title: Micropattern-guided assembly of overlapping pairs of dynamic microtubules.
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.
authors.affiliation: London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Franck J
authors.initials: FJ
authors.lastname: Fourniol
authors.affiliation: Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA.
authors.forename: Tai-De
authors.initials: TD
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Peter
authors.initials: P
authors.lastname: Bieling
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, California, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: R Dyche
authors.initials: RD
authors.lastname: Mullins
authors.affiliation: Department of Bioengineering and Biophysics Group, University of California-Berkeley, Berkeley, California, USA. Electronic address: fletch@berkeley.edu.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Fletcher
authors.affiliation: London Research Institute, Cancer Research UK, London, United Kingdom. Electronic address: thomas.surrey@cancer.org.uk.
authors.forename: Thomas
authors.initials: T
authors.lastname: Surrey
mesh_terms: D000818:Animals; D004867:Equipment Design; D005898:Glass / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure*; D011092:Polyethylene Glycols / Q000737:chemistry; D013499:Surface Properties; D013552:Swine; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism
chemical_list: D008301:Maleimides; C551789:PRC1 protein, Xenopus; D029867:Xenopus Proteins; C043592:maleimide; D011092:Polyethylene Glycols
keywords: Biotin; Dynamic assay; Maleimide; Microtubule; Microtubule overlaps; PEG; Patterning; Streptavidin; Surface chemistry; TIRF microscopy