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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02614

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Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint

Sarah V. Holt, Mailys A. S. Vergnolle, Deema Hussein^*, Marcin J. Wozniak, Victoria J. Allan and Stephen S. Taylor

Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (44K): [in a new window]   Fig. 1. Cenp-F is required for cell division. HeLa cells were transfected with siRNA duplexes then analysed 48 hours later by immunoblotting and immunofluorescence. (A) Immunoblot showing effective repression of Cenp-F, with tubulin acting as a loading control. (B) Quantitation of at least 100 mitotic cells by immunofluorescence scoring the levels of kinetochore-bound Cenp-F as either high, medium or low. (C) Images of mitotic cells showing that Cenp-F and Bub1 colocalize at kinetochores in control cells, but that Cenp-F is absent from kinetochores in Cenp-F RNAi cells; bar, 5 µm. (D) Images of mitotic cells stained to detect Cenp-F and Aurora A showing a tripolar spindle in the Cenp-F-deficient cell; bar, 5 µm. (E) Quantitation of mitotic cells with two or more spindle poles. In the bar graphs, values represent the mean±s.e.m. of three independent experiments in which at least 100 mitotic cells were counted.

View larger version (52K): [in a new window]   Fig. 2. Cenp-F is required for metaphase chromosome alignment. HeLa cells were transfected with siRNA duplexes then analysed 48 hours later by immunofluorescence. (A) Images of mitotic cells stained to detect Cenp-F (green) and the DNA (red), showing normal prophase, prometaphase, metaphase and anaphase configurations in control cells, and aberrant configurations in Cenp-F-deficient cells; bar, 5 µm. (B) Mitotic cells stained to detect Aurora B (green) and the DNA (red). Control cells show that Aurora B localizes to centromeres (panel i) and spindle midzones (panel ii). In Cenp-F-deficient cells, Aurora B localizes to centromeres but midzones and midbodies are rarely seen; bar, 5 µm. (C) Quantitation of pixel intensities showing that Cenp-F is not required for centromere localization of Aurora B. Values represent the mean±s.e.m. from ten well resolved kinetochores in three different cells. (D) Quantitation showing that repression of Cenp-F reduces the frequency of anaphases to 36%; the values represent the mean±s.e.m. of three independent experiments in which at least 100 mitotic cells were scored per experiment. (E) Proportion of prophases (yellow), prometaphases (red), metaphases (green) and anaphases (blue) following exposure to the proteasome inhibitor MG132; the values represent the mean±s.e.m. of three independent experiments in which at least 100 mitotic cells were analysed per experiment.

View larger version (65K): [in a new window]   Fig. 3. Silencing Cenp-F delays mitotic exit. HeLa cells expressing a GFP-tagged histone H2B fusion protein were transfected with siRNA duplexes then analysed 48 hours later by time-lapse microscopy. (A) Images from time-lapse sequences showing normal chromosome alignment and segregation in a control cell, and aberrant mitoses in Cenp-F RNAi cultures. Numbers indicate time in minutes. (B) Graph showing the time in minutes from early prometaphase to mitotic exit showing that Cenp-F repression delays mitotic exit. Ten cells were analysed per group. Note that Cenp-F-deficient cells were identified by their aberrant chromosome morphologies, hence the time-lapse sequences start after prophase.

View larger version (31K): [in a new window]   Fig. 4. Silencing Cenp-F activates the spindle checkpoint. HeLa cells were transfected with siRNA duplexes designed to repress both Cenp-F and BubR1 then analysed 48 hours later by immunoblotting, flow cytometry and time-lapse microscopy. (A) Blot showing efficient corepression of Cenp-F and BubR1. Numbers indicate size markers in kDa. (B) Bar graph quantitating the number of MPM-2-positive cells in asynchronous populations as determined by flow cytometric analysis of 10,000 cells. Values represent the mean±s.e.m. of three independent experiments. (C) Histograms plotting the time from nuclear envelope breakdown to anaphase. Number of cells analysed; Control, 33; BubR1 RNAi, 36; Cenp-F RNAi, 53; Dual RNAi, 74.

View larger version (42K): [in a new window]   Fig. 5. K-fibres form in the absence of Cenp-F. (A) Control and Cenp-F RNAi cells were extracted to preserve K-fibres, then fixed and stained to detect tubulin (green) and the DNA (red). Note that although the spindle appears aberrant in Cenp-F-repressed cells, K-fibres are apparent. (B) Images of nocodazole-arrested cells stained to detect Cenp-F (green) and Hec1 (red) showing that Hec1 localizes to Cenp-F-depleted kinetochores. Scale bars represent 5 µm. (C) Graph plotting inter-kinetochore distances, in µm, using Cenp-A as a marker. Measurements are made using control cells in either prometaphase or metaphase, and Cenp-F deficient cells, horizontal lines represent the means.

View larger version (42K): [in a new window]   Fig. 6. Merotelic attachments form in the absence of Cenp-F. (A) Control metaphase stained to detect Bub1 (green) and tubulin (red), and enlargements showing examples of bioriented kinetochore pairs with K-fibres terminating at kinetochores. (B,C) Cenp-F-deficient cells stained to detect DNA (red), tubulin (green and red), Bub1 (green) and ACA (blue), as indicated. The enlargements show (a) a merotelic oriented kinetochore pair; (b,c) mono-oriented kinetochores lacking sisters; and (d) a merotelic oriented kinetochore that lacks a sister. Bar, 5 µm when the entire spindle is shown, and 1 µm in the enlargements.

View larger version (74K): [in a new window]   Fig. 7. Silencing Cenp-F weakens centromeric cohesion. HeLa cells were transfected with siRNA duplexes designed to repress lamin B1, Cenp-F and Sgo1. (A) Immunoblot showing repression of Cenp-F and Sgo1. (B) Metaphase spreads showing loss of centromere cohesion following repression of Cenp-F and Sgo1. Dashed line in panel (vi) separates chromosomes from two spreads. (C) Images of chromosomes stained with anti-Bub1 and anti-ACA antibodies confirming the presence of separated chromatids. (D) Bar graph quantitating the number of metaphase spreads with separated chromatids. At least 117 cells scored in each group. (E) Bar graph quantitating the number of MPM-2-positive cells in asynchronous populations, showing that repression of Cenp-F and Sgo1 activate the spindle checkpoint. (F) Bar graph quantitating the number of cells with DNA contents greater than 4N, showing that repression of Sgo1, but not Cenp-F, enhances endoreduplication.

View larger version (50K): [in a new window]   Fig. 8. Silencing Cenp-F results in aberrant spindle morphology. (A) Transfected HeLa cells were fixed under conditions that preserve all microtubules and were then stained to detect tubulin and the DNA. Images show representative spindles in control and Cenp-F-deficient cells. Arrowhead identifies an abnormally long microtubule extending beyond the spindle midzone. (B) Histograms plotting tubulin intensity along the spindle axis. (C) Microtubule pelleting assay showing that whereas kinesin heavy chain (KHC) binds taxol-stabilized microtubules, Cenp-F does not. S, supernatant (i.e. the cell lysate used for the assay); C, the sucrose cushion; R, the ATP-release fraction; P, the microtubule pellet. The assay was performed with (+MTs) and without (–MTs) microtubules, confirming that the pelleting of KHC is microtubule dependent.

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