Chapter 67 Isolation of Inner- and Outer-Arm Dyneins

Abstract

Publisher Summary Dyneins are the main constructive body part of the inner and outer arms of eukaryotic flagella and cilia. The composition of axonemal dyneins varies from species to species and within a single axoneme. Within an axoneme, outer-arm dyneins are thought to be homogenous in form, whereas the inner-arm dyneins consist of at least three subforms. Dyneins were first separated from other axonemal components by selective extraction in low-ionic-strength solution containing ethylenediaminetetraacetic acid; however, this procedure causes dynein molecules to dissociate into subunits. It is now most common to use high-ionic-strength (elevated NaCl or KCl) solution to extract dynein from axonemes. Biochemical quantities of dynein can be obtained from five different sources. This chapter describes the procedures for isolation of axonemes and outer-arm dynein from the flagella of the sea urchin Strongylocentrotus purpuratus and for the isolation of inner-arm dynein from the flagella of the green alga Chlamydomonas reinhurdtii . Similar procedures are used to isolate dynein from a number of sources. The chapter discusses isolation of sea urchin sperm axonemes and dynein—collection of sperms, isolation and demembranation of flagella (by osmotic shock and with detergent), and isolation of outer-arm dynein from sea urchin sperm flagella. Because exposure of sea urchin dynein to detergent nonphysiologically activates the dynein ATPase activity, it is advised to demembranate by osmotic shock with high sucrose for investigations into the regulation of axonemes and dynein. There are details on Isolation of Chlamydomonas inner-arm dynein and fractionation of dyneins by sucrose density gradient zonal centrifugation. The existence of numerous outer-arm and inner-arm mutants and the ability to extract both inner and outer arms with high-salt solutions make Chlamydomonas an excellent system for purification of inner-arm dyneins. To isolate inner-arm dyneins from Chlamydomonas , a cell line genetically lacking the entire outer-arm structure is used.