Chapter 69 Reconstitution of Dynein Arms in Vitro

Abstract

Reconstitution experiments have been used in studies of Tetrahymena cilia and Chlamydomonas flagella to begin answering questions of dynein function and regulation, as well as dynein assembly onto doublet microtubules. For these two organisms, reconstitution is difficult as both inner and outer dynein arms are extracted in high-salt buffers. For Chlamydomonas flagella, this difficulty is overcome with the aid of mutants lacking subsets of inner and/or outer dynein arms. This chapter describes the strategies for designing reconstitution experiments and focuses on methods of assessing experimental success. The chapter discusses in detail the methods for isolating axonemes, extracted axonemes, and dynein. Although both outer and inner dynein arms are extracted in high-salt-containing buffers, using mutants that are lacking outer dynein arms and/or subsets of inner dynein arms allows for diversity in experimental design. Similar protocols can be adapted for isolating axonemes and dyneins from other organisms for use in reconstitution. There is discussion on assessing reconstitution—biochemical methods, structural analysis, and functional analysis. The sliding disintegration assay can be employed to assess functional reconstitution in axonemes. Sliding method has been used to assess inner-dynein-arm binding to mutant or extracted axonemes and to determine that dynein is regulated by the radial spokes. For Chlamydomonas , inner dynein arms return only to inner-dynein-arm positions even in excess of inner dynein arms. However, excess dynein added back to extracted axonemes from Tetrahymena binds to both the A- and B-subfibers of doublet microtubules as well as the central pair of microtubules. Inner dynein arms and outer dynein arms of Chlamydomonas return to the original positions, but the inner dynein arms do not rebind if the outer dynein arms are present on the axoneme. For this reason, when reconstituting inner dynein arms, it is necessary to extract the outer dynein arms or make use of outer-dynein-arm-less mutants.