Dynein, 1st ed. 2023 Methods and Protocols Methods in Molecular Biology Series, Vol. 2623

This volume provides a collection of protocols from leading researchers in the dynein field. The enclosed chapters guide readers through a broad range of powerful techniques that can be used to understand the complexities of dynein activity and regulation. These include how to perform live-cell microscopy on various fungi and metazoan cells, how to purify, assemble, and measure the force generation and motility characteristics of various dynein assemblies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Dynein: Methods and Protocols is a valuable resource for both novice and expert scientists in this developing field.

Part I: Methods for the cell biological assessment of dynein function: Fungal systems

1.     Live-cell imaging of dynein-mediated cargo transport in Aspergillus nidulans

 Rongde Qiu, Jun Zhang, Dennis McDaniel, Miguel A. Peñalva and Xin Xiang

2.     Using budding yeast as a model to understand dynein-mediated cargo transport

Part II: Methods for the cell biological assessment of dynein function: Metazoan systems

Sydney E. Cason, Adam R. Fenton, Erika L. F. Holzbaur

4.     Assessment of dynein-mediated nuclear migration in the developing cortex by live-tissue microscopy

Haw-Yuan Cheng, Fang-Shin Nian, Yi-Wen Ou, Jin-Wu Tsai

5.     Using optogenetics to spatially control cortical dynein activity in mitotic human cells

Tomomi Kiyomitsu

6.     Visualization of single dynein molecules in mammalian cells

Nireekshit Addanki Tirumala and Vaishnavi Ananthanarayanan

Part III: At the crossroads: combined in-cell and in vitro methods to measure dynein function

7.     Single-molecule studies of motor adaptors using cell lysates

Adam R. Fenton, Sydney E. Cason, Erika L. F. Holzbaur

8.     Reconstitution of organelle transport along microtubules in vitro

Daniel Beaudet and Adam G. Hendricks

Part IV: Methods for the biochemical reconstitution of active dynein assemblies

9.     Reconstitution of dynein/dynactin transport using recombinant dynein

Clinton K. Lau

10.  Fast and easy transient mammalian cell expression and purification of cytoplasmic dynein

Akane Furuta and Ken’ya Furuta

Part V: Methods to measure motility and force generation of dynein assemblies

11.  High-resolution tracking of dynein-dynactin-BicD2 complexes

Qingzhou Feng, Allison M. Gicking and William O. Hancock

12.  In vivo trapping of latex bead phagosomes for quantitative force measurements

13.  Studying dynein mechanochemistry with an optical trap

Emre Kusakci & Ahmet Yildiz

14.  Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules

Xinglei Liu, Lu Rao, and Arne Gennerich

Part VI: Methods for the structural analysis of dyneins

Kayla A. Jara and Elisar J. Barbar

16.  High-resolution Structural Analysis of Dyneins by Cryo-electron Microscopy

Pengxin Chai, Qinhui Rao, Yue Wang, Kai Zhang

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts