#mitotic exit

Cell Cycle Regulation

The APC/C-Cdh1 complex is required for:

Specificity and timing of APC/C activity arise from sequential association with two adaptors, Cdc20 and Cdh1, both containing seven WD40 repeats forming beta-propeller that binds D-box and KEN motifs. During prometaphase and metaphase, APC/C-Cdc20 polyubiquitinates securin and cyclin B to trigger anaphase and mitotic exit. At telophase, cyclin B degradation reduces CDK1 activity, allowing Cdc14 phosphatase to dephosphorylate Cdh1, enabling its binding to APC/C core. APC/C-Cdh1 then operates from late mitosis through entire G1, continuously ubiquitinating mitotic cyclins A and B, mitotic kinases Polo and Aurora, Geminin inhibitor of licensing, and even Cdc20 itself, ensuring low CDK environment. Low CDK permits pre-replication complex formation consisting of ORC, Cdc6, Cdt1, and double hexamer of MCM2-7 helicase loaded at origins. As cyclin E-CDK2 accumulates at G1/S, it phosphorylates Cdh1 creating 14-3-3 sites, displacing it from APC/C and inactivating ligase, allowing S-phase cyclin accumulation and transition to next cycle. This circuitry is highly conserved across eukaryotes, integrating growth factor signals, DNA damage surveillance, and developmental cues, and its disruption frequently underlies oncogenesis, providing targets for checkpoint inhibitors and cancer therapeutics.

Ref: Peters, Nature Reviews Mol Cell Biol 2006, APC/C Mechanisms. Alberts 7th ed., Chapter 17.

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Cell Cycle Regulation

Which factor regulates the exit from mitosis?

Finishing mitosis demands comprehensive reversal of CDK1 mediated phosphorylations and elimination of cyclin B activity. Cdc14 family phosphatases coordinate this program. In budding yeast, Cdc14 resides sequestered in nucleolus bound to inhibitor Net1 also called Cfi1. Mitotic entry maintains sequestration via high CDK activity. In early anaphase, FEAR pathway comprising separase, Slk19 homodimer, Spo12, Zds1 and Zds2 induces transient release. Subsequently Mitotic Exit Network MEN, Hippo-like cascade of Tem1 GTPase localized at daughter spindle pole body, polo kinase Cdc15 and Dbf2-Mob1 complex, fully liberates Cdc14 through phosphorylation of Net1. Free phosphatase preferentially dephosphorylates CDK sites containing serine-proline followed by lysine, including APC/C coactivator Cdh1 shifting APC/C activity from Cdc20 to Cdh1, transcription factor Swi5 enabling Sic1 CDK inhibitor synthesis, and S-phase regulator Cdc15 reinforcing feedback. Sic1 accumulation plus cyclin B degradation extinguishes CDK1 activity, allowing spindle disassembly, actomyosin ring contraction via Hof1 and Inn1 dephosphorylation, and septum formation. In mammals, orthologous CDC14A/B collaborate with PP2A-B55 for mitotic exit. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Stegmeier & Amon, Closing Mitosis: Regulation of Cdc14 Phosphatase, Annu Rev Genet 2004; Bardin & Amon, MEN and Mitotic Exit.

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Cell Cycle Regulation

Which of the following prevents mitotic exit if chromosomes are not properly aligned?

Accurate mitosis demands anaphase initiation strictly after all chromosomes achieve stable amphitelic attachment generating tension. The spindle assembly checkpoint monitors this criterion. Unattached kinetochores catalyze assembly of Mitotic Checkpoint Complex composed of Mad2 locked as C-Mad2, BubR1 ortholog of yeast Mad3, Bub3 and Cdc20 coactivator of APC/C. Within MCC, BubR1 provides pseudosubstrate motif inserting KEN boxes into Cdc20 binding pockets plus direct inhibition of APC/C central cavity, thereby preventing ubiquitination of securin and cyclin B. BubR1 also functions as protein kinase and scaffold recruiting PP2A-B56 phosphatase counteracting Aurora B at tensionless attachments and promoting establishment of correct bioriented connections. As long as even one kinetochore remains unattached, MCC production continues, suppressing mitotic exit and cytokinesis. Upon microtubule capture, dynein-mediated stripping of Mad1-Mad2 and p31comet-TRIP13 driven MCC disassembly terminates signal, permitting APC/C activation. Persistence of BubR1 mediated inhibition therefore prevents premature exit, safeguarding against aneuploidy, chromosome missegregation and cancer progression. This regulatory circuit illustrates integration of checkpoint kinases, ubiquitin ligases, phosphatases and structural proteins coordinating accurate cell division and preventing aneuploidy associated with tumorigenesis.

Ref: Musacchio, Spindle Assembly Checkpoint Structure of BubR1-MCC, Curr Biol 2015; NCBI Bookshelf, SAC component BubR1.

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Cell Cycle Regulation

Which protein deactivates mitotic CDKs during mitotic exit?

Mitotic exit requires not only destruction of cyclin B but comprehensive reversal of CDK1-Cyclin B phosphorylation on hundreds of substrates. Specialized phosphatases execute this program. In budding yeast, Cdc14 is the master phosphatase. Most of the cell cycle it is sequestered in nucleolus bound to inhibitor Net1 or Cfi1. Two release pathways operate sequentially: the Cdc Fourteen Early Anaphase Release pathway involving separase, Slk19, Spo12 and Zds1/Zds2 provides transient liberation in early anaphase, while the Mitotic Exit Network composed of Tem1, Cdc15 and Dbf2-Mob1 kinases sustains release. Active Cdc14 preferentially dephosphorylates serine followed by proline with basic Lys in +3 position, matching CDK consensus, including targets Cdh1 converting APC/C coactivator from Cdc20 to Cdh1, Swi5 transcription factor enabling Sic1 inhibitor synthesis and Cdc15 reinforcing feedback. In mammals, CDC14A/B and PP2A-B55, regulated by Greatwall-ENSA pathway, fulfill analogous roles. Inhibition traps cells in late anaphase with segregated but condensed chromosomes unable to reform nuclei or undergo cytokinesis. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Stegmeier & Amon, Closing Mitosis: Role of Cdc14 Phosphatases, Annu Rev Genet 2004; Alberts et al., Chapter 17, Mitotic Exit Control.

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Cell Cycle Regulation

Which protein inhibits Tem1 to prevent premature mitotic exit?

Exit from mitosis in Saccharomyces cerevisiae is governed by the Mitotic Exit Network, a conserved Hippo-like kinase cascade initiated by small GTPase Tem1 at spindle pole bodies. Inaccurate spindle positioning would generate anucleate daughters if MEN fired prematurely. The Spindle Position Checkpoint prevents this error. Kin4 kinase, enriched at mother cell cortex and mother spindle pole body, acts as central checkpoint effector. When anaphase spindle fails to penetrate the bud neck, Kin4 phosphorylates Bfa1 subunit of the Bfa1-Bub2 GTPase activating complex. Phosphorylated Bfa1-Bub2 remains active, stimulating Tem1 GTP hydrolysis to GDP-bound inactive state and excluding Tem1 from daughter spindle pole body. This blocks downstream activation of Cdc15 kinase, Dbf2-Mob1 complex and consequent release of Cdc14 phosphatase from nucleolar Net1 sequestration. Upon correct alignment, Lte1 localized in bud antagonizes Kin4, promoting Bfa1 inactivation by polo kinase Cdc5, permitting Tem1-GTP accumulation, Cdc14 release and cyclin B degradation, thereby coupling spatial spindle position to temporal mitotic exit. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Bardin & Amon, Molecular Cell Biology of Spindle Position Checkpoint, Nat Rev Mol Cell Biol 2001; NCBI Bookshelf, MEN and SPOC signaling.

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Cell Cycle Regulation

Which of the following prevents premature mitotic exit?

In budding yeast, mitotic exit is controlled by mitotic exit network, abbreviated MEN, culminating in release of phosphatase Cdc14 from nucleolus to dephosphorylate CDK substrates and activate APC/C-Cdh1 and Sic1 to drive G1 entry. Premature exit would cause cytokinesis before chromosome segregation, generating aneuploid progeny. Two mechanisms prevent this: inhibition of upstream GTPase Tem1 keeps MEN inactive until anaphase when Tem1 is recruited to spindle pole body and activated by Lte1, ensuring that only elongated spindles trigger exit. Separately, Cdh1, alternative activator of APC/C, is kept inactive during early mitosis by CDK1-dependent phosphorylation that prevents its binding to APC/C core, avoiding early degradation of mitotic cyclins Clb2 and polo kinase Cdc5. Phosphorylation also excludes Cdh1 from nucleus. Both restraints must be relieved: Tem1 activation drives Cdc14 release, leading to Cdh1 dephosphorylation and APC/C-Cdh1 activation that clears remaining cyclins. Loss of either brake causes premature Cdh1 activity and MEN firing, explaining why combined inhibition of Tem1 and phosphorylation of Cdh1 acts as dual safeguard against untimely mitotic exit and cytokinesis.

Ref: Visintin et al., Mol Cell 1998, Mitotic Exit Network; Stegmeier & Amon, Ann Rev Genet 2004, Closing Mitosis.

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