Polo-like kinase acts as a molecular timer that safeguards the asymmetric fate of spindle microtubule-organizing centers
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER) / Spanish National Research Council (CSIC) - University of Seville - University Pablo de Olavide, Spain
Figures
Figure 1 with 1 supplement
Cdc5 activity is required for asymmetric SPB inheritance and Spc72 distribution.
(A, B) SPB inheritance in wild-type (F2071) and cdc5-as1 (F2741) cells expressing Spc42-RFP and Spc110-GFP. (A) Representative images of Spc42-RFP (red) and Spc110-GFP (green) localization in cells …
Figure 1—figure supplement 1
SPB inheritance and Spc72 post-translational modification in cells lacking Cdc5 activity.
(A–C) SPB inheritance in wild type (F2276) and cdc5-as1 (F2865) cells expressing both Spc110-dsRed and Spc42-GFP (A, B), as well as in wild type (F2111), cdc5-as1 (F3703), cdc5-2 (F4441), cdc5-77 …
Figure 2 with 1 supplement
Cdc5 regulates Kar9 association with the SPBs.
(A–D) Kar9-sfGFP distribution and SPB inheritance in wild-type (F3754) and cdc5-as1 (F3755) cells also expressing Spc110-dsRed. (A) Representative images showing coincidental or opposite …
Figure 2—figure supplement 1
Kar9 distribution and post-translational modification in cells lacking Cdc5 activity.
(A–C) Kar9-sfGFP distribution in wild-type (F3172), cdc5-as1 (F3753), cdc5-2 (F4498), cdc5-77 (F4704), and cdc5-16 (F4698) cells (A, B), as well as Kar9-mCherry localization in wild-type (F4628) and …
Figure 3 with 1 supplement
Cell cycle analysis of Cdc5 and Spc72 localization.
(A–E) Cells expressing Cdc5-as1-eGFP and Spc72-mCherry (F3053) were arrested in G1 with α-factor and released at 26°C in YPAD medium with (+CMK-C1) or without (+DMSO) CMK-C1. The experiment was …
Figure 3—figure supplement 1
Spc72 and SPB distribution during a synchronous cell cycle.
(A–E) Cells expressing Cdc5-as1-eGFP and Spc72-mCherry (F3053) were grown overnight in YPAD at 26°C, diluted to OD600 = 0.2 in fresh medium, arrested in G1 with 5 µg/ml α-factor and finally released …
Figure 4
BiFC analysis of Cdc5 and Spc72 interaction.
(A, B) Model depicting the role of Cdc5 in the control of Spc72 and Kar9 function on SPB distribution (A) and the consequences of the lack of its kinase activity on the predetermined SPB inheritance …
Figure 5 with 3 supplements
Cdc5 activity is specifically required at metaphase–anaphase transition.
(A-D) Cells expressing Cdc5-as1-eGFP, Spc72-mCherry, and the conditional cdc20-AID allele (F3053) were arrested in G1 with α-factor and released at 26°C in YPAD with 500 µM indole-3-acetic acid …
Figure 5—figure supplement 1
SPB inheritance and Cdc5-as1 localization when Cdc5 activity is inhibited after release from a metaphase arrest.
(A–D) Cells expressing Cdc5-as1-eGFP, the conditional cdc20-AID allele and Spc110-dsRed (F3766) (A, B) or Spc72-mCherry (F3053) (C, D) protein fusions were grown 24 hr (A, B) or overnight (C, D) in …
Figure 5—figure supplement 2
SPB inheritance and Cdc5-as1 localization in cells lacking Cdc5 activity only up to the metaphase-to-anaphase transition.
(A–D) Cells expressing Cdc5-as1-eGFP, the conditional cdc20-AID allele and Spc110-dsRed (F3766) (A, B) or Spc72-mCherry (F3053) (C, D) protein fusions were grown 24 hr (A, B) or overnight (C, D) in …
Figure 5—figure supplement 3
Cdc5 acts as a molecular timer that facilitates the establishment of SPB fate.
(A, B) Cdc5-as1 cells expressing the conditional cdc20-AID and cdc14-1 alleles and the Spc72-mCherry fusion (F4756) were grown for 24 hr in YPAD at 26°C, diluted to OD600 = 0.2 in fresh medium, …
Figure 6
Cdc5 activity facilitates recruitment of new Spc72 protein on the SPBs.
(A, B) SPB inheritance in cdc5-as1 (F3705) and cdc5-as1 tab6-1 (F4140) cells expressing Spc110-dsRed. Graphs show the percentages of cells displaying wild-type (white bars) or reversed (black bars) …
Figure 7 with 1 supplement
Analysis of Spc72 and Kar9 interaction on SPBs.
(A–C) SPB inheritance (A) and Spc72 localization (B) in wild-type (F3712) and kar9Δ (F3774) cells, both expressing Spc42-RFP and Spc72-GFP. Graphs show the percentages of cells displaying wild-type …
Figure 7—figure supplement 1
Cdc5 establishes SPB identity during the prior cell cycle.
(A) Co-immunoprecipitation analysis in cells expressing Kar9-13Myc, Tub4-mScarlet and either Spc72-GFP (F4825), Spc72-AA-GFP (F4829) or untagged Spc72 (No tag, F4823). Cells exclusively expressing …
Author response image 1
Additional files
-
Supplementary file 1
Table S1 including all the strains from the S. cerevisiae W303 genetic background used in this work.
- https://cdn.elifesciences.org/articles/61488/elife-61488-supp1-v2.docx
-
Supplementary file 2
Table S2 detailing the list of antibodies used for immunofluorescence and Western blot.
- https://cdn.elifesciences.org/articles/61488/elife-61488-supp2-v2.docx
-
Supplementary file 3
Excel document including spreadsheets with the source data for each of the figures of the article.
- https://cdn.elifesciences.org/articles/61488/elife-61488-supp3-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/61488/elife-61488-transrepform-v2.pdf
