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Combining Structure–Function and Single-Molecule Studies on Cytoplasmic Dynein

  • Lu Rao
  • Maren Hülsemann
  • Arne Gennerich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1665)

Abstract

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein’s molecular mechanism through the combination of structure–function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure–function studies on dynein.

Key words

Microtubules Microtubule motor proteins Cytoplasmic dynein Recombinant proteins Microtubule immobilization Fluorescence labeling Single-molecule assays Optical tweezers Optical trapping Yeast gene manipulation 

Notes

Acknowledgments

The authors would like to thank Lisa Baker for her help in editing the manuscript. The authors are supported by NIH Grant R01GM098469.

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics CenterAlbert Einstein College of MedicineBronxUSA

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