Chapter Eight - Biochemical and Physiological Analysis of Axonemal Dyneins

Abstract

Axonemal dyneins are highly complex molecular motors that power the beating of cilia/flagella. In addition to the motor subunits, these enzymes contain components that allow for assembly at the correct axonemal location and also enable the motor to respond to a broad array of signals including phosphorylation, Ca(2+), redox changes, and mechanical activation. The green alga Chlamydomonas reinhardtii has become the premier system in which to analyze these motors, as it allows for classical/molecular genetic approaches to be combined with biochemical fractionation, and physiological measurements to gain an integrated view of dynein function. Furthermore, Chlamydomonas provides the opportunity to study axonemal dyneins in the cytoplasm prior to their transport into the cilium/flagellum, thus allowing the nature of the assembly process to be defined. In this chapter, I describe methods used in my laboratory to prepare and fractionate cytoplasmic extracts and to localize axonemal dynein components within the flagellum at both the light microscope level and by biochemical and genetic approaches. Finally, I also detail how to assess dynein-driven flagella motility by measuring beat frequency and propulsive force of both intact cells and reactivated cell models.