Chapter 30 Reactivation of Chlamydomonas Cell Models

Abstract

Unicellular biflagellate alga Chlamydomonas has been used to study how changes in the surrounding medium can alter axonemal motility. This chapter describes a procedure for the preparation of these models, with particular reference to their use in examining the effects of free calcium concentration on axonemal motility. The basic technique is applicable not only to the study of calcium effects, but to the effect of any soluble substance that acts on a detergent-stable component of the axoneme. Similar Chlamydomonas cell models have been useful for investigating the functional rebinding of purified dynein to outer-doublet microtubules. In this approach, the cell's plasma membrane is disrupted by the addition of a nonionic detergent, leaving the cell wall, axonemes, and most internal cell components apparently intact. These resulting cell models are then reactivated by dilution into a reactivation buffer containing ATP. Through the addition of ethylene glycol bis(β-aminoethyl ether)- N,N’ -tetraacetic acid (EGTA) and CaCl 2 , to the reactivation buffer, the amount of free Ca 2+ during reactivation can be carefully regulated. Dark-field light microscopic observation of reactivated cell models allows the integrity of the axonemes, as well as changes in swimming behavior, to be monitored. In studies comparing cell models reactivated at 10 -7 and 10 -9 M Ca 2+ , it has been established that the cell's two axonemes are differentially sensitive to calcium. The chapter describes the solutions and the procedure for the experiment. Cell models are very fragile and easily damaged. Dark-field light microscopy allows the condition of the axonemes to be assessed during the assay. Broken axonemes and axonemes in which the microtubules have splayed apart at the distal end are common forms of damage. Additionally, cell models may be observed in which one axoneme is completely inactivated because of extrusion of the central pair microtubules, which appear coiled at the distal end of the axoneme.