title: Measuring kinetochore-microtubule interaction in vitro.
abstract: Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example, between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule-binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome missegregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Driver
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Powers
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Sarangapani
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. Electronic address: sbiggins@fhcrc.org.
authors.forename: Sue
authors.initials: S
authors.lastname: Biggins
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Asbury
mesh_terms: D001696:Biomechanical Phenomena; D020090:Chromosome Segregation; D004867:Equipment Design; D005656:Fungal Proteins / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D052898:Optical Tweezers; D010770:Phosphotransferases / Q000378:metabolism; D016474:Weight-Bearing; D015003:Yeasts / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D010770:Phosphotransferases
keywords: Chromosomal mis-segregation; Kinase; Laser trapping; Mechanoregulation; Mitosis