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Updated: Jun 2, 2026

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
08:33

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

Timeless links replication termination to mitotic kinase activation

Jayaraju Dheekollu1, Andreas Wiedmer, James Hayden

  • 1The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

Plos One
|May 17, 2011
PubMed

Insights

Human Timeless protein (Tim) coordinates DNA replication termination with mitotic entry. Tim depletion causes mitotic defects and delays kinase activity, revealing its crucial role in cell cycle progression.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The coordination between DNA replication termination and mitosis progression is not fully understood.
  • Human Timeless protein (Tim) is involved in replication fork stability and checkpoint control.

Purpose of the Study:

  • To investigate the role of human Timeless protein (Tim) in coordinating DNA replication termination with mitotic entry.
  • To elucidate the molecular mechanisms by which Tim influences mitotic progression and kinase activity.

Main Methods:

  • Co-immunoprecipitation to identify proteins interacting with Tim.
  • In vitro kinase assays to assess Tim's effect on mitotic kinases.
  • siRNA-mediated Tim depletion to observe mitotic phenotypes.
  • Immunofluorescence microscopy to analyze protein localization and mitotic structures.

Main Results:

  • Human Tim was found in a complex with mitotic kinases CDK1, Auroras A and B, and Plk1.
  • Tim depletion led to loss of sister-chromatid cohesion, defective spindle architecture, and failure to exit mitosis.
  • Tim depletion delayed mitotic kinase activity and reduced histone H3 S10 phosphorylation.
  • Tim is essential for Plk1 recruitment to centromeres and formation of catenated DNA structures.

Conclusions:

  • Human Timeless protein (Tim) plays a critical role in coordinating DNA replication termination with mitotic kinase activation.
  • Tim is a key regulator of mitotic entry, spindle assembly, and sister-chromatid cohesion.
  • These findings highlight Tim's function as a molecular link between DNA replication and mitosis.

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