Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts

PLoS Biol. 2011 Dec;9(12):e1001225. doi: 10.1371/journal.pbio.1001225. Epub 2011 Dec 27.

Abstract

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

Publication types

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

MeSH terms

  • Animals
  • Cell Cycle Proteins / metabolism
  • Cell Cycle Proteins / physiology*
  • Cell Extracts
  • Chromatin / metabolism
  • Chromatin / physiology
  • Guanine Nucleotide Exchange Factors / metabolism
  • Guanine Nucleotide Exchange Factors / physiology*
  • Humans
  • Kinesins / metabolism
  • Kinesins / physiology
  • Microtubules / metabolism
  • Microtubules / physiology
  • Nuclear Proteins / metabolism
  • Nuclear Proteins / physiology*
  • Ovum
  • Spindle Apparatus / metabolism
  • Spindle Apparatus / physiology*
  • Xenopus Proteins / metabolism
  • Xenopus Proteins / physiology*
  • Xenopus laevis

Substances

  • Cell Cycle Proteins
  • Cell Extracts
  • Chromatin
  • Guanine Nucleotide Exchange Factors
  • Nuclear Proteins
  • RCC1 protein, Xenopus
  • Xenopus Proteins
  • Kinesins