 μm/3 min (see arrows). During the same time a kinetochore located at the center of the focal plane (first arrow in the top panel in Z-section 3) lost and regained Mad1 (second arrow in the top panel). Scale bar, 1 μm.

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