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Five factors can reconstitute all three phases of microtubule polymerization dynamics

Takashi Moriwaki1,2, Gohta Goshima3,2

  • 1Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

The Journal of Cell Biology
|November 2, 2016
PubMed

Insights

Researchers reconstituted dynamic microtubule (MT) polymerization cycles in vitro using five core proteins. The mitotic kinase Plk1 switches MT dynamics between interphase and mitotic modes.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cytoplasmic microtubules (MTs) exhibit dynamic polymerization cycles: growth, shrinkage, and pausing.
  • The molecular mechanisms and spatiotemporal regulation of these MT polymerization cycles remain incompletely understood.
  • Previous in vitro studies have not fully recapitulated the complete MT polymerization cycle with defined components.

Purpose of the Study:

  • To reconstitute dynamic microtubule plus-end behavior, including all three phases (growth, shrinkage, pausing), in vitro.
  • To identify the core protein factors essential for generating dynamic MT polymerization cycles.
  • To investigate the regulatory role of the mitotic kinase Plk1 in modulating MT dynamics.

Main Methods:

  • Reconstitution of microtubule dynamics by mixing purified tubulin with five specific Drosophila melanogaster proteins: EB1, XMAP215 (Msps), Sentin, kinesin-13 (Klp10A), and CLASP (Mast/Orbit).
  • In vitro assays to observe and analyze microtubule polymerization, catastrophe, and pausing dynamics.
  • Investigated the effect of CLASP alone and in combination with other factors on MT behavior.
  • Assessed the impact of the mitotic kinase Plk1 (Polo) on the activity of CLASP and kinesin-13, and overall MT dynamics.

Main Results:

  • A minimal set of five proteins (EB1, XMAP215, Sentin, Klp10A, CLASP) reconstituted dynamic MT plus-end behavior, encompassing growth, shrinkage, and pausing.
  • CLASP, when alone, inhibited catastrophe and reduced growth; however, with the other four factors, it induced pausing.
  • The mitotic kinase Plk1 modulated CLASP and Klp10A activities, significantly increasing MT dynamic instability, mimicking mitotic cell behavior.

Conclusions:

  • Five conserved proteins are identified as core factors responsible for generating dynamic microtubule polymerization cycles in cells.
  • Plk1-dependent phosphorylation plays a critical role in switching microtubule dynamics from interphase to mitotic modes.
  • This reconstituted system provides a platform for understanding the molecular regulation of microtubule dynamics.

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