Isolation and analysis of radial spoke proteins.
The 9+2 axoneme that mediates the highly controlled oscillatory beating of cilia and flagella is an elaborate supramolecular complex. Proteomics and genomics have revealed more than 400 distinct polypeptides that presumably are built into axonemal subcomplexes for specific tasks. However, only a handful of proteins can be assigned to the most prominent structural modules visible by electron microscopy. Much less is known about the function and mechanism of individual molecules and complexes. Isolation of intact complexes will hasten discoveries and open the door to a wide range of analyses as showcased by axonemal dynein motors. However, many axonemal components, such as the radial spoke complex, either are not extracted by conditions that solubilize axonemal dynein or at best are only partially released. This chapter discusses strategies and methods to circumvent this problem in order to characterize radial spokes. With appropriate modifications, the lessons learned from the radial spoke complex may be applicable to other axonemal complexes.
Kelekar
Pooja
P
Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, USA.
Wei
Mei
M
Yang
Pinfen
P
D054468:Axoneme / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002696:Chlamydomonas / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D010940:Plant Proteins; D015800:Protozoan Proteins / Q000302:isolation & purification*
D010940:Plant Proteins; D015800:Protozoan Proteins; C060324:radial spoke protein, Chlamydomonas