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.
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Acknowledgements
The phospho-Wee1 antibody was a gift from J.E. Ferrell (Stanford University School of Medicine). The histone expression plasmids were a gift from K. Luger (Colorado State University). The pST39 plasmid was a gift from S. Tan (Pennsylvania State University). The 19 × 601 plasmid was a gift from D. Rhodes (Medical Research Council Laboratory of Molecular Biology). A.G. was supported by a postdoctoral fellowship from the German Research Foundation (DFG), C.J.F. by a Stanford Lieberman Fellowship, and this work was supported by US National Institutes of Health grant no. R01GM074728 to A.F.S.
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A.G., C.J.F. and A.F.S. wrote the manuscript.
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Supplementary Methods
beadMeasurements3.m. MATLAB script for quantifying fluorescence associated with beads in three channels, which implements the analysis described in Box 3. (Documentation on using the script is contained in the comments in the script file.) (ZIP 3 kb)
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Guse, A., Fuller, C. & Straight, A. A cell-free system for functional centromere and kinetochore assembly. Nat Protoc 7, 1847–1869 (2012). https://doi.org/10.1038/nprot.2012.112
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DOI: https://doi.org/10.1038/nprot.2012.112
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