APC/C^Cdc20-mediated degradation of Clb4 prompts astral microtubule stabilization at anaphase onset

Zucca et al. identify in budding yeast two evolutionary conserved mitotic machineries, the Clb4-CDK1 and APC/C^Cdc20 complexes, as central regulators of astral microtubule dynamics.

Abstract

Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/C^Cdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/C^Cdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.

Graphical Abstract

Introduction

Chromosome segregation requires remodeling of the mitotic spindle. The mitotic spindle is composed of microtubules, microtubule-associated proteins (MAPs), and motor proteins (Prosser and Pelletier, 2017). Based on their function, microtubules are divided into three categories: (i) kinetochore microtubules (kMTs), which connect the spindle poles to chromosomes and direct their segregation; (ii) interpolar microtubules (iMTs), which form a bundle of antiparallel microtubules and promote the distancing of the two sister chromatids via spindle elongation; and (iii) astral microtubules (aMTs), which connect the spindle poles to the cellular cortex and guide chromosomes along the polarity axis by dictating spindle positioning and orientation (Winey and Bloom, 2012). Proper spindle positioning is fundamental for the correct segregation of chromosomes, and it is crucial for many cellular processes such as stem cell maintenance, tissue homeostasis, and development (Lechler and Mapelli, 2021). Despite their fundamental role, the molecular mechanisms that regulate aMT dynamics remain elusive and largely overlooked. Here, we investigate aMT dynamics in Saccharomyces cerevisiae and ask whether and how they are coordinated with other cell cycle events.

Chromosome segregation is initiated by the activation of the anaphase promoting complex or cyclosome (APC/C) in complex with its activator subunit Cdc20 (Sudakin et al., 2001; Izawa and Pines, 2014). The APC/C is an E3-ubiquitin ligase whose specificity is dictated by the interaction with its regulatory subunits, Cdc20 and Cdh1 (Visintin et al., 1997). Activation of the APC/C^Cdc20 at the metaphase-to-anaphase transition initiates a three-step signaling cascade that culminates in cohesin cleavage—the point of “no return” for mitotic exit (Fig. 1 A). Cohesin is a protein complex that holds chromosomes together from the moment of their replication up to their separation (Uhlmann et al., 2000). Following cohesin cleavage, the coordination between sister chromatid separation and segregation is directed by changes of mitotic spindle microtubule dynamics. kMTs retract and pull sister chromatids toward the spindle poles while iMTs drive spindle elongation, thereby segregating sister chromatids apart from each other. Consistent with their functions (kMTs search and capture chromosomes and iMTs promote the formation of a short bipolar spindle without forcing elongation), kMTs and iMTs are unstable in metaphase (Higuchi and Uhlmann, 2005). Vice versa, kMTs and iMTs are stabilized in anaphase to preserve proper kMT-chromosome interactions and to promote spindle elongation (Higuchi and Uhlmann, 2005; Mallavarapu et al., 1999). Notably, since budding yeast exhibits a “closed” mitosis—the nuclear envelope does not break down—the mitotic spindle extends within the nucleus between two spindle pole bodies (SPBs), the centrosome equivalent (Byers and Goetsch, 1975). Microtubules extend from both the cytoplasmic (aMTs) and the nuclear (kMTs and iMTs) faces of the SPBs during the cell cycle. By removing the opposing forces to spindle pulling, cohesin cleavage indirectly affects the dynamics of nuclear microtubules.

Figure 1.

aMT dynamics change at the metaphase-to-anaphase transition. (A) Schematic and simplified representation of late mitotic events in S. cerevisiae. Chromosome segregation begins with the activation of the APC/C^Cdc20 complex that unleashes the separase Esp1, which in turn cleaves cohesin. Cohesin cleavage is considered the point of non-return for mitotic exit, hence APC/C^Cdc20 activity is finely regulated and supervised by the DNA damage and spindle assembly checkpoints. Esp1—as a component of the FEAR network—is also required for the initial and partial release of the CDK-counteracting phosphatase Cdc14. One task of FEAR-released Cdc14 is to promote spindle elongation, a duty it shares with the Polo-like kinase Cdc5. Cdc5 and Cdc14 also co-operate to activate MEN, a signaling cascade with the essential function of fully activating the phosphatase. MEN-activated Cdc14 drives CDK inactivation by activating, among others, the APC/C^Cdh1 and reverts CDK-mediated phosphorylation events, thereby triggering mitotic exit. (B and C) Wild-type (Ry1) cells were analyzed in a synchronous cell cycle. (B) At the indicated time points, the percentage of cells with metaphase (light blue circles) or anaphase (dark blue circles) spindles was determined (n = 100 cells), and aMT length and number were measured (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). (C) aMT length and number were measured in metaphase (T₆₀ minutes) or anaphase (T₁₀₀ minutes) cells. We found a mean of 1.2 µm and 1.7 aMT/cell in metaphase cells and of 1.6 µm and 3.2 aMT/cell in anaphase cells. **** = P < 0.0001; asterisks denote significant differences according to two-tailed unpaired t test. Note: Here and throughout the manuscript, aMT length is shown in dot plots while aMT number is shown in bar charts. Each data point in the dot plot represents one single aMT. The median is displayed as a solid line. Error bars in both graphs represent the SEM. (D) Representative images and corresponding diagrams of mitotic spindles in metaphase (light blue) and anaphase (dark blue) cells are shown; scale bar = 5 µm. In the diagram, aMTs are indicated in black, nuclear microtubules (iMTs and kMTs) in gray, and the two SPBs in pink.

Molecularly, the switch in microtubule dynamics from an unstable to a stable state has long been associated with cyclin dependent kinase (CDK) activity. CDK1 activity is high in metaphase and decreases in anaphase due to APC/C-mediated degradation of the cyclin-regulatory subunits and to the activation of the main yeast CDK-counteracting phosphatase Cdc14 (Fig. 1 A). The observation that several motors and MAPs are regulated by phosphorylation and de-phosphorylation events mediated by the antagonistic couple CDK1/Cdc14 gave rise to a model linking spindle microtubules stabilization to the overall change in the phosphorylation landscape in favor of de-phosphorylation (Khmelinskii et al., 2007; Avunie-Masala et al., 2011; Khmelinskii et al., 2009). This view has been recently challenged by an elegant study in budding yeast reporting that the number of phosphorylated or de-phosphorylated residues in late mitosis is similar, thus suggesting that mitotic kinases can compensate for the drop in CDK-mediated phosphorylation (Touati et al., 2018). Supporting this model is the observation that the phosphatase Cdc14 and the Polo-like kinase Cdc5 are redundant in triggering spindle elongation (Roccuzzo et al., 2015). The contribution of spatially defined mechanisms renders the dynamics of microtubule regulation more complex. An example is the phospho-regulation of single kMTs following their binding to kinetochores, which relies on the inhibition of the Aurora B kinase to promote kMT stabilization when the attachment that is limited to a single kinetochore creates tension (Akiyoshi et al., 2010; Sarangapani et al., 2013). Altogether these observations unveiled a sophisticated regulation of nuclear spindle microtubules at the metaphase-to-anaphase transition, which relies both on phosphorylation and de-phosphorylation events—depending on the residue—and takes into account the spatially defined modulation of individual microtubules.

How aMTs fit into this picture remains unknown. Most studies focused on the regulation of aMT binding to the cellular cortex. It emerged that, both in budding yeast and multi-cellular eukaryotes, CDKs negatively affect this interaction. In yeast, the cyclin Clb4 promotes the detachment of aMTs from the cellular cortex in early mitosis by phosphorylating a yet-to-define substrate (Maekawa and Schiebel, 2004). In human, CDK1 reduces aMTs binding to the membrane by phosphorylating the nuclear mitotic apparatus (NuMa; Kotak et al., 2013). NuMa localizes at the cortex and it is a component of the evolutionary conserved cortical machinery essential for spindle orientation (Kiyomitsu and Boerner, 2021). The human Polo-like kinase 1 (Plk1) phosphorylates NuMa (Sana et al., 2018) and contributes to negatively regulating its cortex localization (Kiyomitsu and Cheeseman, 2012). Instead, little is known as to whether aMT dynamics are regulated in a cell cycle dependent manner. The only evidence of a direct regulation of aMT dynamics comes from a recent study highlighting that CDK1-dependent phosphorylation of the plus-end tracking protein GTSE1 is required to destabilize aMTs in prometaphase (Singh et al., 2021).

Here we show that, similar to nuclear microtubules, aMTs dynamics switch from an unstable to a more stable status at anaphase onset. At the heart of this switch, required to maintain aMTs in close proximity of the cellular cortex and ultimately to establish proper spindle positioning, is the APC/C^Cdc20-dependent degradation of the mitotic cyclin Clb4 that likely impacts aMT dynamics by affecting the activity/localization of aMT regulators, of which Kip2 is a prime candidate. Besides identifying the unique function of Clb4 among all cyclins in this process, our data evidence a central role for the APC/C^Cdc20 in choreographing late mitotic events by sequentially instructing the three classes of spindle microtubules.

Results

Astral microtubules are stabilized in anaphase

To gain insights into aMT regulation in mitosis, we probed aMT morphology in wild-type S. cerevisiae cells undergoing a synchronous cycle. At each time-point, cell cycle progression was assessed by monitoring spindle morphologies and aMT length and number, two bona fide indicators of aMT stability (Drechsler et al., 2015). A correlation emerged between the population approaching metaphase and a decrease in both aMT length and number (Fig. 1 B). Contrariwise, these parameters increased when the population entered anaphase (Fig. 1 B). This relationship became particularly evident when we correlated the morphology of aMTs with metaphase and anaphase spindles (Fig. 1, C and D).

Since the distinction between cells in late G2 and in early metaphase, or in late metaphase and in early anaphase, is somewhat arbitrary, we validated our findings by probing aMT dynamics in homogenous populations. More precisely, we assessed aMT morphology in cdc20 and cdc15 mutant cells, which arrest in metaphase and anaphase, respectively (Fig. 1 A and Fig. 2 A). To arrest cells in metaphase, unless otherwise specified, we used a conditional allele of CDC20 where the wild-type CDC20 gene is fused to an Auxin-inducible degron sequence (CDC20-AID, henceforth cdc20; Shetty et al., 2019). To arrest cells in anaphase, we used an ATP analog-sensitive allele of the kinase CDC15 (cdc15-as1; Bishop et al., 2001). cdc20 and cdc15 mutants were released from a G1 block into the restrictive conditions, and aMTs were analyzed at their terminal phenotype (Fig. 2 A). While the arrest uniformed the average aMT length between metaphase and anaphase cells, the number of aMTs remained significantly higher in cdc15 mutants (Fig. 2, B and C). To exclude possible mutant-specific effects, the same assays were performed arresting the cells in metaphase by different mutations (cdc20-1, cdc23-1 [Zachariae and Nasmyth, 1999], GAL-MAD2 [Rossio et al., 2010] and cdc13-1 [Hartwell et al., 1973] cells) or in anaphase (cdc5-as1 [Snead et al., 2007] and cdc14-1 [Hartwell and Smith, 1985] cells). The data obtained confirmed what we found in CDC20-AID and cdc15-as1 cells (Fig. S1 A and Fig. 2, B and C), thus indicating that changes in aMT morphology are determined by the cell cycle phase rather than by the mutant strains used.

Figure 2.

aMTs are stabilized in anaphase. (A) Schematic representation of the experimental setup pertinent to this figure (mutants used and mitotic phase of their terminal arrest). (B and C) cdc20-AID (Ry4853) and cdc15-as1 (Ry1112) cells were analyzed at their terminal arrest (T₁₈₀ minutes). (B) The graphs show aMT length and number of the indicated genotypes (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). The arrests uniformed the average aMT length between metaphase and anaphase cells (2.3 µm in cdc20 cells and 2.1 µm in cdc15 cells), while the number remained significantly higher in cdc15 mutants (from a mean of 3.1 to 4.9 aMT/cell in cdc20 and cdc15 cells, respectively). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (C) Representative images of the terminal phenotype are shown. Scale bar = 2 µm. (D and E) cdc20-AID (Ry7732) and cdc15-as1 (Ry9741) cells harboring a TUB1-GFP fusion were arrested in G1 and released into restrictive conditions. When the majority of the cells reached their terminal arrest (T₁₈₀ minutes), cells were transferred to a CellASIC ONIX plate for live imaging. Individual aMTs were probed. (D) Still images of a representative cell are shown for both strains (scale bar = 1 µm). Graphs of two representative aMTs (1 and 2) per cell are shown. Black and gray arrows indicate the occurrence of catastrophe and rescue events, respectively. (E) Bar charts represent the calculated catastrophe and rescue rates (n = 33 aMTs in cdc20-AID cells and n = 21 aMTs in cdc15-as1 cells were measured). * = P < 0.05; ** = P < 0.01; asterisks denote significant differences according to two-tailed unpaired t test. aMTs of cdc15 cells resulted less dynamic—as assessed by the decrease of both catastrophe (from 0.0044 to 0.0026 event/s) and rescue rates (from 0.004 to 0.0028 event/s)—than aMTs of cdc20 cells. Error bars in graphs represent SEM.

Figure S1.

Analysis of aMTs in different mitotic phases. (A) cdc20-AID (Ry7873), cdc20-1 (Ry586), cdc23-1 (Ry454), cdc13-1 (Ry17), pGAL-MAD2 (Ry10946), cdc14-1 (Ry1574), cdc5-as1 (Ry2446), and cdc15-as1 (Ry1112) cells were synchronized in G1 and released into restrictive conditions. Cells were analyzed at their terminal arrest (T₁₈₀ minutes). The graphs show the aMT length and number of the indicated mutant strains (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). Error bars in graphs equal SEM. (B) Time-lapse images of two representative aMTs of cdc20-AID (Ry7732) cells harboring a TUB1-GFP fusion are shown (T₁₈₀ minutes; scale bar = 2 µm). Black and gray arrows indicate the occurrence of catastrophe and rescue events, respectively.

The observation that upon protracted arrest aMT length uniformed between the two cell cycle phases prompted us to explore aMT dynamics in depth. For this purpose, we moved to live-cell imaging and measured parameters that ultimately define dynamic properties, such as catastrophe and rescues rates (Kosco et al., 2001; Estrem et al., 2016). The length of individual aMTs in cdc20 and cdc15 cells expressing a GFP-tagged Tub1 fusion was measured over time in single cells at their terminal phenotype (Fig. 2 D and Fig. S1 B). In line with previous findings, aMTs of cdc15 cells resulted less dynamic than the ones of cdc20 cells as assessed by the decrease of both catastrophe and rescue rates (Fig. 2 E and Table S1). Taken together, the observations that: (i) aMTs increase in number and length when wild-type cells move from metaphase to anaphase; (ii) aMT number is higher in mutants arrested in anaphase when compared to their metaphase counterpart; and (iii) anaphase aMTs appear less dynamic, support the conclusion that aMTs are stabilized at anaphase onset. Why aMT length is similar in the two conditions remains puzzling.

Stabilization of aMTs in anaphase relies on a specific signature

Having established that aMTs are stabilized at anaphase onset, we wished to characterize the molecular mechanism at the heart of this switch. It is known that anaphase stabilization of iMTs requires the activity of either the phosphatase Cdc14 or the Polo-like kinase Cdc5 (Roccuzzo et al., 2015). To investigate whether aMTs and iMTs share the same regulatory mechanism, we examined the aMT phenotype of cdc14 cdc5 double-mutant cells. These cells arrest in mini-anaphase (after cohesin cleavage, the hallmark of anaphase onset), with short bipolar spindles and undivided nuclei, due to defects in spindle elongation (Fig. 1 A and Fig. 3 A; Roccuzzo et al., 2015).

Figure 3.

aMT stabilization relies on a specific anaphase signature. (A) Schematic representation of the experimental setup pertinent to this figure. (B and C) cdc20-AID (Ry4853), cdc14-1 cdc5-as1 (Ry1602), and cdc15-as1 (Ry1112) cells were analyzed at their terminal arrest (T₁₈₀ minutes). (B) The graphs show aMT length and number of the indicated genotypes (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). aMTs of cdc14 cdc5 cells were longer and more numerous than the ones of cdc20 mutants (from a mean of 2.6 µm and 2.6 aMT/cell in cdc20 cells to a mean of 4 µm and 4.6 aMT/cell in cdc14 cdc5 cells) and similar in number but much longer than the ones of cdc15 cells (from a mean of 2.1 to 4 µm in cdc15 and cdc14 cdc5 cells, respectively). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (C) A representative image of cdc14 cdc5 cells is shown. (D and E) cdc20-AID (Ry7732), cdc15-as1 (Ry9741), and cdc14-1 cdc5-as1 (Ry3256) cells expressing TUB1-GFP fusion protein were arrested in G1 and synchronously released into restrictive conditions. When the arrest was complete (T₁₈₀ minutes), cells were moved to a CellASIC ONIX plate for live imaging. (D) Time-lapse images of a representative cell are shown for the cdc14-1 cdc5-as1 strain (scale bar = 1 µm). Graphs of two aMTs (1 and 2) are shown. The occurrence of catastrophe and rescue events is indicated in the graphs with black and gray arrows, respectively. (E) The bar charts show the catastrophe and rescue rates of the indicated mutant strains. aMTs of cdc14 cdc5 are characterized by a low-catastrophe rate (0.003 events/s, compared to 0.004 and 0.0026 in cdc20 and cdc15 cells, respectively), associated with a high-rescue rate (0.0044 events/s, compared to 0.004 and 0.0028 in cdc20 and cdc15 cells, respectively). n = 33 aMTs in cdc20-AID cells, n = 33 aMTs in cdc14-1 cdc5-as1 cells, and n = 21 aMTs in cdc15-as1. * = P < 0.05; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. Error bars in graphs represent SEM.

aMT dynamics of cdc14 cdc5 cells were probed at their terminal phenotype both by indirect immunofluorescence and live-cell imaging, and compared with the ones of cdc20 and cdc15 arrested cells, which share with the double mutant spindle morphology and mitotic phase, respectively. Consistent with their mini-anaphase arrest, aMTs of cdc14 cdc5 cells were significantly more stable than the ones of metaphase-arrested cdc20 cells: they were both longer and more numerous (Fig. 3, B and C), thus suggesting that the molecular mechanism at the core of the anaphase aMT stabilization differs from the one controlling iMTs, as it is independent of Cdc14 and/or Cdc5 activities.

The aMTs of cdc14 cdc5 arrested cells were also more stable than the ones of late anaphase arrested cdc15 mutants. aMTs of cdc14 cdc5 cells were similar in number, but longer than their cdc15 counterpart (Fig. 3, B and C). This phenotype is not caused by an inappropriate activation of checkpoint pathways since abrogation of checkpoint activities (spindle assembly, DNA damage, and spindle positioning checkpoints) in cdc14 cdc5 cells did not alter aMT morphology (Fig. S2, A–C).

Figure S2.

The aMT phenotype of cdc14-1 cdc5-as1 cells is independent from activation of the spindle assembly checkpoint, the DNA damage response, and the spindle positioning checkpoint SPoC, and it is not a consequence of the spindle elongation defect or the inactivation of Cdc14 and Cdc5. (A) Schematic representation of the experimental setup pertinent to this figure. (B and C) cdc14-1 cdc5-as1 (Ry1602), cdc14-1 cdc5-as1 mad2∆ rad9∆ (Ry3771), and cdc14-1 cdc5-as1 bub2∆ (Ry3346) cells released from a G1 block into restrictive conditions were scored at their terminal arrest (T₁₈₀ minutes). (B) Representative images of the indicated strains are shown (scale bar = 5 µm). (C) The graphs show aMT length and number (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). None of the strains resulted statistically different according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (D and E) cdc15-as1 (Ry1112), cdc15-as1 kar9∆ dyn1-AID (Ry7620), and cdc14-1 cdc5-as1 (Ry1602) cells were treated and analyzed as in B and C. (D) aMT length is shown for the indicated strains. ** = P < 0.01; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (E) Representative images of cdc15-as1 and cdc15-as1 kar9∆ dyn1-AID mutant cells are shown (scale bar = 5 µm). (F) cdc20-AID (Ry4853), cdc20-AID cdc14-1 (Ry4934), and cdc20-AID cdc5-as1 (Ry4936) cells were treated and analyzed as in B and C. None of the strains were statistically different according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (G and H) pMET-CDC20 (Ry1223), pMET-CDC20 cdc14-1 (Ry3204) pMET-CDC20 cdc5-as1 (Ry3209), pMET-CDC20 cdc14-1 cdc5-as1 (Ry3201), and cdc14-1 cdc5-as1 (Ry1602) cells were arrested in G1 with the α-factor pheromone (5 µg/ml) in synthetic complete media lacking methionine and synchronously released into YEPD supplemented with methionine—to repress the expression of CDC20—at 37°C in the presence of CMK (5 µM) to inactivate the cdc14-1 and cdc5-as1 alleles, respectively. (G) aMT length and number were scored 3.5 h after the release. **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. Error bars in graphs represent SEM. (H) Samples were taken at the indicated time points to determine the percentage of cells with metaphase-like (light blue) and anaphase-like (dark blue) spindles (n = 100 cells).

To understand why cdc14 cdc5 and cdc15 cells carry aMTs of different length, we moved to live-cell imaging. It turned out that while the aMT catastrophe and rescue rates of metaphase (cdc20) and anaphase (cdc15) cells always follow the same trend, either by increasing or decreasing simultaneously, thus possibly reaching an equilibrium, the two parameters were uncoupled in the aMTs of the cdc14 cdc5 cells (Fig. 3, D and E). Here, aMTs are characterized by a low catastrophe rate associated with a high rescue rate (Fig. 3, D and E; and Table S1), a combination that favors polymerization and could provide an explanation to the different aMT length observed.

Why catastrophe and rescue rates are uncoupled in cdc14 cdc5 cells remains unclear. Different scenarios can be envisioned: (i) spindle elongation may negatively impact on aMT dynamics (e.g., cross-talk between iMTs and aMTs or spatial constraints imposed by bringing the poles in close proximity to the cellular cortex); (ii) Cdc14 and/or Cdc5 may counteract aMT stabilization; or (iii) a combination of the two. To assess if spindle elongation is impacting aMT dynamics, we allowed cells to elongate their spindles but prevented the establishment of aMT-cortex connections by inactivating the spindle positioning factor Kar9 (Beach et al., 2000) and the minus-end directed motor Dyn1 (Li et al., 1993; Falk et al., 2016) in a cdc15 background. We found that the aMTs of cdc15 kar9 dyn1 cells are significantly longer than the ones of cdc15 cells and resemble aMTs of cdc14 cdc5 cells, indicating that spindle elongation could affect aMT dynamics in an indirect manner likely by bringing these microtubules in the proximity of the cortex (Fig. S2, A, D, and E). To test if Cdc14 and Cdc5 counteract aMT stability, we probed aMT morphology in metaphase-arrested cells lacking either Cdc14 or Cdc5 and found that aMTs of cdc20, cdc20 cdc5, and cdc20 cdc14 cells were similar both in number and length (Fig. S2 F). To exclude redundancy, we looked at the aMTs of a cdc20 cdc14 cdc5 triple mutant and observed that the simultaneous inactivation of Cdc14 and Cdc5 didn’t alter metaphase aMT dynamics (Fig. S2 G). Here, we used an allele of CDC20 whose expression is under the control of the methionine-repressible promoter (pMET-CDC20) because the concomitant inactivation of Cdc14 and Cdc5 is synthetically lethal with all tested CDC20 mutant alleles (cdc20-1, cdc20-3, and cdc20-AID) and with a cdc23 temperature sensitive allele of a gene encoding for a core subunit of the APC/C (Care et al., 1999). aMTs of pMET-CDC20 cdc14, pMET-CDC20 cdc5, and pMET-CDC20 cdc14 cdc5 cells were similar in number to the ones of metaphase cells (pMET-CDC20), albeit slightly longer (Fig. S2 G), a phenotype likely due to the less stringent pMET-CDC20 metaphase arrest (Fig. S2 H) as it was not observed in cdc20 cdc5 and cdc20 cdc14 cells (compare Fig. S2, F with G). All together these data indicate anaphase onset as a critical step for aMT stabilization.

APC/C^Cdc20 activation is sufficient to stabilize aMTs at anaphase onset

To identify the anaphase-specific trait that elicits aMT stabilization, we dissected the signaling cascade directing cohesin cleavage into its three critical steps, namely: (1) the activation of the APC/C^Cdc20; (2) the activation of the separase/Esp1, mediated by the APC/C^Cdc20-dependent degradation of securin/Pds1; and (3) the Esp1-mediated cleavage of the Scc1 subunit of the cohesin complex and, next, probed aMTs in mutant strains impaired in the completion of sequential steps of the cascade: cdc20, esp1, scc1nc (these cells overexpress under the control of the galactose-inducible promoter an uncleavable allele of Scc1—GAL-scc1R180DR268D; Uhlmann et al., 1999; Uhlmann et al., 2000), and cdc14 cdc5 cells (Fig. 4, A and B).

Figure 4.

APC/C^Cdc20 activation stabilizes aMTs. (A) Schematic representation of the three-steps signaling cascade defining the metaphase to anaphase transition: (1) activation of the APC/C^Cdc20; (2) activation of the separase/Esp1, mediated by the APC/C^Cdc20-dependent degradation of securin/Pds1; and (3) Esp1-mediated cleavage of the Scc1 subunit of the cohesin complex. (B) Schematic representation of the experimental setup pertinent to this figure. (C–E) cdc20-AID (Ry4853), esp1-1 (Ry9490), pGAL-SCC1nc (Ry8210), and cdc14-1 cdc5-as1 (Ry1602) cells were synchronized in G1 and released in restrictive conditions. Note: In cdc20 mutants, the APC/C^Cdc20 is inactive, all its substrates are present, separase is inactive, and cohesin is bound to chromatin; in esp1 mutant cells, the APC/C^Cdc20 is active and all its substrates are removed, including Pds1, but separase remains inactive and cohesin is still bound to chromatin; in scc1nc mutants, not only all the APC/C^Cdc20 substrates are removed, but also all separase substrates are properly cleaved, with the only exception of cohesin, which remains intact and bound to chromatin; and, finally, in cdc14 cdc5 cells, all the steps of the cascade are completed. (C) Representative images of esp1-1 and pGAL-SCC1nc cells with short bipolar spindles are shown (T₁₄₀ minute; scale bar = 5 µm). (D) Samples were taken at the indicated time points to determine the percentage of cells with short bipolar spindles (n = 100 cells). (E) aMT length and number were scored T₁₄₀ minutes after the release, the time point before spindle collapse in esp1-1 and pGAL-SCC1nc cells (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). aMTs of both esp1 and scc1nc cells were slightly more numerous and longer than aMTs of cdc20 cells (from 2.3 µm and 2.4 aMT/cell in cdc20 cells to 2.9 µm and 2.8 aMT/cell, and 3 µm and 3.2 aMT/cell in esp1 and scc1nc cells, respectively). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test; the arrow indicates the time point when the analysis was carried out. Error bars in graphs represent SEM.

To assess aMT morphology and dynamics, the mutants of interest were released from the G1 arrest into restrictive conditions. During the analysis, we noticed that, differently from cdc20 and cdc14 cdc5 cells, which maintained a stable short bipolar spindle for the entire duration of the experiment, both esp1 and scc1nc cells disassembled their mitotic spindle soon after reaching a short bipolar spindle configuration (metaphase-like morphology; Fig. 4, C and D). This observation is consistent with the ability of these cells to pull the nucleus, hence the spindle, into the bud and, as a consequence, exit from mitosis prematurely, albeit having failed to separate sister chromatids (Campbell et al., 2019). To perturb the system as little as possible, we initially analyzed aMTs of esp1 and scc1nc cells at the time point preceding spindle disassembly, with the caveat that we might have a mixed population of cells, some at the terminal phenotype and others still in metaphase (Fig. 4 D). Consistent with this possibility, the difference in aMT length and number between cdc14 cdc5 and cdc20 cells was less obvious when these parameters were measured at the time point chosen for esp1 and scc1nc cells (compare Fig. 4 E with Fig. 3 B). Nevertheless, aMTs of esp1 and scc1nc cells were slightly more numerous and longer than aMTs of cdc20 cells and resembled the ones of cdc14 cdc5 cells, suggesting that APC/C^Cdc20 activation is the molecular event triggering aMT stabilization (Fig. 4 E).

To confirm this observation, we engineered esp1 strains to prevent spindle collapse. Since spindle collapse is caused by the movement of the nucleus into the bud and the consequent activation of the mitotic exit network (MEN; Campbell et al., 2019), a signaling cascade that triggers mitotic exit through the phosphatase Cdc14 (Fig. 1 A), we: (i) precluded spindle movement into the bud by disrupting Dyn1-mediated pulling forces; (ii) impaired MEN activity by inhibiting Cdc15 or Cdc5 (Campbell et al., 2019); and (iii) inactivated Cdc14 (Fig. 1 A and Fig. 5, A and B). In agreement with our hypothesis, most esp1 dyn1, esp1 cdc15, esp1 cdc5, and esp1 cdc14 cells retained a short bipolar spindle for the entire duration of the experiment (Fig. S3) and exhibited, at their terminal arrest, aMTs significantly longer and more numerous than the ones of cdc20 cells, pheno-copying the ones of the cdc14 cdc5 mutants (Fig. 5, C and D). These data further support that the activation of the APC/C^Cdc20 is the molecular event required to dictate aMT stabilization at anaphase onset.

Figure 5.

APC/C^Cdc20 activation is sufficient to stabilize aMTs at anaphase onset. (A) Schematic representation of the experimental setup pertinent to this figure. (B) Schematic representation of the pathways explaining spindle collapse in esp1-1 cells. Spindle collapse/disassembly is triggered by CDK inactivation. In yeast, CDK inactivation requires the activity of the MEN, a Ras-like GTPase signaling pathway localized on the SPB. MEN activation requires SPB movement into the bud, where the inactive cascade meets its activator, asymmetrically localized there. For this step, wild-type cells relies on spindle elongation. (C and D) On the contrary, esp1-1 cells may activate the MEN through a Dyn1-dependent pulling of the spindle, hence nucleus, into the bud before cohesin cleavage. esp-1 (Ry9490), esp1-1 dyn1∆ (Ry9516), esp1-1 cdc15-as1 (Ry9512), esp1-1 cdc5-as1 (Ry9134), esp1-1 cdc14-1 (Ry9131), esp1-1 cdc14-1 cdc5-as1 (Ry9128), and cdc14-1 cdc5-as1 (Ry1602) cells of the indicated phenotype were synchronized in G1 and released into restrictive conditions. Cells were scored at their terminal arrest. (C) Representative images of the indicated mutants at their terminal arrest are shown (scale bar = 5 µm). (D) For the mutants of interest, aMT length is shown in dot plots while aMT number is shown in bar charts (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). aMTs of esp1 mutants were significantly longer and more numerous than aMTs of cdc20 cells, resembling the ones of cdc5 cdc14 cells (with an average length of 4, 4.2, 4, 4.5, and 4.2 µm, and an average number of 3.8, 4.4, 4.1, 4.7, and 4.3 aMT/cell in esp1 cdc15, esp1 cdc5, esp1 cdc14 esp1, cdc5 cdc14, and esp1 dyn1 cells, respectively; compared to cdc20 and cdc14 cdc5 cells that showed an average aMT length of 2.6 and 4 µm, and an average number of 3.2 aMT/cell and 4.3 aMT/cell, respectively). ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. Error bars in graphs equal SEM.

Figure S3.

Preventing MEN activation or nuclear movement into the bud stops spindle collapse in esp1-1 cells. cdc20-AID (Ry7873), esp1-1 (Ry9490), esp1-1 dyn1∆ (Ry9516), esp1-1 cdc15-as1 (Ry9512), esp1-1 cdc5-as1 (Ry9134), esp1-1 cdc14-1 (Ry9131), esp1-1 cdc14-1 cdc5-as1 (Ry9128), and cdc14-1 cdc5-as1 (Ry1602) cells were arrested in G1 and released into restrictive conditions for the alleles used. Samples were taken at the indicated time points to determine the percentage of cells with short bipolar spindles (n = 100 cells).

The APC/C^Cdc20 stabilizes aMTs through the degradation of the mitotic cyclin Clb4

The APC/C^Cdc20 complex targets proteins for proteasomal degradation (Pines, 2011); hence, to elucidate the molecular mechanism driving aMT stabilization, we searched among its substrates for the one(s) whose degradation impacts on aMT dynamics. Given that critical APC/C^Cdc20 metaphase substrates are securin (Pds1 in yeast; Cohen-Fix et al., 1996; Yamamoto et al., 1996) and cyclin B (Clb cyclins in yeast; Sullivan and Morgan, 2007), we asked whether their removal could prompt aMT stabilization in phases of the cell cycle when they are normally unstable.

As Pds1 degradation is sufficient to trigger spindle elongation and chromosome segregation in cdc20 mutants (Cohen-Fix et al., 1996; Yamamoto et al., 1996), to avoid interference by the elongated spindle, we analyzed the impact of Pds1 deletion on aMTs in cdc20 mutant cells also defective in spindle elongation (pMET-CDC20 cdc14 cdc5; Fig. S4 A). We found that the aMTs of pMET-CDC20 pds1 cdc14 cdc5 cells are similar in length and number to the ones of pMET-CDC20 cdc14 cdc5 cells, and significantly less and shorter than the ones of cdc14 cdc5 pds1 cells (Fig. S4 B), thus indicating that Pds1 removal is not sufficient to promote aMT stabilization and pointing toward other putative candidates.

Figure S4.

The individual removal of Pds1 and the majority of known or putative APC/CCdc20 substrates does not alter aMT dynamics. (A) Schematic representation of the experimental setup pertinent to this figure. (B) cdc14-1 cdc5-as1 (Ry1602), pMET-CDC20 cdc14-1 cdc5-as1 (Ry3201), pds1∆ cdc14-1 cdc5-as1 (Ry2143), and pMET-CDC20 pds1∆ cdc14-1 cdc5-as1 (Ry8969) cells were arrested in G1 with α-factor (5 µg/ml) in synthetic complete media lacking methionine and released into YEPD media lacking the pheromone and supplemented with methionine and CMK (5 µM) to repress the expression of CDC20 and to inactivate the cdc5-as1 allele, respectively. The culture was incubated at 37°C to inactivate the cdc14-1 allele. aMT length and number were analyzed at the terminal arrest (∼3.5 h after the release; for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (C) cdc20-AID and clb2∆ synthetic lethality as evidenced by tetrad dissection. -LEU means synthetic complete media lacking leucine; only cells with the clb2Δ allele can grow. G418 means YEPD added with Geneticin; only cells carrying the cdc20-AID allele can grow. (D) cdc20-AID (Ry7873), cdc20-AID kip1∆ (Ry9294), cdc20-AID acm1∆ (Ry10025), cdc20-AID dbf4-1 (Ry9877), cdc20-AID alk2∆ (Ry9880), cdc20-AID alk2∆ alk1∆ (Ry9883), cdc20-AID clb3∆ (Ry10738), and cdc14-1 cdc5-as1 (Ry1602) cells were arrested in G1 and released in YEPD into restrictive conditions. cdc20-AID dbf4-1 cells were treated as the other strains, but incubated at 37°C when the majority of the cells reached metaphase (∼2 h after the release). aMT length and number were scored at the terminal arrest. ** = P < 0.01; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test against the control strain cdc20-AID. Error bars in graphs equal SEM.

We then assessed whether Clb cyclins could be such a candidate. Since most cdc20 mutant alleles are synthetically lethal with CLB2 deletion (Costanzo et al., 2016 and Fig. S4 C), to avoid issues of synthetic lethality we tested the consequences of deleting individual cyclin subunits in cells arrested in S-phase with hydroxyurea (HU; Koç et al., 2004; Chabes et al., 2003). Of all tested cyclins, only deletion of Clb4 significantly altered aMT length and number, pinpointing Clb4 as the critical substrate of the APC/C^Cdc20 affecting aMT stabilization (Fig. 6, A and B).

Figure 6.

Systematic analysis of putative APC/C^Cdc20 substrates involved in anaphase aMT stabilization unveils the key role of B-Cyclin Clb4. (A and B) Wild-type (Ry1), clb1∆ (Ry5976), clb2∆ (Ry20), clb3∆ (Ry10927), clb4∆ (Ry10924), and clb5∆ (Ry445) cells were arrested in S-phase and analyzed at their terminal phenotype. (A) The graphs show aMT length and number of the indicated genotypes (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). Among the different clb mutants, only clb4 cells showed longer and more numerous aMTs than wild-type cells (from 1.6 µm and 2.1 aMT/cell in wild-type cells to 2.6 µm and 3.2 aMT/cell in clb4 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). (B) Representative images of wild-type and clb4∆ arrested in S-phase are shown (scale bar = 5 µm). (C) Schematic representation of the experimental setup pertinent to panel D. (D) cdc20-AID (Ry7873), cdc20-AID clb4∆ (Ry10741), and esp1-1 cdc15-as1 (Ry9512) cells were released from a G1 block into restrictive conditions and analyzed at their terminal arrest (T₁₈₀ minutes). Graphs for aMT length and number are shown. Similarly to the ones of esp1 cdc15 cells, aMTs of cdc20 clb4 cells resulted more stable than the aMTs of cdc20 cells (from 2.4 µm and 3.2 aMT/cell in cdc20 cells to 3.9 µm and 4.5 aMT/cell in cdc20 clb4 cells and 3.5 µm and 4.4 aMT/cell in esp1 cdc15 cells; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test). Error bars in graphs equal SEM. (E) pGAL-3HA-CDC20 CLB4-13myc (Ry10889) cells were arrested in S-phase with HU (10 mg/ml) in YEP media supplemented with Raffinose. Upon reaching the arrest (around 180 min from HU addition), the culture was split in two. One half was maintained in the same conditions, whereas 2% galactose was added to the other half to induce the expression of Cdc20. Clb2 and Clb4 protein levels were probed by Western blot analyses at the indicated timepoints. Pgk1 protein was used as an internal loading control in immunoblots. Size markers on the sides of the gel blots indicate relative molecular mass. Source data are available for this figure: SourceData F6.

To validate this result, we deleted CLB4 in a cdc20 background and analyzed aMTs of cdc20 clb4 cells at the metaphase arrest. We found that lack of Clb4 led to abnormally stable metaphase aMTs. In particular aMTs of cdc20 clb4 cells were as stable as the ones of esp1 cdc15 (Fig. 6, C and D). As Clb4 was never formally ascertained as an APC/C^Cdc20 substrate, we next probed Clb4 levels in: (i) wild-type cells overexpressing Cdc20 (GAL-CDC20; Visintin et al., 1997) and (ii) wild-type, cdc20, cdc15, and cdc14 cdc5 cells undergoing a synchronous cell cycle. As reported for bona fide APC/C^Cdc20 substrates (e.g., Pds1 [Visintin et al., 1997]), we found that: (i) in S-phase arrested cells, high levels of Cdc20 led to fast Clb4 degradation, while Clb2, whose degradation is mainly dependent on the APC/C^Cdh1 complex (Visintin et al., 1997; Schwab et al., 1997), remained stable and eventually accumulated (Fig. 6 E); and (ii) Clb4 protein levels remained high in cdc20 cells, but dropped when cells entered anaphase in wild-type, cdc14 cdc5, and cdc15 cells (Fig. 7, A–C). Additionally, the kinetics of Clb4 degradation resembled that of Pds1 degradation and differed from that of Clb2 degradation, which has delayed degradation with respect to Clb4 in cycling cells (Fig. 7 B); and was only partially degraded in anaphase arrested cells (Fig. 7 C; Baumeret al., 2000).

Figure 7.

B-Cyclin Clb4 is degraded at anaphase onset. (A) Schematic representation of the experimental setup pertinent to this figure. (B) Wild-type (Ry10891) cells expressing Clb4-13myc were arrested in G1 and synchronously released into fresh YEP media with glucose. At the indicated time points, the percentage of cells containing metaphase (light blue circles) and anaphase (dark blue circles) spindles was determined (n = 100 cells), and protein samples were taken to follow Clb4, Pds1, and Clb2 protein levels. (C) cdc20-AID (Ry10967), cdc14-1 cdc5-as1 (Ry10973), and cdc15-as1 (Ry10970) cells expressing Clb4-13myc were synchronized in G1 with α-factor (5 µg/ml) and synchronously released into restrictive conditions. At the indicated time points, the percentage of cells containing metaphase (light blue circles) and anaphase (dark blue circles) spindles was determined (n = 100 cells) and protein samples were taken to probe Clb2 and Clb4 protein levels by Western blot analyses. Pgk1 was used as a loading control. Size markers on the sides of the gel blots indicate relative molecular mass. Source data are available for this figure: SourceData F7.

To complete our analysis and to exclude the contribution of other APC/C^Cdc20 targets, we also tested the kinesin motor protein Kip1 (Gordon and Roof, 2001), the Haspin-like kinase Alk2 (Nespoli et al., 2006), the APC/C^Cdh1 inhibitor Acm1 (Enquist-newman et al., 2008), and the DNA replication-promoting kinase Dbf4 (Ferreira et al., 2000). Individual proteins were depleted in a cdc20 mutant background, and the resulting double mutants were next investigated for aMT morphology. Of all the proteins tested, only deletion of Kip1 or Acm1 exhibited aMTs with an intermediate phenotype—slightly increased in length than their cdc20 counterpart (Fig. S4, A and D).

The findings that: (i) aMTs of cdc20 clb4 cells phenocopy the ones of esp1 cdc15, (ii) this phenotype is recapitulated only by the removal of Clb4, and finally (iii) Clb4 is an APC/C^Cdc20 substrate, identify Clb4 as the main target of the APC/C^Cdc20 whose degradation is required for aMT stabilization in anaphase and highlight a unique and specialized function of Clb4 among cyclin subunits.

Multiplexed quantitative phospho-proteomics associates aMT stabilization to de-phosphorylation

To gain insights into the complex molecular mechanism underlying aMT dynamics, we interrogated the proteome and phospho-proteome of cdc20, cdc14 cdc5, and cdc15 cells representing the metaphase, mini-anaphase, and anaphase cell cycle phase, respectively. Samples were collected at the terminal phenotype and subjected to tandem mass tags-based multiplexed quantitative phospho-proteomics. Our dataset covered 4,641 proteins (Table S3) and identified 5,324 phospho-sites (Table S4). To validate our experimental setup, we looked at CDK1 substrates (Fin1 [Bouchoux and Uhlmann, 2011], Ase1 [Khmelinskii et al., 2007], and Orc6 [Bouchoux and Uhlmann, 2011]) whose de-phosphorylation status is known to change from the metaphase-to-anaphase transition and throughout anaphase with different kinetics (Fig. S5, A–C).

Figure S5.

The phospho-proteomic analyses unveiled numerous residues that may be involved in anaphase aMT stabilization and recapitulated the de-phosphorylation kinetic of different known CDK substrates. (A–C) As a proof of concept that our phospho-proteomic analysis is a good proxy for cell cycle progression, we hand-picked and scored the phosphorylation status of residues in three CDK substrates, namely Fin1 (A), Ase1 (B), and Orc6 (C), whose kinetics of de-phosphorylation differs in anaphase. More precisely, we identified two out of five putative CDK1 residues reported for Fin1 (S36, T68), four out of seven for Ase1 (T55, S64, S198, S803), and three out of four for Orc6 (S106, S116, T146). Consistent with Fin1 being de-phosphorylated in early anaphase, Ase1 in mid-anaphase, and Orc6 at mitotic exit, when we compared the cdc20 and cdc15 datasets, we found that all Fin1 residues, two out of four Ase1 residues, and no Orc6 residues were de-phosphorylated. Moreover, when the cdc14 cdc5 dataset was included in the analysis and compared to the other two datasets individually, the residues whose de-phosphorylation is Cdc14-dependent were appreciated (i.e., Ase1S803, whose phosphorylation status is high in both cdc20 and cdc14 cdc5 cells, but low in cdc15 cells). Above each graph, a schematic representation shows the identified putative CDK-phosphorylated residues covered by our analysis (highlighted in red), and the residues that were not identified in the dataset (highlighted in black). The graphs show the log₂ fold change (FC) of each residue generated comparing each mutant with each other. The log₂ fold changes of each residue are normalized by the log₂ fold changes of the respective proteins to account for changes in protein abundance. (D and E) The log₂ fold changes of the phospho-residues obtained with this analysis were plotted against the log₂ fold changes of the corresponding proteins. The graph in D is pertinent to the global analysis shown in Fig. 8 A, while the one in E is pertinent to the global analysis shown in Fig. 8 B. (F) The graph shows the phospho-residues that belong to proteins already linked to the regulation of aMT dynamics and their log₂ fold changes as calculated above and normalized by the log₂ fold change of the protein to account for protein abundance change. Only residues with a log₂ fold change of at least 0.4 and a significant P value are shown. The residues that belong to the minimal CDK consensus are shown in red. ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001.

Having established that the chosen mutants are a good proxy for the mitotic cell cycle phases of interest (Fig. S5, A–C), we assessed global phosphorylation changes. We identified 974 phosphosites that changed their status when going from metaphase to anaphase, as assessed by comparing cdc20 with cdc15 cells. Among the 974 phosphosites, 197 were phosphorylated (69 of which reside in the minimal CDK1 consensus site) and 777 were de-phosphorylated (of which 407 are putative CDK substrates; Fig. 8 A). Since aMT stabilization occurs at anaphase but is independent of Cdc14 and Cdc5, we restricted the analysis by comparing the phospho-proteome of cdc20 cells with the combination of the datasets of cdc14 cdc5 and cdc15 cells. By these means, we identified 340 phospho-sites that changed from metaphase to anaphase, of which 81 were phosphorylated (34 of which reside in the minimal CDK1 consensus site) and 259 were de-phosphorylated (of which 160 are putative CDK substrates; Fig. 8 B). The changes of these phosphorylation sites were unlikely caused by changes in protein abundance as the respective protein amounts remained largely unchanged (Fig. S5, D and E). Taken together, our data evidence a bias towards de-phosphorylation for anaphase cells.

Figure 8.

Modeling the metaphase-to-anaphase transition by phospho-proteomics unveiled a switch toward de-phosphorylation. (A and B) cdc20-AID (Ry4853), cdc14-1 cdc5-as1 (Ry1602), and cdc15-as1 (Ry1112) cells were arrested in G1 and released into restrictive conditions. Cells were harvested at their terminal arrest (T₁₈₀ minutes) to extract proteins to process for TMT MS analysis—three biological replicates for cdc20-AID and cdc14-1 cdc5-as1 cells and two biological replicates for cdc15-as1 cells. The volcano plot represents the log₂ fold change (FC), and the respective –log₁₀ P values were calculated comparing the values obtained for each phospho-residue in cdc20-AID and cdc15-as1 cells. To identify the phospho-residues that were significantly different between the two mutant cells (blue dots), an FDR of 0.001 (S0 at 0.05) was applied, with an additional cut-off of a log₂ fold change <−0.5 or >0.5. (C) Schematic representation of the kinesin protein Kip2; highlighted in red are the Kip2 residues with a CDK1 (S/TP) binding motif, in yellow the one with the GSK3 consensus motif (multiple residues within a continuous S/TxxxS/T pattern; adapted from Drechsler et al. [2015]). Of note, S63 and T275 are the residues mutated in the kip2-2A allele. S28 is a residue dephosphorylated at anaphase onset, identified by our analysis. (D) Schematic representation of the experimental setup pertinent to panel E. (E) cdc20-AID kip2∆ pCEN-KIP2 (Ry10858), cdc20-AID kip2∆ pCEN-KIP2S63AT275A (Ry10861), cdc14-1 cdc5-as1 kip2∆ pCEN-KIP2 (Ry10857), cdc14-1 cdc5-as1 kip2∆ (Ry10844), and cdc14-1 cdc5-as1 (Ry1602) cells were released from a G1 block into restrictive conditions and analyzed at their terminal arrest (T₁₈₀ minutes). aMT length and number are shown in the graphs (for aMT length, n = 100 aMTs; for aMT number, n = 100 cells). Similarly to cdc14 cdc5 kip2 pCEN-KIP2 cells, cdc20 kip2 pCEN-KIP2-2A showed longer and more numerous aMTs than cdc20 kip2 pCEN-KIP2 cells (from 1.9 µm and 2.1 aMT/cell in cdc20 kip2 pCEN-KIP2 cells to 3.9 µm and 3.6 aMT/cell in cdc20 kip2 pCEN-KIP2-2A and 3 µm and 3.2 aMT/cell in cdc14 cdc5 kip2 pCEN-KIP2 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. On the same line, cdc14 cdc5 kip2 cells showed shorter and less aMTs than cdc14 cdc5 cells (from 3.9 µm and 4.5 aMT/cell in cdc14 cdc5 cells, to 1.3 µm and 1.6 aMT/cell in cdc14 cdc5 kip2 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. Error bars in graphs represent SEM. (F) clb4∆ pCEN-KIP2 (Ry10963), pGAL-3HA-CLB4 pCEN-KIP2 (Ry10894), clb4∆ pCEN-KIP2S63AT275A (Ry10965), and pGAL-3HA-CLB4 pCEN-KIP2S63AT275A (Ry10896) were arrested in S-phase. At the arrest samples were taken at the indicated times to probe Kip2 and Clb4 levels and mobility. Pgk1 was used as an internal loading control in immunoblots. Size markers on the sides indicate relative molecular mass. (G) cdc20-AID pCEN-KIP2 (Ry10955), cdc20-AID clb4∆ pCEN-KIP2 (Ry10959), cdc20-AID pCEN-KIP2S63AT275A (Ry10957), and cdc20-AID clb4∆ pCEN-KIP2S63AT275A (Ry10961) cells were arrested in G1 and released into restrictive conditions. Samples were taken at the indicated times for Western blot analyses. Kar2 is an internal loading control. Size markers on the sides of the gel blots indicate relative molecular mass. Source data are available for this figure: SourceData F8.

To identify possible regulators of aMT dynamics, we scrutinized the phosphorylation status of residues within proteins known to be involved in microtubule regulation (de Gramont et al., 2007; Yeh et al., 2000; Kosco et al., 2001; Blake-Hodek et al., 2010), namely: (i) five kinesin motor proteins (Cin8 [Hoyt et al., 1992], Kip1 [Hoyt et al., 1992], Kip2 [Roof et al., 1992], Kip3 [DeZwaan et al., 1997], and Kar3 [Meluh and Rose, 1990]), (ii) the single minus-end directed Dyn1 protein (Li et al., 1993), and (iii) five MAPs (Bim1 [Schwartz et al., 1997], Bik1 [Berlin et al., 1990], Ase1 [Pellman et al., 1995], Stu1 [Pasqualone and Huffaker, 1994], and Stu2 [Wang and Huffaker, 1997]). Since the activity of these proteins is often modulated by microtubule-cortex interaction (Yeh et al., 2000; Omer et al., 2018; Hoopen et al., 2012), we extended our analysis to proteins that mediate the connection between aMTs and the cellular cortex, such as the cortical receptor Num1 (Farkasovsky and Küntze, 1995), the actin motor protein Myo2 (Beach et al., 2000), and the actin nucleator Bni1 (Yeh et al., 2000). Interestingly, we found that Cin8, Kip2, Bim1, Bik1, Ase1, Stu1, Num1, Myo2, and Bni1 showed at least one residue with a different phosphorylation status between metaphase and anaphase arrested cells with a bias in favor of de-phosphorylation in anaphase (Fig. S5 F).

To validate our analysis and as a proof of concept we chose Kip2, a kinesin with a microtubule polymerization function (Fig. 8 C). Kip2 is inhibited by GSK3-dependent phosphorylation. This event likely requires priming at the S63 and T275 Kip2 sites by an additional kinase, of which CDK1 is one candidate (Drechsler et al., 2015; Chen et al., 2019). Notably, the S28 residue highlighted by our phospho-proteomic analysis falls into a stretch of residues within the GSK3 consensus motif (Fig. 8 C). To test if CDK-mediated phosphorylation of Kip2 contributes to keep aMTs unstable in metaphase, we, first, probed aMTs in cdc20 kip2-S63AT275A mutant cells (henceforth kip2-2A) and found that they pheno-copy the ones of cdc14 cdc5 double mutant cells (Fig. 8, D and E). Next, we confirmed that anaphase stabilization of aMTs requires Kip2 activity, as deleting KIP2 in cdc14 cdc5 double mutant cells reduced both aMT length and number (Fig. 8, D and E). To establish if Kip2 is a substrate of Clb4, we compared the phosphorylation profiles of Kip2 wild-type and 2A allelic variants in S-phase-arrested cells following Clb4 overexpression. Since we didn’t observe significant differences when protein samples were run on regular acrylamide gels, to exacerbate differences among the phospho-species, we moved to Phos-tag gels. We found that high levels of Clb4 alter the phosphorylation pattern of both Kip2 allelic variants (Fig. 8 F). Indeed, we observed the appearance of a slower migrating smear above wild-type Kip2 and the disappearance of one fast migrating band (band b) concomitantly with the enrichment of a slower migrating one (band c) in Kip2-2A. Next, we compared the phosphorylation profiles of the two Kip2 allelic variants in cdc20 metaphase-arrested cells in the presence or absence of Clb4 at physiological levels (Fig. 8 G). Deletion of CLB4 led to the appearance of a faster migrating band (band a) in wild-type Kip2. In Kip2-2A, when Clb4 was present, the phosphorylation profile of the kinesin pheno-copied the one triggered by Clb4 overexpression, vice versa when the cyclin was absent the slower migrating band (band c) collapsed with concomitant increase of the faster one (band b; Fig. 8 G). Taken together these data identify Kip2 as a bona fide Clb4 substrate and pave the way for further testing of putative substrates/sites within other candidates including Ase1 (S834), Myo2 (T1097), and Bni1 (T1918).

Dynamic aMTs and Cdc5 guide proper spindle orientation

The identification of Clb4 as central to the regulation of aMT dynamics contributes to our understanding of the mechanism by which budding yeast properly aligns its mitotic spindle along the mother-bud axis, an essential requisite for survival. Schiebel and colleagues proposed that Clb4 in complex with CDK1 facilitates spindle alignment by mediating the interactions of aMTs with the bud cortex and enables the turnover of established aMT-bud cortex attachments (Maekawa and Schiebel, 2004). Our data are now suggesting that it does so by controlling aMT stability. This model allows clear predictions: (i) stable microtubules should exhibit an increased residence time at the cortex and (ii) premature aMT stabilization should result in an increased number of wrong attachments.

To assess if the increased aMT stability observed in cdc14 cdc5 double mutant cells reflects changes in the dynamics of aMT-cortex interactions, we analyzed the behavior of aMTs at the cellular cortex in cdc20 and cdc14 cdc5 arrested cells, and measured the time that each individual aMT spent in proximity of the cellular cortex. In agreement with our hypothesis, aMTs of cdc14 cdc5 cells remained close to the cellular cortex longer than aMTs of cdc20 cells, thus suggesting that the dynamics of aMT-cortex connections are altered in double mutant cells (Fig. 9, A and B).

Figure 9.

Dynamic aMTs and Cdc5 guide spindle positioning. (A and B) cdc20-AID (Ry7732) and cdc14-1 cdc5-as1 (Ry3256) cells harboring a TUB1-GFP fusion were synchronized in G1 and released into restrictive conditions. Approximately 3 h after the release, cells were moved to a CellASIC ONIX plate for live imaging. (A) Time-lapse images from a representative cell are shown for the cdc20-AID and cdc14-1 cdc5-as1 strains (scale bar = 1 µm). Black arrows indicate the frames when the highlighted aMTs were in close proximity with the cellular cortex. (B) The bar charts show the quantification of the time that each aMT spent in proximity with the cortex. On average, aMTs of cdc14 cdc5 and cdc20 cells remained close to the cortex during 77 and 39% of the analyzed time, respectively (n = 10 aMTs in cdc20-AID cells and n = 15 aMTs in cdc14 cdc5 cells). **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test. (C) Schematic representation of the experimental setup pertinent to panels E and F. (D) Representative images of cells with proper or anomalous aMTs, a distinction based on aMT direction, are shown (scale bar = 2 µm). (E) cdc20-AID (Ry4853), cdc14-1 cdc5-as1 (Ry1602), esp1-1 cdc15-as1 (Ry9512), and cdc20-AID clb4∆ (Ry10741) cells were synchronized in G1 and released into restrictive conditions. Cells were analyzed at their terminal arrest (T₁₈₀ minutes). The bar charts show the quantification of cells with anomalous aMTs (as shown in D) in the four strains. On average, 22% of cdc20 cells, 58% of cdc14 cdc5, 47% of esp1 cdc15 cells, and 50% of cdc20 clb4 cells showed anomalous aMTs (n = 100 cells). * = P < 0.05; ** = P < 0.01; *** = P < 0.001; asterisks denote significant differences according to two-tailed unpaired t test. (F) cdc20-AID (Ry4853), cdc14-1 cdc5-as1 (Ry1602), esp1-1 cdc15-as1 (Ry9512), cdc20-AID clb4∆ (Ry10741), pMET-CDC20 cdc5-as1 (Ry3209), pMET-CDC20 cdc14-1 cdc5-as1 (Ry3201), pMET-CDC20 cdc14-1 (Ry3204), esp1-1 cdc14-1 (Ry9131), esp1 cdc5-as1 (Ry9134), and esp1-1 cdc14-1 cdc5-as1 (Ry9128) cells were treated as in E. The dot plot shows the bud-neck/spindle angles measured in the cells of the indicated genotype (n = 100 cells). On the right, a schematic representation of the angles generated by the bud-neck and the spindle, an indicator of spindle orientation, is shown. The closer the angle is to 90°, the more the spindle is properly oriented toward the mother-bud axis; vice versa, the more the angle is close to 0°, the more it is misoriented. Each data point in the dot plot represents a single cell. Note that an average angle of 45° means a complete randomization of spindle orientation. * = P < 0.05; **** = P < 0.0001; asterisks denote significant differences according to ordinary One-Way ANOVA and Tukey’s multiple comparisons test, using cdc20-AID cells as control. Error bars in graphs represent SEM.

To assess if premature aMT stabilization favors wrong attachments, we probed aMT directionality. Proper cortical attachments foresee that aMTs originating from the bud-directed SPB enter the bud, while the others point to the cortex of the mother cell. If stabilization prevents error correction, then mutants with prematurely stable microtubules should manifest directionality problems, with aMTs originating from two different SPBs pointing to the same cellular compartment, and aMTs originating from a single SPB pointing to both mother and daughter cells (in our analysis, both categories were grouped as abnormal aMTs). Consistent with our hypothesis, we found that all mutants with stable aMTs (cdc20 clb4, esp1 cdc15, and cdc14 cdc5) showed an increased percentage of abnormal aMTs when compared to cdc20 mutants (Fig. 9, C–E).

Since proper attachment of aMTs with the bud cortex directs the spindle toward the emerging bud and orients it to the polarity axis (Shaw et al., 1997; Maddox et al., 2000; Segal et al., 2002), we asked whether anomalous aMTs affect spindle orientation. We probed spindle orientation by measuring the angle formed by the spindle and the bud-neck in cdc20, cdc14 cdc5, esp1 cdc15, and cdc20 clb4 cells. We found that, while most metaphase arrested cells correctly orient their spindle, cdc14 cdc5 cells do so in a random manner suggesting that stable aMTs may lead to spindle positioning defects. The finding that in esp1 cdc15 and cdc20 clb4 cells spindles were properly oriented indicates that aMT stability may predisposes to, but it is not sufficient to compromise spindle orientation (Fig. 9 F). Since among the tested mutants only cdc14 cdc5 manifested spindle orientation defects, we wondered if Cdc14 and/or Cdc5 inactivation could account for the phenotype. To test this hypothesis, we analyzed spindle orientation in cdc20 cdc14, cdc20 cdc5, and cdc20 cdc14 cdc5 cells, in which aMTs are unstable, and in esp1 cdc5, esp1 cdc14 cdc5, and cdc20 clb4 cdc5 cells, in which aMTs are stable. We found that all tested strains with unstable aMTs or with stable aMTs but active Cdc5 carry properly oriented spindles, instead cells in which aMTs are stable and Cdc5 is inactivated, showed orientation defects (Fig. 9 F). These data indicate that unstable aMTs and Cdc5 activity are redundantly required for proper spindle orientation.

Discussion

Despite the importance of aMTs in controlling spindle stability and orientation, their regulation remains largely understudied. Here we report that in budding yeast, similarly to kMTs and iMTs, aMT dynamics are also intrinsically regulated in a cell cycle dependent manner. More precisely, they remain unstable up to metaphase and are stabilized as soon as cells enter anaphase. This finding is consistent with their role in searching for cortical anchor sites and correcting erroneous attachments in metaphase and next in stabilizing the binding of the mitotic spindle with the cortex to properly guide spindle positioning and elongation in anaphase.

Our study in budding yeast identifies two evolutionary conserved mitotic machineries, the Clb4-CDK1 and APC/C^Cdc20 complexes as central regulators of aMT dynamics. The presence of Clb4 is sufficient to render aMTs unstable up to metaphase, likely by introducing one or multiple inhibitory phosphorylation events in factors affecting microtubule stability, of which Kip2 is an example. Vice versa, aMT stabilization, observed at anaphase, is dictated by the APC/C^Cdc20-mediated degradation of the mitotic cyclin Clb4. Of note, this function of Clb4 in aMT regulation is unique as its removal is sufficient to stabilize aMTs in cell cycle phases when they are normally unstable, whereas the same does not stand true for the other mitotic cyclins. Finally, aMT stability in late anaphase is counteracted by a mechanism that likely integrates two anaphase specific requirements, namely spindle elongation and stable aMT-cortex links.

The identification of a kinase as central to aMT regulation immediately calls for the phosphatase required to reverse these Clb4-CDK1–mediated phosphorylation events. If in yeast the main mitotic CDK-counteracting phosphatase is Cdc14, our data indicate that at least one additional phosphatase is involved. A possible candidate is Glc7 (the sole PP1 catalytic subunit in yeast) in combination with its regulatory subunit Bud14. Indeed, overexpression of Bud14 increases aMT length in a way reminiscent of cells lacking Clb4 (Knaus et al., 2005). The involvement of the Glc7-Bud14 complex is also supported by its localization. Both Glc7 and Bud14 accumulate at bud cellular cortex, where they could counteract the activity of the Clb4-CDK1 complex, which mainly localizes at the plus-end of aMTs directed toward the bud (Ni and Snyder, 2001; Knaus et al., 2005; Maekawa and Schiebel, 2004). Importantly, this interplay between Clb4-CDK1 and Bud14-Glc7 at the bud cellular cortex would not only satisfy the requisite of the yet-to-identify “spatial cue” necessary for the specific stabilization of bud-directed aMTs as reported by Barral and colleagues (Lengefeld et al., 2018), but also integrates intrinsic (e.g., cell cycle regulation of cyclin levels) and extrinsic signals (e.g., interaction between bud-directed aMTs and the bud cellular cortex).

The mechanisms of regulation of aMT dynamics by CDK1 and APC/C^Cdc20 activities that this work identified in yeast are likely retained in vertebrates. At least three pieces of evidence support this hypothesis. First, in all vertebrates CDK1-CyclinB activity is high in metaphase and decreases at anaphase onset due to APC/C^Cdc20-mediated degradation of the cyclin B subunit (Sullivan and Morgan, 2007). Similarly to the Clb4-CDK1 complex in yeast, CDK1-CyclinB activity destabilizes aMTs in prometaphase by phosphorylating the EB1-dependent microtubule plus-end tracking protein GTSE1 in human cells (Singh et al., 2021). Second, in mammalian cells, Dyn1 activity is low in metaphase and high in anaphase, and, since, in budding yeast, the increase in Dyn1 activity is directed by aMT stabilization, it is possible that aMTs are stabilized in anaphase also in vertebrates (Kiyomitsu and Cheeseman, 2012; Kiyomitsu and Cheeseman, 2013; Kotak et al., 2013; Estrem et al., 2016). Third, aMTs are often reported to shrink during late anaphase in human cells, thus suggesting that a mechanism counteracting aMT stability connected to spindle elongation/cortex proximity may also exist in these cells.

This work identifies budding yeast Cdc5 as a novel regulator of spindle positioning redundant to aMT stabilization. Interestingly, Plk1, the human homologue of Cdc5, has also been intensively linked to the regulation of spindle positioning (di Pietro et al., 2016). Indeed, Plk1-dependent phosphorylation of several components of the cortex platform that bind to aMTs is required to correctly orient and position the spindle along the cleavage plane (Bergstralh et al., 2017). Investigating how Cdc5 influences these processes may further clarify the role of this kinase in multi-cellular eukaryotes and shed light into how it coordinates spindle positioning with the plethora of mitotic events under its control. Taken together, our work legitimizes yeast as an optimal model system to investigate aMT regulation and spindle positioning at molecular level. Besides the fact that each yeast cell has few aMTs (1–6 aMTs per cell; Shaw et al., 1997), easily distinguishable from other types of microtubules and singularly traceable over time (Fees et al., 2017), our mass spectrometric analysis clearly illustrates the advantages of exploiting the power of yeast genetics to clarify complex molecular processes.

An important corollary of our study in yeast is uncovering the APC/C^Cdc20 complex as the master “choreographer” of late mitotic events, where it precisely coordinates, in time and space, the sequence of events required for the faithful execution of mitosis by sequentially impacting on the stability of the three classes of spindle microtubule: astral, kinetochore, and interpolar. More precisely, active APC/C^Cdc20 targets several substrates for degradation, including Clb4, Pds1, and other mitotic cyclins (Fig. 10, Step 1). Degradation of Clb4 is the signal for aMT stabilization that drives proper spindle positioning (Fig. 10, Step 2). Instead, Pds1/Securin degradation unleashes the protease Esp1/Separase (Fig. 10, Step 2). Esp1 has a dual role. On the one hand it indirectly affects kMT dynamics by cleaving the cohesin subunit Scc1 (Uhlmann et al., 1999; Baskerville et al., 2008) that leads to chromosome separation and movement to the poles (Asbury, 2017; Fig. 10, Step 3). On the other hand, as a component of the cdc14 Early Anaphase Release (FEAR) network (Stegmeier et al., 2002), it promotes the transient activation of Cdc14 (Fig. 10, Step 3). In turn, Cdc14 acts on motors and MAPs to stabilize iMTs (Khmelinskii and Schiebel, 2008; Roccuzzo et al., 2015; Higuchi and Uhlmann, 2005), thereby prompting spindle elongation and ultimately chromosome segregation (Fig. 10, Step 4). The time delay imposed by the incremental number of molecular events impacting specific classes of spindle microtubules guarantees that proper spindle positioning precedes sister chromatid separation (cohesin cleavage) which in turn precedes their segregation (spindle elongation; Fig. 10).

Figure 10.

A model for the timely regulation of late mitotic events by the APC/C^Cdc20 in S. cerevisiae. Proper rearrangement of spindle microtubule dynamics coordinates sister chromatid segregation with late mitotic events. The APC/C^Cdc20 directs this process by triggering a signaling cascade in which individual steps lead to the regulation of a single class of spindle microtubules. Step 1: The APC/C^Cdc20 targets for degradation different substrates, including the mitotic cyclin Clb4 and the securin Pds1. Step 2: Clb4 degradation by de-phosphorylating—directly or indirectly—the kinesin-like protein Kip2 promotes aMT stabilization. Instead, Pds1 degradation unleashes the separase Esp1. Step 3: Esp1 cleaves the cohesin subunit Scc1 and activates the Cdk-counteracting phosphatase Cdc14; meanwhile, kMTs retract to the poles. Step 4: Cdc14-mediated de-phosphorylation of different motors and MAPs triggers stabilization of iMTs.

Several observations suggest that this logic of mitotic progression stands true in vertebrates. While clear differences exist between budding yeast and multi-cellular eukaryotes mitosis (i.e., budding yeast cells do not break the nuclear envelope during cell division, DNA is only partially condensed, etc.), we already mentioned data supporting a regulation of aMTs mediated by CDK1 activity (Singh et al., 2021). In respect to chromosome movement toward the poles (anaphase A), evidence exists that kMT de-polymerization is coupled to cohesin cleavage (Oliveira et al., 2010). Finally, spindle elongation, hence chromosomes segregation, relies on the assembly of the central spindle (the Metazoa equivalent of the yeast spindle midzone), and this process requires, as in yeast, iMT stabilization and bundling. The latter is promoted by the centralspindlin complex comprising the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP), which in turn is regulated by phosphorylation and de-phosphorylation events of the kinesin motor domain (Mishima et al., 2004). In yeast, the phospho-regulation controlling spindle midzone assembly is mediated by a proline-directed kinase-phosphatase switch, with Cdc14 being a likely candidate (Mishima et al., 2004). Interestingly, the two human homologues of Cdc14 bind to microtubules and promote both their stabilization and bundling activity during mitosis (Cho et al., 2005). This finding in addition to the knowledge that, in human cells, a CDK counteracting phosphatase(s), other than Cdc14 homologues, whose activation necessitates proteasomal activity (Skoufias et al., 2007), drives mitotic exit, makes it likely that the stepwise regulation of late mitotic events dictated by the APC/C^Cdc20 is an evolutionary conserved mechanism of regulation.

Materials and methods

Yeast strains

All strains used in this study are isogenic to W303 and are listed in Table S2.

Growth conditions

Cell cycle arrest and synchronization experiments were performed as previously described (Amon, 2002). Yeast cells were grown in yeast extract peptone (YEP) media unless differently specified in the figure legend. Carbon sources (glucose, raffinose, or galactose) were used at a final concentration of 2%. In all experiments cells were pre-arrested in G1 phase with 5 µg/ml α-factor (RP01002; Genscript) and released in a synchronous cell cycle or into the arrest of interest. To release cells from G1, cells were washed with 10 volumes of fresh medium and transferred into medium lacking the pheromone. When pertinent, drug(s) were added and restrictive conditions applied.

In detail: Temperature-sensitive alleles were inactivated by incubating the culture at the restrictive temperature of 37°C. cdc5-as1 and cdc15-as1 alleles were inhibited by adding to the media 5 µM of CMK (custom-made, Accendatech; Snead et al., 2007) or of 1NM-PP1 analogue 9 (A603003; Toronto Research Chemicals), respectively. Cells carrying an AID degron were inactivated with Auxin (I5148; Sigma-Aldrich) at a final concentration of 500 µM. Methionine (M9625; Sigma-Aldrich) was added at a final concentration of 8 mg/ml to inactivate the MET-CDC20 construct. HU (H8627; Sigma-Aldrich) was used at a final concentration of 10 mg/ml to arrest cells in S-phase.

Indirect in situ immunofluorescence

1 ml of cell culture OD₆₀₀ = ∼0.4–0.6 was incubated overnight in fixing solution (3.7% formaldehyde in 0.1 M potassium phosphate buffer, pH 6.4) at 4°C. Next, cells were washed three times in 0.1 M potassium phosphate buffer and once in sorbitol buffer (1.2 M sorbitol, 0.33 M citric acid, pH 5.9). Cells were lysed with 0.1 mg/ml Zymolyase 100 T in sorbitol buffer for ∼30 min at 30°C. When digestion was complete spheroplasts were washed in sorbitol buffer and deposited on a poly-L-lysine coated multi-wells slide for 10 min. Slides were soaked in −20°C cold methanol for 3 min and in −20°C cold acetone for 10 s.

For spindle microtubule visualization spheroplasts were: (a) incubated 90 min with rat anti-tubulin alpha YOL34 monoclonal antibody (MCA78G; AbD Serotec) diluted 1:100 in PBS/BSA (1% crude BSA, 0.04 M K₂HPO₄, 0.01 M KH₂PO₄, 0.15 M NaCl, 0.1% NaN₃); (b) washed five times in PBS/BSA; (c) incubated 90 min with FITC-conjugated donkey anti-rat antibody (712-095-153; Jackson ImmunoResearch Laboratories) used at 1:100 dilution; (d) washed five times in PBS/BSA; and (e) added with a DAPI mounting solution (90 ml of glycerol 100%, 10 ml of KPBS, 100 mg of p-phenylenediamine, 5 μg of DAPI). The KPBS solution is 0.04 M K₂HPO₄, 0.01 M KH₂PO₄, 0.15 M NaCl. Slides were closed with a cover slip and sealed with nail polish (Senic-Matuglia and Visintin, 2017).

Astral microtubule analysis in fixed cells

Images of stained cells were acquired at room temperature with an upright LEICA DM6 B microscope with a 100X/1.40 oil UPlanSApo ∞/0.17/DFN 25 Olympus objective and Andor Zyla.4.2P camera using Leica Application Suite X software. Optimized z-stacks were taken to cover a thickness of 6.1 µm. Image analysis was performed using the “Fiji Is Just ImageJ” (FIJI) software. aMT length was measured in three dimensions using the FIJI plug-in “simple neurite tracer.” To properly score aMT number, the presence of abnormal aMTs and the bud-neck/spindle angles, Z-series were collapsed into a maximum-intensity two-dimensional projection using the Z-project function. Note that the aMT number is likely underestimated due to the difficulty to separate two or more aMTs (bundle) close to each other. The bud-neck was defined based on the Differential Interference Contrast (DIC) image of the cell.

Astral microtubule analysis in live cells

50 µl of arrested cells carrying a GFP-tagged Tub1 fusion were collected at OD₆₀₀ = 0.2–0.4 and loaded in a CellASIC ONIX plate for haploid yeast cells (Millipore). This procedure allows a constant addition of fresh medium to the culture and prevents cellular movements during image acquisition. Cells were incubated in filtered YEP media with glucose (YEPD) media at 37°C. Images were acquired every 10 s for a total of 5 min using a Nikon Eclipse Ti inverted microscope with a 100X/1.40 oil Olympus objective and an Andor Zyla sCMOS camera using the NIS software version 5.10.00. At each time point, 17 z-stack images were taken (0.4 µm apart) covering a total thickness of 6.4 µm. Image acquisition was followed by a deconvolution process automatically performed by the Huygens software. Image analysis was performed using FIJI. To reduce the complexity and the noise of the analysis, aMT length measurements were performed using the FIJI plug-in “simple neurite tracer” in maximum-intensity two-dimensional projection using the Z-project function. As described in Fees et al. (2017), different events were identified: (i) polymerization events—defined as an increase in microtubule length by at least 0.5 µm across a minimum of three time points; (ii) depolymerization events—defined as a decrease in microtubule length by at least 0.5 µm across a minimum of three time points; and (iii) pause events—defined as net changes in microtubule length <0.5 µm across a minimum of three time points. Next, catastrophe frequencies were calculated by dividing the number of polymerization/pause after polymerization-to-depolymerization transitions by the total time of all growth events. Rescue frequencies were calculated by dividing the number of depolymerization/pause after depolymerization-to-polymerization transitions by the total time of all shrinkage events. Polymerization rates were calculated by dividing the net change in length by the change in time for each polymerization event. Depolymerization rates were calculated by dividing the net change in length by the change in time for each depolymerization event. Dynamicity was calculated by dividing the sum of the absolute value of all length changes (in µm) by the total duration of the image acquisition (in seconds), and by multiplying the resulting values by 1690 (number of tubulin subunits corresponding to 1 µm of microtubule length) to obtain tubulin subunits per second (Toso et al., 1993). aMT-cortex connection was evaluated based on the DIC image of the cell.

Definition of astral microtubule stability

In the literature, the term “stability” is often used to express two slightly different concepts: (i) the tendency of aMTs to polymerize and elongate, instead of depolymerize and shorten; and (ii) the capacity of aMTs to maintain a determined length over time, without growing or shrinking. The first one can be depicted by different indicators, like aMT length and number in fixed cells, or the maximum length reached by each single aMT in live cell imaging. The second one can be measured using the parameter “dynamicity” in live cell imaging, which indicates the net exchange of tubulin subunits over time—independently of polymerization or depolymerization. In this manuscript, the term “stability” refers to the first definition.

Immunoblot analysis

Cells were lysed in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM p-nitrophenyl phosphate, 50 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride, and 2 µg/ml pepstatin with glass beads and boiled in 1× sample buffer. The primary antibodies used were: mouse 9E10 monoclonal anti-myc (CVMMS-150R-1000, used at 1:1,000 dilution; Covance) to detect Clb4-13myc and Kip2-13myc; mouse 16B12 anti-HA (901515, used at 1:1,000; Bio Legend) to detect 3HA-Clb4; rabbit anti-Clb2 (Y-180; SC-9071, used at 1:1,000 dilution; Santa Cruz Biotechnology) for Clb2; mouse anti-Pgk1 (A-6457, used at 1:5,000 dilution; Molecular Probes) for Pgk1; rabbit anti-Pds1 (used at 1:1,000 dilution) for Pds1 was a kind gift of Adam Rudner (Ottawa Institute of Systems Biology, Ottawa, Canada); rabbit anti-Kar2 (used at 1: 200,000 dilution) for Kar2 was a kind gift of Mark D. Rose (Princeton University, Princeton, NJ); goat anti-rabbit IgG (H + L)–HRP conjugate (170-6515, used at 1:5,000 dilution; Bio-Rad) and goat anti-mouse IgG (H + L)–HRP conjugate (170-6516, used at 1:10,000 dilution; Bio-Rad) were used as secondary antibodies to visualize proteins using chemiluminescence (ECL; GE Healthcare). Phos-tag gels were obtained adding Phos-tag AAL-107 (304-93521, Fujifilm Wako Chemicals Europe GmbH, used at the final concentration of 50 µm) and MnCl₂ 10 mM in 6% Acrylamide gels.

Statistical analysis

In Figs 1 C, 2, B and C, and 9 B, P values were determined by unpaired two-tailed Student’s t test using the GraphPad Software. In Figs. 3, B and E, 4 E, 5 D, 6, A and D, 8 E, 9, E and F, S1 A, S2, C, D, F, and G, and S4, B and D, P values were determined by One-Way Anova-Tukey’s multiple comparisons test using the GraphPad Software. Data distribution was assumed to be normal, but this was not formally tested. P value of <0.05 was considered statistically significant (∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001; ∗∗∗∗ = P < 0.0001). In graphs, averages ± SEM is normally shown.

Repeatability of the experiments

Each experiment has been repeated at least three times. The results were highly reproducible.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis

Sample preparation followed a previously reported procedure (Li et al., 2019). Digested samples were labeled with Tandem Mass Tag (TMT)-11plex reagents (90406, A34807; Thermo Fisher Scientific) in the following order: 126 (cdc15-as1), 127n (cdc15-as1), 129n (cdc20-AID), 129c (cdc20-AID), 130n (cdc20-AID), 130c (cdc14-1 cdc5-as1), 131 (cdc14-1 cdc5-as1), and 131c (cdc14-1 cdc5-as1). Proteomic and phospho-proteomic data were collected on an Orbitrap Fusion and an Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific), respectively. An online real-time search algorithm (Orbiter) was used in proteomic data collection as described previously (Li et al., 2020; Schweppe et al., 2020). MS raw files were initially converted to mzXML and monoisotopic peaks were re-assigned using Monocle (Rad et al., 2020). Database searching with SEQUEST included all entries from the Saccharomyces Genome Database (2014). Peptide-spectrum matches were adjusted to 1% false discovery rate (FDR; Elias and Gygi, 2007). Protein-level FDR was filtered to the target 1% FDR level. Phosphorylation site localization was determined using AScore algorithm (Beausoleil et al., 2006) and filtered at 13 (95% confidence). For TMT reporter ion quantification, a 0.003-Da window around the theoretical m/z of each reporter ion was scanned, and the most intense m/z was used. Reporter ion intensities were adjusted to correct for the isotopic impurities of the TMT reagents according to the manufacturer’s specifications. Peptides were filtered for a summed signal-to-noise of 100 across all channels. For each protein, peptide TMT values were summed to create protein quantifications. To compensate for differential protein loading within a TMT plex, the summed protein quantities were adjusted to be equal within the plex. Phosphorylation site quantifications were also normalized by correction factors generated in this process to account for protein loading variance. For each protein or phosphorylation site within a TMT plex, the signal-to-noise value was scaled to sum to 100 for subsequent analysis. Unpaired student’s t test was performed in Perseus (version 1.6.15.0) to identify the phospho-residues that were significantly different between the different mutant cells. An FDR of 0.001 (S0 at 0.05) was applied, with an additional cut-off of a log₂ fold change <−0.5 or >0.5. To identify the phospho-residues likely phosphorylated by CDK, and the phospho-peptides were filtered using the minimal CDK consensus (S/TP).

Online supplemental material

Fig. S1 shows aMT length and number of different mutant strains that arrest either in metaphase or in anaphase and individual frames of two aMTs of a cdc20 mutant cell analyzed by live-cell imaging; Fig. S2 shows experiments performed to explain the aMT phenotype of cdc14 cdc5 cells; Fig. S3 shows spindle kinetics of different esp1 mutants; Fig. S4 shows experiments to identify the APC/C^Cdc20 substrate(s) whose degradation is required for anaphase aMT stabilization; Fig. S5 shows details of the phospho-proteomic analysis performed; Table S1 contains aMT dynamic parameters; Table S2 lists the strains used in this study; Table S3 is available as an Excel file and contains the protein quantification obtained by the proteomic analysis; Table S4 is available as an Excel file and contains phospho-site quantification obtained by the phospho-proteomic analysis.

Supplementary Material

Table S1

contains aMT dynamic parameters.

Table S2

lists the strains used in this study.

Table S3

is available as an Excel file and contains the protein quantification obtained by the proteomic analysis.

Table S4

is available as an Excel file and contains phospho-site quantification obtained by the phospho-proteomic analysis.

SourceData F6

contains original blots for Fig. 6.

SourceData F7

contains original blots for Fig. 7.

SourceData F8

contains original blots for Fig. 8.

Data availability

Strains, reagents, and protocols used in the manuscript are available to the scientific community upon request. The MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD028828.