VISUALIZATION OF CALCIUM TRANSIENTS CONTROLLING ORIENTATION OF CILIARY BEAT

To image changes in intraciliary Ca controlling ciliary motility, we m icroinjected Ca Green dextran, a visible wavelength fluorescent Ca ind icator, into eggs or two cell stages of the ctenophore Mnemiopsis leid yi. The embryos developed normally into free-swimming, similar to 0.5 mm cydippid larvae with cells and ciliary comb plates (similar to 100 mu m long) loaded with the dye. Comb plates of larvae, like those of a dult ctenophores, undergo spontaneous or electrically stimulated rever sal of beat direction, triggered by Ca influx through voltage-sensitiv e Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluore scence intensity peaks rapidly (34-50 ms), then slowly falls to restin g level in similar to 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulu s pulse train, and slowly decline in 1-2 s. In both spontaneous and el ectrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms ( 1 video field) and to a similar relative extent above resting level fr om base to tip of the cilia. The decline of fluorescence intensity als o begins simultaneously and proceeds at similar rates along the ciliar y length. Ca-free sea water reversibly abolishes spontaneous and elect rically stimulated Ca Green ciliary emissions as well as reversed beat ing. Calculations of Ca diffusion from the ciliary base show that Ca m ust enter the comb plate along the entire length of the ciliary membra nes. The voltage-dependent Ca channels mediating changes in beat direc tion are therefore distributed over the length of the comb plate cilia . The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat c ycle.