Combining structure–function and single-molecule studies on cytoplasmic dynein

Lu Rao, Maren Hülsemann, Arne Gennerich

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages53-89
Number of pages37
Volume1665
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1665
ISSN (Print)1064-3745

    Fingerprint

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Rao, L., Hülsemann, M., & Gennerich, A. (2018). Combining structure–function and single-molecule studies on cytoplasmic dynein. In Methods in Molecular Biology (Vol. 1665, pp. 53-89). (Methods in Molecular Biology; Vol. 1665). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7271-5_4