Chapter 31 Reactivation of Extracted Paramecium Models

Abstract

Paramecium exhibits a swimming behavior because of the ciliary activity regulated by membrane electrogenesis. A depolarizing receptor potential evoked by stimulation of the front end of Paramecium electrotonically spreads over the entire cell membrane to activate (open) depolarization-sensitive Ca 2+ channels in the ciliary membrane. Activation of the channels causes regenerative entry of Ca 2+ ions into the cilia, producing a sudden increase in the intraciliary Ca 2+ concentration and an action potential. The Ca 2+ increase activates a mechanism for reversing the direction of the effective power stroke of cilia to cause backward swimming of Paramecium. A hyperpolarizing receptor potential evoked by stimulation of the posterior end of Paramecium also spreads electrotonically over the entire cell membrane. Hyperpolarization of the membrane brings about an increase in the intracellular cyclic adenosine monophosphate (cAMP) concentration, which activates the ciliary beating mechanism to cause an increase in the frequency of ciliary beating in the normal direction. This brings about rapid forward swimming of the specimen. In a permeabilized cell, functions of the membrane as a signal transducer and a diffusion barrier are disrupted, while the motor function of the ciliary apparatus is kept normal. In addition to ATP, Mg 2+ , and KCl, other chemicals in the reactivation solution are important to keep the reactivated ciliary activity stable. The chapter discusses extraction of paramecium —Triton X-100 and modified Triton X-100 extraction (where Mg 2+ ions to the Triton X-100-containing extraction solution), glycerol extraction, Triton-glycerol extraction, modified Triton-glycerol extraction (with a solution having a higher concentration of Triton X-100); the processes of collecting, extracting, washing and reactivating for each extraction. When Triton X-100 is used to obtain permeabilized seaurchin spermatozoa, almost 100% of them showed ATP-Mg 2+ -reactivated flagellar motion. The chapter also provides a process for isolation of ciliated cortical sheets from triton-extracted paramecium .