QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Decottignies, A. Articles by Nurse, P. Search for Related Content PubMed PubMed Citation Articles by Decottignies, A. Articles by Nurse, P.

In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis

Cell Cycle Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
^* Author for correspondence (e-mail: a.decottignies{at}icrf.icnet.uk )

View larger version (59K): [in a new window]   Fig. 1. Analysis of cdc2- and cdc13-YFP proteins. (A) Co-immunoprecipitation assay of either cdc13-YFP or cdc1381-YFP and cdc2p. Cells expressing cdc13-YFP (AD178, lanes 1,3) or cdc1381-YFP (AD203, lanes 2,4) were grown for 20 hours at 32°C in EMM2 without thiamine. Immunoprecipitation from cell extract proteins was performed with polyclonal anti-cdc2p serum. Cdc13p (tagged or untagged) was detected in total cell extracts (lanes 1,2) or immunoprecipitated complexes (lanes 3,4) by western blot using anti-cdc13p mAb. Lanes 1,2, 50 µg of cell extract proteins. Lanes 3,4, immunoprecipitation. (B) pREP5 plasmids with the cdc2- (lanes 1-3) and cdc13-YFP (lanes 5-6) fusions were integrated at the gene locus as shown on the cartoon and strains were grown for 20 hours in either EMM2 without thiamine (-T) or YES (+T) medium. 50 µg of cell extract proteins were loaded and detected using mAbs against cdc2p (1-3) and cdc13p (4-6). Lane 1, pREP5::cdc2-YFPint (AD143) -T; lane 2, pREP5::cdc2-YFPint (AD143) +T; lane 3, pREP5::cdc2-YFPint nmtcdc2::kanR (AD185) -T; lane 4, control WT strain (PN745); lane 5, pREP5::cdc13-YFPint (AD112) -T; and lane 6, pREP5::cdc13-YFPint (AD112) +T.

View larger version (69K): [in a new window]   Fig. 3. Cdc2-YFP leaves the mitotic spindle in early anaphase A, before sister chromatid separation. Time-lapse series showing cdc13-YFP (A, AD112) and cdc2-YFP/cen1-GFP (B, AD265) localisation at the metaphase/anaphase transition. Pictures from cells were taken every 1 or 2 minutes as indicated. Cdc2-YFP fluorescence disappeared from the spindle (B, 2') immediately prior to separation of cen1-GFP dots (arrowheads) on sister chromatids (B, 3'). Bar, 5 µm.

View larger version (74K): [in a new window]   Fig. 5. Cdc2-YFP accumulates on the SPB of cells arrested at the G2/M transition or treated with HU. (A) Cdc2-YFP (AD185, a-e) fluorescence is very low on the SPB of septated cycling cells growing at 32°C in YES medium (c-e). SPB fluorescence was not observed in binucleate cells before septation (a-b). Bar, 5 µm. (B) Cdc2-YFP (AD185, a) and cdc13-YFP (AD112, b) accumulate in the nucleus and on the SPB of cells treated with 11 mM HU for 4 hours at 32°C. YFP-cig2 fluorescence was also enriched on the SPB of cells grown for 16 hours in the absence of thiamine at 32°C and incubated with 11 mM HU for 4 hours (AD179, c). Bar, 5 µm. (C) SPB accumulation of cdc2-YFP (AD189, a) and cdc13-YFP (AD226, b) is stronger in cdc25-22 ts cells after incubation for 4 hours at 36°C. Aggregation of cytoplasmic cdc2-YFP is observed at 36°C (see Fig. 4 legend). Bar, 5 µm. (D) cdc25-22 cells were arrested at the G2/M transition by incubation for 4 hours at 36°C and released into mitosis at RT: cdc13-YFP (AD226, a-e), cdc2-YFP (AD189, f-j), GFP-2-tubulin (AD244, k-o) or both cdc2-YFP and GFP-2-tubulin (AD268, p-t). G2/M boundary (a f,k,p) and mitosis (b-e,g-j,l-o,q-t). Bar, 5 µm.

View larger version (62K): [in a new window]   Fig. 2. Cellular localisation of cdc2-YFP and cdc13-YFP in the mitotic cell cycle. Cells were grown exponentially in YES medium, layered onto solid medium-coated slides and observed by LFM at RT. (A) Fluorescence of cdc2-YFP (AD185) and cdc13-YFP (AD112) was observed in different cells at various stages of the cell cycle: G2 interphase (a); G2/M boundary (b); mitosis from prophase to anaphase B (c-k); G1 and S phase (1-m). Bar, 5 µm. (B) Quantification of nuclear and cytoplasmic cdc2-YFP. Pictures from cells (strain AD185) at different stages of the cell cycle were taken and analysed using PhotoShop 5.5 to estimate the total cdc2-YFP fluorescence in the nucleus and the cytoplasm. Image fields were focused with non-fluorescent optics and fluorescence was observed only by the camera, using the same exposure time in each case. 15-25 cells were analysed at each stage and the bars give the s.d. Fluorescence measurements are in a single focal plane with the diameter of the nucleus at its maximum. Therefore, the graph gives the maximum proportion of nuclear cdc2-YFP at each stage of the cell cycle. The line shows the average cell length. Bar, 5 µm. (C) Cdc13-YFP fluorescence (strain AD112) was enriched at the nuclear periphery of cells exiting from mitosis in (a) to (i). Bar, 5 µm. (D) Cdc2-CFP and cdc13-YFP fluorescence were observed separately in strain AD117 using laser scanning confocal microscopy: anaphase B (a); S phase (b); G2 interphase (c); and mitosis (d). Bar, 5 µm. In addition, the black and white cdc2-CFP and cdc13-YFP pictures are shown below the colour pictures.

View larger version (80K): [in a new window]   Fig. 4. Cyclin B-dependent accumulation of cdc2-YFP into the nucleus after mitotic exit. (A) Cells from strains AD205 (a-b), AD207 (c, f), AD210 (d) and AD245 (e) were grown overnight at 25°C in the absence of thiamine. Cultures were incubated at 36°C for 4 hours to block the cells at the G2/M transition (time 0). In d-f, the cdc13 gene expression was switched off by addition of 15 µM of thiamine after either 3 hours (d) or 4 hours (e-f) at 36°C. Cells were observed by LFM for 2 hours 30 minutes at RT. Cdc2-YFP nuclear fluorescence was similar during the first 30 minutes of release (from G2/M to metaphase) in all strains (a). From anaphase B to post-cytokinesis, cdc2-YFP nuclear fluorescence (b-f, arrowheads) was dependent on the cyclin B present in the strain (cdc13p, cig1p and cig2p (b), cdc13p (c), cig2p (d), cig1p (e) or none of them (f)). Bar, 5 µm. (B) In the same experiments, DNA content was measured by FACS on fixed cells: asynchronous cultures at 25°C (AS), after 4 hours at 36°C (0) and at different time points during the release at 25°C as indicated. AD207 (a and d), AD210 (b) and AD245 (c). (C) Cdc2- and cdc13-YFP fluorescence were not observed in the nucleus of cdc10-V50 ts cells incubated for 4 hours at 36°C (AD192 and AD212). Note the cdc2-YFP aggregation in the cytoplasm of cells incubated at 36°C. This aggregation at high temperatures has also been reported recently for the GFP alone (Fukuda et al., 2000). Bar, 5 µm.

View larger version (108K): [in a new window]   Fig. 8. Localisation of cdc2-YFP during mating, karyogamy and meiosis. (A) Cartoon summarizing the localisation of chromosomes, centromeres (), telomeres () and SPB () during mating, karyogamy and meiotic prophase in S. pombe as described (Chikashige et al., 1994; Chikashige et al., 1997). (B) A cdc2-YFP-expressing strain (AD185) and a cdc2+ strain (PN745) were crossed on nitrogen-depleted medium and observed by LFM. Conjugating cells (a-b). Both nuclei become fluorescent (b). During karyogamy, cdc2-YFP is enriched in bright dots (c-f, arrowheads). In the horse-tail nucleus, cdc2-YFP is concentrated into 1-3 bright dots (g-k). Nucleus movement stops and meiosis I starts (l-s). We followed cdc2-YFP fluorescence in the same diploid cell for 70 minutes from meiotic prophase (i) to metaphase of the first meiotic division (s). Meiosis I (s-u). Meiosis II (v-x).

View larger version (42K): [in a new window]   Fig. 9. Cdc2-YFP co-localises with cen1-GFP during meiotic prophase. (A) A cdc2-YFP-expressing strain (AD185) and a cdc2+ strain (PN745) were crossed on nitrogen-depleted medium. A time-lapse series showing cdc2-YFP fluorescence in the horse-tail nucleus of meiotic prophase is presented (time is indicated in minutes). Cdc2-YFP was found in the nucleoplasm and was concentrated into 2 or 3 bright dots during nuclear movement. (B) A cdc2-YFP-expressing strain (AD185) and a cen1-GFP strain (MKY7A-4) were crossed and a cell in meiotic prophase was observed in vivo using a laser scanning confocal microscope to distinguish cdc2-YFP (a) from cen1-GFP (b) fluorescence. In all cases, one cdc2-YFP dot (arrows) co-localised with the GFP fluorescence associated with the centromere of chromosome I (arrowheads) (c).

View larger version (28K): [in a new window]   Fig. 10. Cdc2-YFP is enriched in the cluster of SPB-centromeres-telomeres in response to P-factor, in the absence of a mating partner. In the nucleus of h- cyr1sxa2 cells responding to P-factor, telomeres (), centromeres () and SPB () cluster together at one end of the nucleus (Chikashige et al., 1997) (cartoon). The h- cyr1sxa2 cells expressing cdc2-YFP (AD257) were grown exponentially in EMM2 medium at 32°C before incubation for 8 hours at 30°C in the presence of 0.5 µg/ml P-factor. Arrows indicate cdc2-YFP-enriched dots. Arrowheads show the dark portion of the nucleus that corresponds most probably to the rDNA and, therefore, to the telomeres of chromosome III.

View larger version (58K): [in a new window]   Fig. 6. Cdc2-YFP does not leave the mitotic spindle if cdc13p is not recognized by the APC. Fluorescence of cdc2- and cdc13-YFP was observed in the nucleus and on the SPBs and spindle of ts mutants defective in either proteasome (mts2) or APC (cut4-533) function after incubation for 3 hours at 36°C. AD157 (a), AD213 (b), AD152 (c) and AD267 (d). Aggregation of cytoplasmic cdc2-YFP is observed at 36°C (see Fig. 4 legend). Upon overexpression of indestructible cdc13p (lacking the first 81 aa) after 16 hours incubation at 32°C in thiamine-free medium, cdc2-YFP was present in the nucleus and on the SPBs and spindle of anaphase-arrested cells (AD266, e). Overexpressed cdc1381-YFP showed similar localisation (AD217, f). Bar, 5 µm.

View larger version (79K): [in a new window]   Fig. 7. Association of cdc2-YFP and cdc13-YFP with mitotic microtubules does not require dis1p function. (A) The endogenous dis1+ gene was replaced by the dis1-GFP fusion in a cdc25-22 background (AD219). Cells were arrested at the G2/M transition after 4 hours incubation at 36°C and dis1-GFP fluorescence was observed in vivo at RT. Dis1-GFP was found on cytoplasmic MTs in the block (a) and then relocalised to discrete dots along the forming spindle (b-e). Fluorescence was enriched at the extremities of the elongating spindle (f-j). c-f show a time-lapse series of the same mitotic cell as it progresses through mitosis. Bar, 5 µm. (B) In cold-sensitive dis1-203 cells incubated for 8 hours at 20°C, cdc2-YFP (AD199, a) and cdc13-YFP (AD264, b) were still associated with the unsegregated chromosomes, the SPBs and the spindle of mitosis-arrested cells.