The beat pattern and beat frequency of cilia and flagella

in many kinds of eukaryote are controlled by changes in intracellular Ca2+ concentration. Ciliary movement in Paramecium comprises two kinds of ciliary strokes, the effective stroke and the recovery stroke, and shows metachronal coordination (Machemer, 1972). When Paramecia meet an obstacle, their cilia reverse the direction of their beat, allowing the organism to swim backwards (Jennings, 1906). Collision of a Paramecium cell with an obstacle is believed to mechanically stimulate the anterior portion of the cell. This stimulation is then thought to induce activation of mechano-sensitive Ca2+ channels in the cell membrane, resulting in depolarization of the cell (Naitoh and Eckert, 1969; Naitoh et al., 1972). This depolarization causes an increase in Ca2+ influx into cilia through the activated, voltage-sensitive Ca2+ channels in the ciliary membrane (Ogura and Takahashi, 1976; Dunlap, 1977; Machemer and Ogura, 1979). Using permeabilized P. caudatum cells, Nakaoka and Ooi (1985) found that cAMP inhibits Ca2+-induced ciliary reversal. Several investigators identified a 29 kDa axonemal protein that copurified with 22S outer-arm dynein, which is phosphorylated in a Ca2+and cAMP-dependent manner (Hamasaki et al., 1989, 1991; Bonini and Nelson, 1990). Brief digestion of demembranated cilia with trypsin suppressed not only the inhibitory effect of cAMP on Ca2+-induced ciliary reversal but also the phosphorylation of the 29 kDa axonemal protein (Noguchi et al., 2000). These results suggest that the 29 kDa protein may be a mediator of the effect of Ca2+ on ciliary reversal. If this were the case, Ca2+ would control the direction of ciliary beat not by acting on the ciliary base, but throughout the cilium, because the 29 kDa dynein-associated protein is present throughout the cilium. To clarify the region of Ca2+ sensitivity with respect to ciliary reversal, Hamasaki and Naitoh (1985) iontophoretically applied Ca2+ to detergent-permeabilized cilia of P. caudatum. Their study revealed that the basal region of the permeabilized cilium is the most sensitive to Ca2+ in inducing ciliary reversal. In contrast, Tamm and Tamm (1989) showed that the Ca2+ sensitivity extended the length of cilia, using detergentpermeabilized macrocilia of Beroë mitrata. In an effort to more faithfully represent the natural conditions of cilia, however, we used living Paramecia, rather than a detergent-permeabilized system in the present study, to prevent any possible loss of Ca2+ sensitivity through the permeabilization procedure. We adopted a photolysis method, instead, in which caged calcium (o-nitrophenyl EGTA [NP-EGTA]) was first injected into intact P. caudatum cells, followed by application of a UV-light pulse to a restricted area to release Ca2+ from the NP-EGTA. The results of this study suggest strongly that Ca2+ causes ciliary reversal by acting upon the entire cilium above the basal body. 1163 The Journal of Experimental Biology 206, 1163-1170 © 2003 The Company of Biologists Ltd doi:10.1242/jeb.00219