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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.002758

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Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle

M. Angeles Juanes^*, Ethel Queralt^*,, M. Carmen Bañó and J. Carlos Igual

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, 46100 Burjassot (Valencia), Spain

View larger version (54K): [in this window] [in a new window]   Fig. 1. Characterization of the tetO7:ROT1 mutant strain. (A) Rot1 protein levels in extracts from cultures of the ROT1-HA (MCY10) and the tetO7:ROT1-HA (MCY192) strains grown on YPD and incubated in the presence of 5 µg/ml doxycycline were examined by western analysis. A control extract from the wild-type strain (CML240) is included (no tag). A non-specific band that crossreacts with the antibody is shown as loading control (*). (B) Tenfold serial dilutions from exponentially growing cultures of the wild-type strain (CML240) and the tetO7:ROT1 (JCY216) strain transformed with a control vector or a centromeric plasmid containing the ROT1 gene, were spotted onto YPD medium with or without 5 µg/ml doxycycline (dox) and incubated at 28°C for 3 days. (C) Exponentially growing cultures of the wild-type (CML240) and the tetO7:ROT1 (JCY216) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Graph shows the distribution of cells at different cell cycle stages. Pictures show DIC images, DAPI staining of DNA and staining of spindle by indirect immunofluorescence using anti-tubulin antibody. A collection of rebudded cells is shown in for the tetO7:ROT1 strain (see text). Values are the mean+s.d. of three experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Analysis of cell shape in rot1 strains. Exponentially growing cultures of the wild-type (W303), tetO7:ROT1 (MCY68), clb2 (CD104-2c) and clb2 tetO7:ROT1 (JCY255) strains were synchronized by -factor arrest in the presence of 5 µg/ml doxycycline. After release, the bud length/bud width ratio in anaphase and telophase cells (as deduced by the presence of segregated nuclei after DAPI staining of DNA) was measured. Values are the mean+s.d. of three experiments. Images of representative cells for each strain are shown.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Genetic interaction between ROT1 and CDC42. (A) The cdc42-1 (DJTD2-16D) and cdc42-1 tetO7:ROT1 (MCY92) strains transformed with a centromeric plasmid containing the ROT1 gene where indicated, were streaked onto YPD plates with or without 5 µg/ml doxycycline. Plates were incubated for 3 days at 28°C or 37°C. (B) The cdc42-1 (DJTD2-16D) strain transformed with an empty vector or a multicopy plasmid containing the ROT1 gene was streaked onto YPD plates and incubated at 35°C for 3 days.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Connection between Rot1 and the cell integrity pathway. (A) Tenfold serial dilutions of exponentially growing cultures of the wild-type (W303, upper panel; 1783, middle panel; and OHNY1, lower panel), tetO7:ROT1 (MCY68, upper panel; MCY186, middle panel; and MCY200, lower panel), pkc1 (JC6-3a), tetO7:ROT1 pkc1 (JCY433), slt2 (DL454), tetO7:ROT1 slt2 (JCY431), rho1 (HNY21) and tetO7:ROT1 rho1 (MCY158) strains were spotted onto YPD medium with or without 0.5 µg/ml doxycycline and incubated at 28°C for 3 days. (B) Western analysis of the phosphorylation of Slt2 in extracts from wild-type (CML240) and tetO7:ROT1 (JCY216) cells grown on YPD and incubated in the presence of 5 µg/ml doxycycline for 8 hours. A loading control of total protein is shown (* lower panel).

View larger version (47K): [in this window] [in a new window]   Fig. 5. Organization of the actin cytoskeleton in the tetO7:ROT1 mutant strain. (A) Exponentially growing cells of the wild-type (CML240) and the tetO7:ROT1 (JCY216) strains were incubated in the presence of doxycycline for 8 hours and fixed. Pictures show DIC images, DAPI staining of DNA and Alexa Fluor 498-labeled phalloidin staining of F-actin. (B) Wild-type (CML240) and tetO7:ROT1 (JCY216) cells were synchronized by addition of -factor in the presence of 5 µg/ml doxycycline. After release, cells at the different cell cycle stages were analyzed as described above.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Subcellular localization of polarity proteins in the tetO7:ROT1 mutant strain. Exponentially growing cells of the tetO7:ROT1 (JCY216) strain transformed with a plasmid expressing a GFP-tagged version of the Spa2 (A), Cdc24 (B) or Cdc42 (C) proteins were synchronized by addition of -factor in the presence of 5 µg/ml doxycycline. DIC images and GFP signal of a collection of cells at the different cell cycle stages are shown.

View larger version (54K): [in this window] [in a new window]   Fig. 7. Effect of ROT1 inactivation on septum formation. (A) Exponentially growing cultures of the tetO7:ROT1 (JCY216) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Cell were fixed and digested with zymolyase (zym) in the presence of sorbitol. Graph shows the distribution of cells at different cell cycle stages. Values are means+s.d. of three experiments. (B) Exponentially growing cells of the MYO1-GFP (MCY198) strain were synchronized by addition of -factor in the presence of 5 µg/ml doxycycline. DIC images and GFP signal of a collection of cells at the different cell cycle stages are shown. (C,D) Exponentially growing cells of the tetO7:ROT1 (JCY216) strain were incubated in the presence of 5 µg/ml doxycycline for 10 hours. Cells were fixed and stained with the membrane-specific dye DiI and chitin dye Calcofluor White (CFW) to examine membrane continuity and septum formation in rebudded cells (see text). In C, samples were analyzed by wide-field fluorescence microscopy. A collection of rebudded cells in which a complete septum could not be detected is shown. Picture to the right end shows a control of a rebudded cell with a complete septum. In D, samples were analyzed by confocal microscopy with a series of 140 nm sections. Discontinuous signals were evident in a large fraction of rebudded cells in the case of CFW staining or in all rebudded cells in the case of DiI staining. A selection of the optical sections obtained with one of these cells is shown for each case.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Genetic interaction between ROT1 and CLB2. (A) Clb2 protein level in extracts from cultures of the CLB2-HA (JCY285) and the CLB2-HA tetO7:ROT1 (JCY286) strains grown on YPD and incubated in the presence of 5 µg/ml doxycycline for 8 hours, were examined by western analysis. A control extract from the wild-type strain is included. A loading control of total protein is shown in the lower panel. (B) Protein kinase activity in HA immunoprecipitates from extracts were assayed using histone H1 as a substrate. Lower panels show the Clb2 and Cdc28 protein levels in the immunoprecipitates. A control extract from the wild-type untagged strain is included. (C) Tenfold serial dilutions from exponentially growing cultures of the wild-type (CML240) and tetO7:ROT1 (JCY216) strains transformed with an empty vector or a plasmid expressing the CLB2 gene under the control of the GAL1 promoter were spotted onto YPD or YPGal medium containing 0.5 µg/ml doxycycline and incubated at 28°C for 3 days. (D) Tenfold serial dilutions from exponentially growing cultures of the wild-type (W303), tetO7:ROT1 (MCY68), clb2 (CD104-2c) and clb2 tetO7:ROT1 (JCY255) strains were spotted onto YPD medium with or without 5 µg/ml doxycycline and incubated at 28°C for 3 days. (E) Exponentially growing cultures of the wild-type (W303), tetO7:ROT1 (MCY68), clb2 (CD104-2c) and clb2 tetO7:ROT1 (JCY255) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Graph shows the distribution of cells at different cell cycle stages. Values are the mean+s.d. of three experiments. DIC images show DAPI staining of DNA and Alexa Fluor 498-labeled phalloidin staining of F-actin of the clb2 tetO7:ROT1 cells.

View larger version (36K): [in this window] [in a new window]   Fig. 9. Genetic interactions between the ROT1 gene and genes of the ubiquitin-proteasome pathway. (A) Tenfold serial dilutions from exponentially growing cultures of the wild type (W303 or WCG4 in the upper panel), tetO7:ROT1 (MCY68 or MCY125 in the upper panel), pre1 pre2 (WCG4-11/22), pre1 pre2 tetO7:ROT1 (MCY32), cdc16, cdc16 tetO7:ROT1 (JCY553), apc2 (KTM200U) and apc2 tetO7:ROT1 (JCY555) were spotted onto YPD medium with or without 0.5 µg/ml doxycycline (Dox) and incubated at 30° for 3 days. (B) Tenfold serial dilutions from exponentially growing cultures of the cdc20 and cdc20 tetO7:ROT1 (MCY139) strains transformed with a centromeric plasmid containing the ROT1 gene as indicated, were spotted onto YPD medium with or without 5 µg/ml doxycycline and incubated at 28°C or 37°C for 3 days.

View larger version (64K): [in this window] [in a new window]   Fig. 10. Control of Clb2 protein stability by Rot1. (A) Wild-type (CML240) and tetO7:ROT1 (JCY216) cells transformed with a plasmid bearing a GAL:CLB2-HA gene were grown on raffinose (raf), incubated in galactose (gal) for 30 minutes and transferred to glucose medium. Clb2 protein level decay was examined at the indicated times by western analysis. Cdc28 protein level is shown as a loading control. (B) Exponentially growing cultures of the cdc15 (MCY123) and cdc15 tetO7:ROT1 (MCY151) cells expressing HA-tagged Clb2 and Myc-tagged Sic1 were arrested by incubation at 37°C in the presence of 5 µg/ml doxycycline. After 4 hours (>95% of telophase cells in both cases), cells were transferred to 28°C and Clb2 and Sic1 protein levels were analyzed at the indicated times by western analysis. (C) Wild-type (CML240) and tetO7:ROT1 (JCY216) cells bearing the GAL:CLB2-HA gene were grown on raffinose and arrested by -factor in the absence or presence of 5 µg/ml doxycycline. After 4 hours (>96% of unbudded cells in all the cases), galactose was added to the arrested cells and, after 30 minutes, protein synthesis was blocked by the addition of cycloheximide (chx). Clb2 protein level decay was examined at the indicated times by western analysis. Cdc28 protein level is shown as a loading control.