Chapter 52 Isolation of Radial Spoke Heads from Chlamydomonas Axonemes

Abstract

Publisher Summary There are three binding sites in the radial spoke heads of Chlamydomonas axonemes. They are bound to the central microtubule complex, paired by a thin fiber, and connected through a stalk to the A-tubule of the outer-doublet microtubules. The interactions occurring at these binding sites result in a modification of axonemal waveforms that is necessary for efficient swimming and displacement of the cell body. The radial spoke heads of Chlamydomonas are considered as a model system for studying organelle assembly. They can be partially purified as a protein complex and are composed of six distinct proteins. Five components of the radial spoke head complex are missing from flagella of distinct Chlamydomonas mutants. These mutants are paralyzed and have radial spoke components as defective gene products. Rescue of radial spoke heads in axonemes of dikaryons between a radial spoke mutant and a wild-type strain requires coassembly of radial spoke proteins from both strains. The procedure described in this chapter was developed for partially purifying radial spoke heads from Chlamydomonas axonemes. It should be applicable to the isolation of radial spoke heads from axonemes of other systems, assuming that in these systems radial spoke head components form a complex and can be separated from radial spoke stalks as in Chlamydomonas axonemes. The procedure for isolation of radial spoke heads from Chlamydomonas axonemes are based on the preferential solubilization of radial spoke heads. That solubilization occurs under conditions of low ionic strength. The axonemes are previously extracted with 0.5 M NaCl and 1 m M ATP-Mg to remove the outer and inner dynein arms. The final step of the procedure consists of a sedimentation of solubilized radial spoke heads in a sucrose gradient. This step does not separate the radial spoke heads from the tubulin subunits. Column chromatography would be necessary.