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Methods Enzymol. 2014;540:339-60. doi: 10.1016/B978-0-12-397924-7.00019-4.

Micropattern-guided assembly of overlapping pairs of dynamic microtubules.

Author information

1
London Research Institute, Cancer Research UK, London, United Kingdom.
2
Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA.
3
Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA; Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, California, USA.
4
Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, California, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
5
Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA. Electronic address: fletch@berkeley.edu.
6
London Research Institute, Cancer Research UK, London, United Kingdom. Electronic address: thomas.surrey@cancer.org.uk.

Abstract

Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein.

KEYWORDS:

Biotin; Dynamic assay; Maleimide; Microtubule; Microtubule overlaps; PEG; Patterning; Streptavidin; Surface chemistry; TIRF microscopy

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