Molecular Tools for Studying the Radial Spoke

Abstract Studies of cilia and flagella often entail biochemical analysis of axonemal complexes that either associate with the nine outer doublet microtubules or the two singlet microtubules in the 9 + 2 axoneme. Each complex contains multiple subunits, a few of which are ubiquitous vital proteins, while many are novel with prevalent domains that remain to be characterized. Investigation of axoneme biochemistry will continue providing insights into flagellar biology as well as molecular complexes in general. Yet, the complicated contents and extensive molecular interactions pose significant challenges in experimentation. As such, most biochemical studies remain limited to dynein motors and often require extensive training and expensive equipment. The rapid accumulation of high-throughput database and versatile research tools has now lessened the obstacles significantly. Here, we describe the strategies and methods that were used to circumvent some of the common difficulties to characterize the radial spoke in Chlamydomonas axoneme, some of which were tailored to students with little research experience. They could be adapted for the study of many other axonemal complexes and for classroom settings as well.