A cell-free system for functional centromere and kinetochore assembly

Nat Protoc. 2012 Oct;7(10):1847-69. doi: 10.1038/nprot.2012.112. Epub 2012 Sep 20.

Abstract

This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantigens / chemistry
  • Autoantigens / metabolism
  • Cell-Free System*
  • Centromere / metabolism*
  • Centromere / ultrastructure
  • Centromere Protein A
  • Chromatin / metabolism
  • Chromosomal Proteins, Non-Histone / chemistry
  • Chromosomal Proteins, Non-Histone / metabolism
  • Fluorescent Antibody Technique / methods
  • Kinetochores / metabolism*
  • Kinetochores / ultrastructure
  • Xenopus

Substances

  • Autoantigens
  • Centromere Protein A
  • Chromatin
  • Chromosomal Proteins, Non-Histone