Related Experiment Video

Updated: Nov 5, 2025

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
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Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Published on: December 5, 2017

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Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1

Elyse S Fischer1, Conny W H Yu1, Dom Bellini1

  • 1MRC Laboratory of Molecular Biology, Cambridge, UK.

EMBO Reports
|May 20, 2021
PubMed

Insights

The spindle assembly checkpoint ensures proper cell division. This study reveals how Mad1 and Bub1 interact at kinetochores, crucial for regulating cell cycle progression and preventing errors.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Structural Biology

Background:

  • The spindle assembly checkpoint (SAC) prevents errors in chromosome segregation during cell division.
  • The mitotic checkpoint complex (MCC) inhibits the anaphase-promoting complex/cyclosome (APC/C) to arrest mitosis.
  • Mps1 kinase initiates MCC assembly at kinetochores via phosphorylation-dependent signaling.

Purpose of the Study:

  • To elucidate the structural basis of Mad1-Bub1 interaction at kinetochores.
  • To understand the role of this interaction in MCC assembly and SAC function.

Main Methods:

  • X-ray crystallography to determine the structure of Mad1 C-terminal domain (Mad1CTD) bound to phosphorylated Bub1CD1 peptides.
  • Biochemical analysis to study the binding stoichiometry and mechanism.

Main Results:

  • The crystal structure of Mad1CTD bound to two phosphorylated Bub1CD1 peptides was determined at 1.75 Å resolution.
  • Phosphorylated Bub1 Thr461 directly interacts with Mad1 Arg617 and caps the CD1 α-helix.
  • Mad1 homodimer binds only one Bub1CD1 peptide in solution, suggesting asymmetric binding.

Conclusions:

  • The Mad1-Bub1 interaction is critical for targeting the Mad1-Mad2 complex to kinetochores for MCC assembly.
  • The observed binding stoichiometry, influenced by Mad1CTD asymmetry, may optimize MCC formation.
  • This structural insight advances our understanding of SAC regulation and its role in maintaining genomic stability.
Keywords:
Bub1Cell cycleMad1Mitotic checkpoint complexSpindle assembly checkpoint

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