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Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips

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Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1486))

Abstract

Kinetochores are large multiprotein complexes that drive mitotic chromosome movements by mechanically coupling them to the growing and shortening tips of spindle microtubules. Kinetochores are also regulatory hubs, somehow sensing when they are erroneously attached and, in response, releasing their incorrect attachments and generating diffusible wait signals to delay anaphase until proper attachments can form. The remarkable ability of a kinetochore to sense and respond to its attachment status might stem from attachment- or tension-dependent changes in the structural arrangement of its core subcomplexes. However, direct tests of the relationship between attachment, tension, and core kinetochore structure have not previously been possible because of the difficulties of applying well-controlled forces and determining unambiguously the attachment status of individual kinetochores in vivo. The recent purification of native yeast kinetochores has enabled in vitro optical trapping-based assays of kinetochore tip-coupling and, in separate experiments, fluorescence imaging of single kinetochore particles. Here we introduce a dual instrument, combining optical trapping with multicolor total internal reflection fluorescence (TIRF) imaging, to allow kinetochore structure to be monitored directly with nanometer precision while mechanical tension is simultaneously applied. Our instrument incorporates differential interference contrast (DIC) imaging as well, to minimize the photo-bleaching of fluorescent tags during preparative bead and microtubule manipulations. A simple modification also allows the trapping laser to be easily converted into a real-time focus detection and correction system. Using this combined instrument, the distance between specific subcomplexes within a single kinetochore particle can be measured with 2-nm precision after 50 s observation time, or with 11-nm precision at 1 s temporal resolution. While our instrument was constructed specifically for studying kinetochores, it should also be useful for studying other filament-binding protein complexes, such as spindle poles, cortical microtubule attachments, focal adhesions, or other motor–cytoskeletal junctions.

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Authors and Affiliations

  1. Department of Physiology & Biophysics, University of Washington, 1959 NE Pacific Street, Box 357290, Seattle, WA, 98195, USA

    Yi Deng & Charles L. Asbury

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  1. Yi Deng
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  2. Charles L. Asbury
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Correspondence to Charles L. Asbury .

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Editors and Affiliations

  1. Department of Anatomy and Structural Biology & Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Arne Gennerich

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Deng, Y., Asbury, C.L. (2017). Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_17

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  • DOI: https://doi.org/10.1007/978-1-4939-6421-5_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6419-2

  • Online ISBN: 978-1-4939-6421-5

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