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Farnesylation of Cenp-F is required for G2/M progression and degradation after mitosis

School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (33K): [in a new window]   Fig. 1. The C-terminal 630 amino acids of Cenp-F localises to kinetochores. (A) Schematic of Cenp-F showing the domain encoded by C630 and the fragment used to generate the anti-Cenp-F antibody, SCF.1. (B) HeLa cells stained to detect endogenous BubR1 (red), myc-tagged C630 (green) and DNA (blue). During interphase C630 localises to the nucleus and in prometaphase it co-localises with BubR1 at kinetochores. Bars, 5 µm. (C) Immunoblot of pellets (P) and supernatants (S) following extraction of interphase and mitotic HeLa cells showing that SCF.1 recognises a single band significantly greater than 200 kDa.

View larger version (27K): [in a new window]   Fig. 2. Expression of C630 inhibits proliferation and delays progression through G2/M. HeLa cells were infected with either a control retrovirus or retroviruses encoding GFP, myc-tagged C630 and N-mBub 1. Puromycin-resistant cells were selected, expanded then analysed. (A) Timeline used to generate cells expressing the cDNA of interest. (B) Fluorescence histograms showing that 95% of the cells infected with GFP retroviruses express GFP. (C) Immunofluorescence analysis of HeLa cells infected with retroviruses encoding C630 immediately after or 12 hours after release from a G1/S block indicating that expression levels of C630 are low at G1/S but high in late G2. (D) Growth curves showing that expression of C630 results in a growth defect. (E) DNA content histograms of asynchronous cultures plus the number of cells in each phase of the cell cycle. The mitotic index was determined by quantitating MPM-2-positive cells. The values represent the average of three independent cultures indicating that expression of C630 results in accumulation of cells in G2/M. (F) DNA content histograms of infected HeLa cells following release from a G1/S block showing that expression of C630 delays progression through G2/M. The cells were analysed every 3 hours but only the key time points are shown.

View larger version (40K): [in a new window]   Fig. 3. Expression of C630 delays entry into mitosis. HeLa cells infected with either a control retrovirus or retroviruses encoding myc-tagged C630 were analysed by flow cytometry and fluorescence microscopy in the presence and absence of nocodazole. (A) Graph plotting the mitotic index following treatment of asynchronous populations with nocodazole for the times indicated showing that the C630 population accumulates mitotic cells slower than the control population. Also shown is data from cells expressing the four CAAX mutants, C:S, V:D, 4 and 150 (see text for details). Each point represents the mean and s.e.m. derived from three independent experiments. (B) Graph plotting the mitotic index following release of cells synchronised at G1/S into nocodazole showing that the C630 population accumulates mitotic cells slower than the control population. (C) Bar graph plotting the proportions of cells in each phase of mitosis showing that expression of C630 does not appear to effect progression through mitosis. Each value is expressed as a percentage of the number of cells in mitosis and represents the mean and s.e.m. derived from three independent experiments in which at least 50 mitotic cells were counted. (D) Representative immunofluorescence images of metaphase and anaphase cells typically observed in control and C630 cultures showing that expression of C630 does not appear to disrupt chromosome alignment or segregation. Tubulin is shown in green, myc-tagged proteins in red and the chromosomes in blue.

View larger version (33K): [in a new window]   Fig. 4. The CAAX motif of C630 is required for delaying progression through G2/M. HeLa cells were infected with a control retrovirus, retroviruses encoding myc-tagged C630 and the four CAAX mutants, C:S, V:D, 4 and 150. Puromycin-resistant cells were analysed to determine protein expression, localisation, relative growth rates and cell-cycle timing. (A) Growth curves showing that the CAAX mutants induce a less severe growth defect relative to C630. (B) DNA content histograms of asynchronous cultures showing that the CAAX mutants induce a less profound G2/M accumulation relative to C630. (C) Graph plotting the 4n/2n DNA content ratio, as determined by DNA content flow cytometry, following release from a G1/S block. Unlike C630, the CAAX mutants do not induce a significant G2/M delay. (D) Immunoblot showing the relative expression levels of C630 and the CAAX mutants. The arrow shows the position of C630 while the asterisk indicates a nonspecific background band that serves as a convenient loading control. (E) Immunofluorescence analysis 12 hours after release from a G1/S block showing that the C630 CAAX mutants localise to the nucleus during interphase.

View larger version (62K): [in a new window]   Fig. 5. Cenp-F localises to the nuclear periphery at the G2/M transition. Deconvolved images showing HeLa cells stained to detect Cenp-F (green) and DNA (blue). The cells were also stained to detect phospho-histone H3 (A,B) and Bub1 (C-E). Bars, 10 µm. (A) Interphase and prometaphase cells showing localisation of Cenp-F to the nucleus and kinetochores, respectively. (B) Two interphase cells; the one on the left is in late G2 and shows Cenp-F localising to the nucleus and the nuclear envelope. A small fraction is present in the cytoplasm. (C) Prometaphase cell showing colocalisation of Cenp-F and Bub1 at kinetochores. (D) Late prophase cell showing Cenp-F in the nucleus, cytoplasm and at the nuclear envelope. (E) A cell at the G2/M transition showing early signs of chromosome condensation (see inset), a few Bub1 foci and Cenp-F at the nuclear envelope.

View larger version (36K): [in a new window]   Fig. 6. Cenp-F localises to the nuclear envelope in G2. Deconvolved images showing HeLa cells stained to detect Cenp-F (green), lamin B2 (red) and DNA (blue). (A) G2 cell showing localisation of Cenp-F and lamin B2 to the nuclear envelope. Note that Cenp-F also localises to an invagination within the nucleus (arrowhead). (B) Enlarged view of a G2 cell showing that Cenp-F and lamin B2 localise to different regions of the nuclear envelope.

View larger version (114K): [in a new window]   Fig. 7. The CAAX motif of Cenp-F is required for nuclear envelope and kinetochore localisation. (A) HeLa cells stained to detect myc-tagged proteins, Cenp-F and DNA as indicated. Nuclear envelope and kinetochore localisation of Cenp-F is diminished in the presence of C630 but is readily apparent when the C:S mutant is expressed. (B) Bar graph quantitating the number of cells with Cenp-F detectable at the nuclear envelope 12 hours after release from a G1/S block. Nuclear envelope staining is less frequent in cells expressing C630. (C) Projections of deconvolved image stacks showing HeLa cells stained to detect myc-tagged proteins (red), Bub1 (green) and DNA (blue). The C:S and V:D mutants localise poorly to kinetochores.

View larger version (39K): [in a new window]   Fig. 8. Farnesyl transferase activity is required for Cenp-F localisation. (A) The percentage of cells with Cenp-F at the nuclear envelope in the presence or absence of SCH 66336 at various times following release from a G1/S block. (B) Early prophase HeLa cells cultured in the presence or absence of SCH 66336 stained for Cenp-F (green), lamin B2 (red) and DNA (blue) showing that nuclear envelope localisation of Cenp-F is not apparent in the presence of the FTI. (C) Nocodazole-arrested mitotic HeLa cells cultured in the presence or absence of SCH 66336 were isolated by shake off, centrifuged onto glass slides and then stained for Cenp-F (green) and phospho-histone H3 (red). Kinetochore staining is not apparent in the presence of SCH 66336.

View larger version (56K): [in a new window]   Fig. 9. Farnesyl transferase activity is required for Cenp-F degradation. (A) DNA content histograms of HeLa cells cultured in the presence or absence of FTIs following release from a G1/S block. (B) Immunofluorescence images of the cells 15 hours after release from G1/S stained to detect phospho-histone H3 (red), Cenp-F (green) and DNA (blue). In the presence of SCH 66336, Cenp-F is detectable in all the interphase nuclei. (C) Immunoblots of asynchronous or G1/S blocked HeLa cells showing that Cenp-F is present in G1/S cells only when cultured in the presence of SCH 66336. (D) HeLa cells infected with retroviruses encoding C630 or the C:S mutant stained to detect myc-tagged proteins (red), Cenp-F (green) and DNA (blue). The cells were synchronised at G1/S using a single thymidine block. The cells were then analysed immediately following release (i.e. at G1/S) or 12 hours after release (i.e. in G2). Like endogenous Cenp-F, C630 is only detectable in G2 cells, whereas the C:S mutant is also detectable in G1/S cells.

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