TY - CHAP
T1 - Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules
AU - Liu, Xinglei
AU - Rao, Lu
AU - Gennerich, Arne
N1 - Funding Information:
X. Liu, L. Rao, and A. Gennerich are supported by the National Institutes of Health grant R01GM098469.
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein’s molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein’s multiple AAA+ ATPase domains regulate dynein’s attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.
AB - Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein’s molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein’s multiple AAA+ ATPase domains regulate dynein’s attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.
KW - Cytoplasmic dynein
KW - Fluorescence labeling
KW - Microtubule immobilization
KW - Microtubule motor proteins
KW - Microtubules
KW - Optical trapping
KW - Optical tweezers
KW - Recombinant proteins
KW - Single-molecule assays
KW - Yeast gene manipulation
UR - http://www.scopus.com/inward/record.url?scp=85145668804&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85145668804&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-2958-1_14
DO - 10.1007/978-1-0716-2958-1_14
M3 - Chapter
C2 - 36602689
AN - SCOPUS:85145668804
T3 - Methods in Molecular Biology
SP - 221
EP - 238
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -
