The circadian bioluminescence rhythm of Gonyaulax is related to daily variations in the number of light-emitting organelles LAWRENCE FRITZ, DAVID MORSE and J. W. HASTINGS The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA Summary The number of scintillons, which are cellular or- ganelles responsible for light emission in the mar- ine alga Gonyaulax, were counted by both immuno- fluorescence and electron microscopic methods and found to vary tenfold between subjective day and subjective night. The number of scintillons peaks during the subjective night, as does stimulated bioluminescence (flashing). Furthermore, the num- ber drops sharply at the time of the maximal spontaneous bioluminescence (glow), which occurs at the end of the night phase, suggesting that the breakdown of scintillons may be responsible for this mode of emission. Key words: circadian rhythms, bioluminescence, dinoflagellates. Introduction Bioluminescence in the marine dinoflagellate Gonyaulax polyedra occurs in two different modes, both of which exhibit daily rhythmicity (Hastings and Dunlap, 1986). Light emission following stimulation occurs as brief (100 ms) bright flashes (flash peak intensity ~10 9 quanta s" 1 cell" 1 ); it is maximal in the middle of the night and minimal during the day. The second mode, a low intensity bioluminescent glow (~10 4 quanta s~ cell" ), is maximal just prior to dawn. These rhythms continue even in cells maintained under conditions of constant light and temperature and are thus regulated by the circadian clock (Hastings, 1959; Johnson and Hastings, 1986; Sweeney, 1987). In Gonyaulax, flashing has been shown by image- intensified video microscopy to emanate from discrete subcellular organelles termed scintillons (Johnson et al. 1985). Scintillons can also be observed in the living cell by the endogenous fluorescence of luciferin, the substrate in the bioluminescent reaction. The cellular localization and ultrastructural features of the organelles have re- cently been elucidated using immunocytochemical tech- niques (Nicolas et al. 1987). The small (—0.5 [J,m) spherical organelle has a specialized dense matrix and is topologically a part of the cytoplasmic compartment, occurring, in effect, as an evagination protruding into the cell vacuole (Fig. 1). Gonyaulax luciferase, the enzyme involved in the bioluminescent reaction, is rhythmic in its activity (McMurry and Hastings, 1972), and the changes are related to its actual de novo synthesis and destruction (Dunlap and Hastings, 1981; Johnson et al. 1984). A Journal of Cell Science 95, 321-328 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 second protein associated with the luminescent reaction, Gonyaulax luciferin binding protein (LBP), has also been shown to exhibit a circadian rhythm, in its activity (Sulzman et al. 1978), its synthesis, and its abundance (Morse et al. 1989). Initial attempts to relate the circadian rhythm to ultrastructural changes in the amounts of these proteins in the scintillons were unsuccessful: the immunocyto- chemical labeling of scintillons with antibodies to lucifer- ase and LBP appeared equally strong in cells fixed at different times of day. As described here, the difference between day- and night-phase cells lies instead in the number of the organelles. There are approximately 10 times more scintillons in cells fixed at mid-night than in those fixed during mid-day. This change in the number of scintillons continues under constant conditions and thus also constitutes a circadian rhythm. The results further suggest that the daily breakdown of scintillons may account for the low-intensity glow emission. Materials and methods Gonyaulax polyedra (strain 70) cells were grown in alternating light-dark cycles of 12h each (LD, 12:12) in 1.51 volumes of f/2 medium (Guillard and Ryther, 1962) in 2.81 Fernbach flasks with illumination provided by cool white fluorescent lights (100ftEinsteinsM~ 2 s~') at 20°C±2deg. C. Living cells were examined for luciferin fluorescence with a Zeiss epifluorescence microscope using a Zeiss no. 487718 filter set (excitation, 395 nm; emission, 450nm). Bioluminescence was detected with a photomultiplier photometer and recorded graphically (Sweeney and Hastings, 1958); light intensity is expressed in quanta/s (Hastings and Weber, 1963). 321