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First published online 30 January 2007
doi: 10.1242/jcs.03380

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Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 in S. cerevisiae

Raïssa Eluère¹, Nicolas Offner^2,*, Isabelle Varlet¹, Olivia Motteux¹, Laurence Signon¹, André Picard², Eric Bailly¹ and Marie-Noëlle Simon^1,

¹ Genome Instability and Carcinogenesis, CNRS FRE 2931, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France
² Laboratoire Arago, UMR7628, 66650 Banyuls sur Mer, France

View larger version (68K): [in this window] [in a new window]   Fig. 1. (A,B) Cellular localization of the Clb2 mutants when expressed from the CLB2 endogenous promoter. WT, clb2NLS and clb2L303 cells in which a GFP tag was introduced at the CLB2 locus were grown to mid-log-phase on YPD plates supplemented with adenine. All images were taken with the same optical settings and processed with identical parameters into Adobe PhotoShop. (A) Cellular distribution of Clb2NLS and Clb2L303A compared with the wild-type cyclin. (B) Representative images of the pattern of distribution of Clb2NLS-GFP in late anaphase. Bar, 2µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. (A,B) Cell cycle progression of cells expressing mutant forms of CLB2. The indicated cells were synchronously released from an -factor-induced G1 arrest at 30°C. Aliquots were taken every 15 minutes. (A) DNA content was analyzed by flow cytometry and (B) the percentage of budded (crosses), pre-anaphase (triangles) and binucleate (squares) cells determined microscopically after a brief sonication. Pre-anaphase cells were defined as having the nucleus located at the bud neck. Note that in our strain background, about 20% of clb2 cells had the nucleus at the bud side of the neck just before anaphase and were classified as pre-anaphase cells. Each panel is representative of at least two independent experiments. More than 200 cells were counted for each time point. (C) Cells from the strain W303 clb1,3,4 clb2ts (Amon et al., 1993) were transformed with the indicated vectors and assayed for growth at 36°C. Serial 5x dilutions of saturated cultures were spotted on YPD plates and incubated at the indicated temperatures for 36 hours.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Delayed phosphorylation of Net1 in clb2NLS-expressing cells. (A) clb2NLS but not clb2L303A exacerbates the temperature-sensitive growth phenotype of cdc15-2. Serial 10x dilutions of saturated cultures were spotted on YPD plates and incubated at the indicated temperatures for 3-4 days. (B) The synthetic effect of the clb2NLS allele is recessive. The same experiment as in A was repeated with cdc15-2/cdc15-2 diploid cells expressing the indicated CLB2 alleles. (C) The kinetics of Net1 phosphorylation was analyzed in extracts from clb2NLS clb1 and clb1 cells expressing NET1-Myc and PDS1-HA alleles. Cells were released from an -factor-induced G1 arrest at 25°C and aliquots taken every 10 minutes. Total cellular extracts were separated by a long migration on 8% SDS-polyacrylamide gels to detect Net1-Myc, and by 10% SDS-PAGE for Clb2, Pds1 and Cdc28. To better compare the Net1 migration profiles, clb2NLS clb1 and clb1 extracts were run on the same gel. Clb2 and Pds1 were used as markers of cell cycle progression.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Degradation profile of the Clb2NLS mutant protein. (A) Comparison of Clb2 levels in metaphase and telophase-arrested cells. WT and cdc15-2 cells expressing the indicated CLB2 alleles were exponentially grown at 25°C and transferred at 37°C with (WT background, metaphase arrest) or without (cdc15-2 background, telophase arrest) 15 µg/ml nocodazole for 2 hours. Metaphase arrests were performed at 37°C to normalize the temperature shift required to block cells in telophase. Cellular extracts were analyzed by western blot with the indicated antibodies. Signals from Alexa Fluor-coupled secondary antibodies were quantified with an Odyssey scan. Clb2 signals were normalized using Cdc28 as a loading control. Signals from metaphase-arrested cells were considered as 100%. (B) Kinetics of Clb2 and Clb2NLS degradation during mitotic exit. cdc15-2/cdc15-2 CLB2/clb2NLS diploid cells were released from a temperature shift-induced telophase arrest as described in Materials and Methods. Clb2 signals were normalized using Cdc28 as a loading control and are expressed as the percentage of the signals measured at t0 (100%).

View larger version (54K): [in this window] [in a new window]   Fig. 5. clb2NLS cells display a premature switch of growth polarization. (A) Cell cycle regulation of Bem1-GFP localization. (B) The percentage of cells with Bem1-GFP polarized at the bud tip was monitored in the indicated strains in log-phase cultures at 25°C. Medium and large buds were 2,5-3,5 µm and 3,5-5 µm in length respectively. Large budded cells with Bem1-GFP localized at the mother-bud neck were not scored. Bars indicate the standard deviation of at least three independent experiments with more than 500 cells counted for each strain. (C) Cells from the indicated strains were arrested in G1 with 5 µg/ml -factor, washed and synchronously released in fresh YPD medium containing 15 µg/ml nocodazole. The cells were fixed after 75 minutes at 30°C and processed for actin staining with Rhodamine-phalloidin. The percentage of cells displaying a polarized distribution of cortical actin patches was quantified for each strain by counting at least 150 cells. Bars, 2 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 6. The hyperpolarized phenotype of clb1 clb2L303A cells is independent of CLN1,2. (A) clb1 and clb1 clb2L303A cells expressing a CLN2-HA3 allele were released from an -factor arrest at 30°C and analyzed at the indicated times for the amount of Cln2-HA, Clb2 and Cdc28 by western blot analysis. (B) clb1 clb2L303A and clb1 clb2L303A cln1 cln2 cells were processed as described in the legend of Fig. 5C before staining for actin with Rhodamine-phalloidin. Bar, 2µm.

View larger version (93K): [in this window] [in a new window]   Fig. 7. Additive effects of GAP and CLB2 deletions on cell elongation. (A,B) The indicated strains were grown to exponential phase on YPD plates at 25°C and examined by DIC microscopy. Bar, 3µm.

View larger version (67K): [in this window] [in a new window]   Fig. 8. Mutation of a C-terminal KY motif affects bud neck localization of Clb2. (A) A GFP tag was appended to the clb2K459A,Y460C allele at the CLB2 locus. Cells were processed for microscopic observations as described in Fig. 1 and are shown in comparison with CLB2-GFP cells before (a) and during (b-d) nuclear division. (B) The indicated mitotic cyclins were overexpressed as GFP fusion under the control of the GAL1 promoter. Cells grown in SC-raffinose were collected on nitrocellulose filters and induced for 2 hours on YEP-galactose plates before microscopic observation. (C) The percentage of cells with Bem1-GFP polarized at the bud tip was monitored in clb2K459A,Y460C and clb2K459A,Y460C,NLS cells as described in Fig. 5. Bar, 2µm.