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Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins

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Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2478))

Abstract

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP–MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Suvranta K. Tripathy and Vladimir M. Demidov have equal first-author contribution.

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Abbreviations

AOD:

Acousto-optic deflector

BG:

Biotinylated benzylguanine

b-PEG:

Biotin polyethylene glycol

BSA:

Bovine serum albumin

DTT:

Dithiothreitol

FPGA:

Field-programmable gate array

GBP:

GFP-binding protein

MAP:

Microtubule-associated protein

MT:

Microtubule

QPD:

Quadrant photo-detector

SD:

Standard deviation

UFFC:

Ultrafast force-clamp

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Acknowledgments

We gratefully thank Drs. Yale E. Goldman and Michael Ostap, and their lab members, for their continuous support and excellent suggestions over the years as we developed the protocols and methods detailed herein. We also thank members of our lab for stimulating discussions and technical assistance. This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R35-GM141747 to E.L.G, and the National Science Foundation (grant #2029868). Development of protocols to functionalize pedestals and microscopy chambers was supported in part by a grant from the Russian Science Foundation (project No. 21-45-00012).

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Author notes

  1. Suvranta K. Tripathy

    Present address: Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI, USA

  2. Shaowen Wu

    Present address: Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, China

  3. Fazly I. Ataullakhanov and Ekaterina L. Grishchuk contributed equally to developing and implementing this work.

Authors and Affiliations

  1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Suvranta K. Tripathy, Vladimir M. Demidov, Shaowen Wu, Fazly I. Ataullakhanov & Ekaterina L. Grishchuk

  2. Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russian Federation

    Ivan V. Gonchar & Fazly I. Ataullakhanov

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  1. Suvranta K. Tripathy
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Correspondence to Ekaterina L. Grishchuk .

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  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Arne Gennerich

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Tripathy, S.K., Demidov, V.M., Gonchar, I.V., Wu, S., Ataullakhanov, F.I., Grishchuk, E.L. (2022). Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 2478. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2229-2_22

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