The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Abstract

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.

Figure 1. The SAC controls Kar9 asymmetry independently of Cdk1/Clb4, but in a Cdk1/Clb5-dependent manner. (A) Representative images of metaphase cells expressing Kar9-YFP, Clb4-Venus or Clb5-Venus together with CFP-Tub1. Scale bar is 2 µm. (B) Representative images of metaphase cells expressing Kar9-YFP in various mutant backgrounds. (C and D) Quantification of Kar9 asymmetry in nnf1–17 mutant background in combination with mutations affecting Cdk1/Clb4-dependent phosphorylation of Kar9 (C) or Cdk1/Clb5 activity (D). Cells coexpressed Kar9-YFP and CFP-Tub1 and were either MAD2 or mad2∆. Quantification as described in the text, cells were shifted to 37°C for 50 min before imaging. Stars indicate p-values obtained from one-way ANOVA comparing “strong asymmetry” (3 clones with total n > 115). Scale bar is 2 µm. (E) Quantification of metaphase arrest in nnf1–17 and clb5∆ cells in either MAD2 or mad2∆ background. Percentage of metaphase cells over the total number of cells was determined. Stars indicate p-values obtained from one-way ANOVA comparing to WT or as indicated (three clones with total n > 1100).

Figure 2. The SAC inhibits the MEN during metaphase. (A) and (B) Cells expressing the nnf1–17 allele in combination with mutations affecting Dbf2-dependent phosphorylation of Kar9. Cells coexpressed Kar9-YFP and CFP-Tub1 and were either MAD2 or mad2∆, as well as CLB5 or clb5∆. All quantifications were performed as in Figure 1C, cells were shifted to 37°C for 50 min before imaging. Stars indicate p-values obtained from one-way ANOVA comparing “strong asymmetry” (three clones with total n > 130). (C) Analysis of Kar9 asymmetry in FEAR mutant cells at 22°C as in (A).

Figure 3. SAC activation perturbs faithful segregation of the old SPB to the bud. (A) Metaphase cells expressing nnf1–17, tem1–3 or deletions of the cyclins CLB4 and CLB5 in combination with either MAD2 or mad2∆. In addition, cells expressing dbf2–2 dbf20∆ or Kar9-R194A S332A S429A in either NNF1 or nnf1–17 background as well as FEAR mutant cells were analyzed. Cells coexpressed a switchable mCherry/GFP-tag allowing distinction of old and new SPB, as well as Kar9-YFP and CFP-Tub1. Stars indicate p-values obtained from one-way ANOVA comparing to WT or as indicated (three clones with total n > 125). Asterisks mark the new SPB, yellow arrows indicate Kar9 fluorescence. Scale bar is 2 µm. (B) Same strains and conditions as for (A), but anaphase cells were imaged. Three clones with total n > 87 cells. (C) Schematic drawing of the pathways controlling Kar9 asymmetry and SPB specification in metaphase.