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In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends

Susana Montenegro Gouveia1, Kris Leslie, Lukas C Kapitein

  • 1Department of Cell Biology, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands.

Current Biology : CB
|September 21, 2010
PubMed

Insights

Mitotic centromere-associated kinesin (MCAK) normally destabilizes microtubules. In vitro, end-binding protein 3 (EB3) targets MCAK to growing microtubule ends, enhancing catastrophe rates and promoting microtubule remodeling.

Area of Science:

  • Cell Biology
  • Molecular Motors
  • Cytoskeleton Dynamics

Background:

  • Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerase.
  • MCAK accumulates at growing microtubule plus ends in cells, a behavior dependent on end-binding proteins (EBs).
  • The functional significance of this localization and EB influence on MCAK activity remained unclear.

Purpose of the Study:

  • To investigate the functional interplay between MCAK and EB3.
  • To determine how EB3 affects MCAK's microtubule-destabilizing activity.
  • To elucidate the role of EB3-mediated MCAK accumulation at growing microtubule tips.

Main Methods:

  • Reconstitution of EB3-dependent MCAK activity on dynamic microtubules in vitro.
  • Assays to measure microtubule assembly, growth, and shortening dynamics.
  • Analysis of MCAK association rates with microtubule tips.

Main Results:

  • MCAK alone inhibits microtubule assembly, but EB3 addition restores growth, independent of MCAK-EB3 binding.
  • EB3 targets MCAK to growing microtubule ends by increasing its association rate, requiring direct interaction.
  • EB3-mediated accumulation enhances MCAK's catastrophe-inducing capacity without altering growth or shortening velocities.

Conclusions:

  • EB3 directs MCAK to growing microtubule plus ends, modulating its activity.
  • The MCAK-EB3 complex promotes rapid switching between microtubule growth and shortening.
  • This dynamic regulation is crucial for microtubule cytoskeleton remodeling during processes like mitosis.

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