Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.
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.
Department of Molecular & Cell Biology, University of California-Berkeley, Berkeley, California, United States of America.
David
D
Halpin
Petr
P
Kalab
Jay
J
Wang
Karsten
K
Weis
Rebecca
R
Heald
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016547:Kinesins / Q000378:metabolism / Q000502:physiology; D008870:Microtubules / Q000378:metabolism / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010063:Ovum; D008941:Spindle Apparatus / Q000378:metabolism / Q000502:physiology*; D029867:Xenopus Proteins / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C499304:RCC1 protein, Xenopus; D029867:Xenopus Proteins; D016547:Kinesins