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A cell-free system for functional centromere and kinetochore assembly

Nature Protocols volume 7, pages 1847–1869 (2012)Cite this article

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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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Figure 1: A cell-free system for centromere and kinetochore reconstitution.
Figure 2: The cell-free system can be used to analyze centromere assembly, kinetochore assembly, microtubule polymerization and mitotic checkpoint activation.
Figure 3: Generation and analysis of biotinylated DNA arrays for chromatin reconstitution.
Figure 4: Experimental apparatus for assembling chromatin arrays by salt dialysis.
Figure 5: Analysis and quality control of the chromatin array assemblies.
Figure 6: Setup of an immunofluorescence experiment and of spin-down tubes for the analysis of microtubule polymerization.
Figure 7: Representative images of anticipated results using the cell-free system to analyze centromere and kinetochore assembly and function.

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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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  1. Department of Biochemistry, Stanford Medical School, Stanford, California, USA

    Annika Guse, Colin J Fuller & Aaron F Straight

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  1. Annika Guse
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A.G., C.J.F. and A.F.S. wrote the manuscript.

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Correspondence to Aaron F Straight.

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Supplementary information

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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