The circadian bioluminescence rhythm of Gonyaulax is related to daily variations in the number of light-emitting organelles

Size: px

Start display at page:

Download "The circadian bioluminescence rhythm of Gonyaulax is related to daily variations in the number of light-emitting organelles"

Cassandra Webster
5 years ago
Views:

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.

1 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 organelles responsible for light emission in the marine alga Gonyaulax, were counted by both immunofluorescence 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 number 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 imageintensified 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 recently been elucidated using immunocytochemical techniques (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, (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 immunocytochemical labeling of scintillons with antibodies to luciferase 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 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

2 Cytoplasmic membrane Theca Cytoplasm neutral ph Action potential triggers proton flux H + Fig. 1. Schematic representation of scintillon structure and localization, along with the proposed mechanism for triggering the bioluminescent flash of Gonyaulax. The individual organelles (scintillons) responsible for bioluminescence protrude as cytoplasmic outpocketings into the vacuole, which is highly complex and branching. A stimulus elicits an action potential, which is propagated in the vacuolar membrane and causes a proton flux from the low ph vacuole into the scintillon. The lowered ph within the scintillon triggers the reaction and a flash of light is emitted (Hastings and Dunlap, 1986; Nicolas et al. 1987). For immunofluorescence microscopy, cells were harvested by centrifugation, fixed in 3 % glutaraldehyde in 0.4M-phosphate buffer, ph7.4, for lh followed by 2min in buffered 1% osmium tetroxide. Cells were dehydrated through an ethanol series and embedded in LR white resin. Sections (0.2 fim thick) were cut on a diamond knife, placed on glass slides in an antigen blocking buffer (10mM-Tris-HCl, ph7.4, ISOmM-NaCI, 1.5% bovine serum albumin (BSA)) for 30min, followed by overnight incubation at 4 C in a primary antibody (anti-lbp) diluted 1/50 in the blocking buffer. After washing with the same buffer to remove unbound antibody, the sections were incubated for 2h in a 1/100 dilution of secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG), washed three times in buffer and examined under the fluorescence microscope with the Zeiss filter set no (450 nm excitation/520nm emission). For electron microscopy, the same fixation and labeling procedures were used, but 0.1 fun sections were cut and placed on 150 mesh nickel electron microscopy grids, with goldconjugated (15 nm) goat anti-rabbit as the secondary antibody. Results and discussion The endogenous blue fluorescence of Gonyaulax cells, which is due to the substrate molecule luciferin, is much greater at night than by day (Fig. 2; and Johnson et al. 1985). The possibility that this might be attributable to differences in the numbers of scintillons, which are located cortically, was difficult to evaluate in living cells, because fluorescence emission from out-of-focus scintillons above and below the focal plane obscured, especially at night-time, those scintillons in focus. Quantitative measurements were obtained by labeling thin sections with a polyclonal antibody against LBP followed by a fluorescein (FITC)-labeled secondary antibody. With cells maintained under a LD12:12 regime, and fixed at mid-night and at mid-day, the number of scintillons per cell section was determined to be 3.2±0.08 (S.E., n-143) and 0.32±0.05 (s.e., «=118). Selected examples of cell sections treated in this way are shown in Fig. 3. On the basis of the average cell diameter (40 Um), section thickness (0.2 ^m) and approximate scintillon diameter (0.5 fim), there are approximately 320 scintillons per night-phase cell and 32 per day-phase cell, a tenfold variation. The calculation was made by assuming that a given scintillon would be seen in two sections, and that there are 200 such sections. Since sections are labeled on only one side, and the antibody does not penetrate the section, this number could be lower than the actual value. Similar experiments were performed using a polyclonal rabbit anti-luciferase (Fig. 4). In this case, however, scintillon numbers are confounded by the presence of the numerous trichocysts (about 70 per section), which are also immunoreactive (Nicolas et al. 1985, 1987). The trichocysts are cigar-shaped ejectile organelles, probably used for defense, and are not believed to play a role in bioluminescence. In cross-section, a stained trichocyst has a dark central core, where the non-immunoreactive trichocyst protein is located. But those sectioned at the tips cannot be readily distinguished from scintillons. As seen in Fig. 4, their numbers remain constant with time. Counts of immunolabeled sections gave 74±7 and 66±8 trichocysts per section («=11 for each) from night and day phases, respectively. Scintillons are characteristically electron-dense organelles, and occur primarily along the cell periphery just internal to the cell's amphiesma (cortex). Since the antibody utilized in the immunolabeling recognizes a protein present within the scintillon and not the organelle itself, the change in the number of the organelles was confirmed by direct counts in cell sections, using the transmission electron microscope. Night- and day-phase scintillons appeared similar in size and electron density, but very different in numbers. An average of 6.7±1.1 (s.e., n = l) scintillons were counted in sections of nightphase cells (Fig. 5), whereas very few were observed in day-phase cells 0.57±0.3 (s.e., n = l) (Fig. 6). These numbers correspond to 540 and 46 scintillons per cell in night-phase and day-phase cells, respectively, calculated by assuming that a given scintillon is seen in five sections and that there are 400 such sections per cell. The identity of the dense bodies as scintillons was confirmed by immunogold labeling with anti-lbp (Fig. 5, inset); only the dense bodies were specifically 322 L. Fritz et al.

3 Fig. 2. In vivo fluorescence of cells from night (A) and day (B) phase, grown in LD, 12: 12. The blue fluorescence of the scintillons, which is due to the substrate (luciferin), co-localizes with in vivo bioluminescence. Day-phase cells are less fluorescent and appear to possess fewer scintillons than night-phase cells. The red background is due to the fluorescence of chlorophyll. Bar, 15 jxm.

5 Fig. 3. Cell sections from night- (A) and day- (B) phase cells, immunolabeled with a polyclonal antibody to LBP. Gonyaulax cells from LD, 12: 12 were fixed at mid-night and mid-day, respectively. Thin sections of cells were immunolabeled with rabbit anti-lbp followed by FITC-conjugated goat anti-rabbit secondary antibody. Night-phase cells show numerous immunoreactive particles (scintillons) at the cell periphery, whereas day-phase cells do not. Scintillon numbers per cell are based on counts of more than 100 cell sections such as these. The reason for the staining of the nucleus is not known. Bar, 10 f.lm. Circadian rhythm in numbers of scintillons 323

6 Fig. 4. Cell sections from night- (A) and day- (B) phase cells immunolabeled with polyclonal anti-luciferase. Cells were harvested and fixed as in Fig. 3, but here are labeled with a primary antibody against luciferase. Scintillons are difficult to identify because of the many trichocysts, which also label. Trichocysts are seen in both cross- and longitudinal sections; their labeling does not vary with time of day; counts of night- and day-phase cell sections averaged 74±7 (» = 11) and 66±7 (;?=11), respectively. The luciferase antibody thus recognizes, in addition to luciferase in scintillons, a different and non-circadian protein in the trichocysts. Bar, 10;ttm. 324 L. Fritz et al.

7 Fig. 5. Electron micrographs of cells maintained under LD, 12:12 and fixed at mid-night. A. Low magnification; bar, 5jUm. B. High magnification; bar, 1 jim. Numerous scintillons are observed along the cell periphery (arrowheads). Inset: scintillons from cell sections labeled with rabbit anti-lbp followed by gold-conjugated goat anti-rabbit. Bar, 0.5 ftm. Scintillons (s, arrowheads); nucleus («), with permanently condensed chromosomes, typical in dinoflagellates (Spector, 1984); trichocysts (t); chloroplasts (c); cell wall (w). Circadian rhythm in numbers of scintillons 325

8 Fig. 6. Electron micrographs of cells maintained under LD, 12: 12 and fixed at mid-day. A. Low magnification; bar, 5 j.im. B. High magnification; bar, 1 jiim. No scintillons are evident. Labeling as in Fig. S. 326 L. Fritz et al.

300' a 200-100- a Flash Bioluminescence 9 Scintillons o Glow Bioluminescence 12 24 36 48 Hours under continuous light Fig. 7. Circadian rhythms in bioluminescence and the number of scintillons.

9 300' a a Flash Bioluminescence 9 Scintillons o Glow Bioluminescence Hours under continuous light Fig. 7. Circadian rhythms in bioluminescence and the number of scintillons. Cells were grown in LD, 12:12 and transferred to constant light and temperature (18 C) at zero time. At the times indicated, samples were removed for measurements of glow (O O) and others for counting. For each point, between 30 and 100 sections were counted ( ). The rhythm of bioluminescent flashing (D D), was measured under similar conditions. Glow bioluminescence: one major division equals 10 7 quantas" 1 ; flash bioluminescence: arbitrary units. labeled. These observations thus confirm that the daily variations in numbers of scintillons, as also determined by immunofluorescence, are attributable to variations not only in the protein contents of this organelle, but to the actual numbers of the organelles responsible for bioluminescence. In order to establish the rhythm as circadian, as opposed to direct effects of the light-dark regime, cells were maintained under constant dim light for over 48 h and harvested for fixation and immunolabeling at the times indicated in Fig. 7. During the latter part of the first subjective night (hours 18-24), the number of scintillons per cell decreased dramatically from 260±20 to 8±3. A second peak, only half the amplitude of the first (120+30), was paralleled by the diminished amplitude of the second glow peak. In measurements of the rhythm of photosynthetic 14 CC>2 uptake using the same cell culture, a similarly diminished amplitude of the second photosynthesis peak was also observed (data not shown). Circadian control of physiological processes has generally been viewed as a phenomenon involving a control of reaction rates. In the case of the Gonyaulax bioluminescence rhythm, the circadian rhythm involves the de novo synthesis and degradation of the proteins involved in the light-emitting reaction (Johnson et al. 1984; Morse et al. 1989). The present study shows that the turnover of these proteins actually involves the disappearance and reformation of the entire organelle responsible for bioluminescence. Although this may seem a remarkable or even extreme mechanism, the dissolution of the organelle 60 would have as an expected consequence the dumping and probable breakdown of the proteins it contains. By contrast, trichocyst labeling remains constant between day and night, as visualized with the anti-luciferase antibody, while the luciferase protein itself fluctuates 10- fold in amount (Dunlap and Hastings, 1981; Johnson et al. 1984). Since the membrane of the scintillon is continuous with the vacuolar membrane (tonoplast), the organelle could be readily disposed of by its pinching off and disposal within the vacuole. Alternatively, its disappearance might involve release of its contents back into the cytoplasm, in a process analogous to, but topologically different from, the final stages of exocytosis. Although both possibilities seem reasonable, we favor the latter. However, we have not yet obtained EM images of the stages in the dissolution of scintillons. The time at which the maximum number of scintillons per cell occurs is coincident with the time at which the bioluminescent flashing capacity is maximal, indicating a relationship between the two. In addition to the flashing, cells spontaneously emit a low-intensity glow, which peaks several hours after the maximum in bioluminescent flashing (Brodaeia/. 1986; Johnson and Hastings, 1986). Although cell divisions occur at about the same time (Sweeney and Hastings, 1958), the glow intensity is independent of the number of cells dividing (Homma and Hastings, 1989«,6), and fewer than 10% of the cells actually divided at each cycle in the present experiments. When plotted together with the scintillon numbers (Fig. 7), the glow appears to be correlated with the decrease in the number of scintillons, suggesting that the breakdown of scintillons may give rise to the glow. This work was supported in part by grants to J.W.H. from the National Institutes of Health (GM 19536) and National Science Foundation (DMB-8616S22). References BRODA, H., GOOCH, V. D., TAYLOR, W., AIUTO, N. AND HASTINGS, J. W. (1986). Acquisition of circadian bioluminescence data in Gonyaulax and an effect of the measurement procedure on the period of the rhythm. J. biol. Rliyth. 1, DUNLAP, J. C. AND HASTINGS, J. W. (1981). The biological clock in Gonyaulax controls luciferase activity by regulating turnover. J. biol. Chem. 256, 10S GUILLARD, R. AND RYTHER, J. (1962). Studies on marine phytoplankton diatoms. Can. J. Microbiol. 8, HASTINGS, J. W. (1959). Unicellular clocks. A. Rev. Microbiol. 13, HASTINGS, J. W. AND DUNLAP, J. C. (1986). Cell-free components in dinoflagellate bioluminescence: The paniculate activity: scintillons; the soluble components: luciferase, luciferin, and luciferin binding protein. Meth. Enzym. 133, HASTINGS, J. W. AND WEBER, G. (1963). Total quantum flux of isotropic sources. J. opt. Soc. Am. 53, HOMMA, K. AND HASTINGS, J. W. (1989a). The S phase is discrete and is controlled by the circadian rhythm in the dinoflagellate Gonyaulax polyedra. Expl Cell Res. 182, HOMMA, K. AND HASTINGS, J. W. (19896). Cell growth kinetics, division asymmetry, and volume control at division in the marine dinoflagellate Gonyaulax polyedra: A model of circadian clock control of the cell cycle. J. Cell Sci. 92, JOHNSON, C. H. AND HASTINGS, J. W. (1986). The elusive mechanisms of the circadian clock. Atn. Scient. 7, Circadian rhythm in numbers of scintillons 327

10 JOHNSON, C. H., INOUE, S., FLINT, A. AND HASTINGS, J. W. (1985). Compartmentation of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulax as studied with image intensified video microscopy and flow cytometry. J. Cell Biol. 100, JOHNSON, C. H., ROEBER, J. AND HASTINGS, J. W. (1984). Circadian changes in enzyme concentration account for rhythm of enzyme activity in Gonyaulax. Science 223, KRASNOW, R., DUNLAP, J. C, TAYLOR, W., HASTINGS, J. W., VETTERLING, W. AND'GOOCH, V. D. (1980). Circadian spontaneous bioluminescent glow and flashing of Gonyaulax polyedra. J. comp. Phys. 138, McMuRRY, L. AND HASTINGS, J. W. (1972). Circadian rhythms: Mechanism of luciferase activity changes in Gonyaulax. Biol. Bull, mar. biol. Lab. Woods Hole 143, MORSE, D., MILOS, P. M., Roux, E. AND HASTINGS, J. W. (1989). Circadian regulation of the synthesis of substrate binding protein in the Gonyaulax bioluminescent system involves translational control. Proc. natn. Acad. Sci. U.S.A. 86, NICOLAS, M.-T., JOHNSON, C. H., BASSOT, J.-M. AND HASTINGS, J. W. (1985). Immunogold labeling of organelles in the bioluminescent dinoflagellate Gonyaulax polyedra with antiluciferase antibody. Cell biol. Int. Rep. 9, NICOLAS, M.-T., NICOLAS, G., JOHNSON, C. H., BASSOT, J.-M. AND HASTINGS, J. W. (1987). Characterization of the bioluminescent organelles in Gonyaulax polyedra (dinoflagellates) after fast-freeze freeze fixation and antiluciferase immunogold staining. J. Cell Biol. 105, SPECTOR, D. L. (1984). Dinoflagellate nuclei. In Dinoflagellates (ed. D. L. Spector), pp Academic Press, NY. SULZMAN, F. N., KRIEGER, N. R., GOOCH, V. D. AND HASTINGS, J. W. (1978). A circadian rhythm of the luciferin binding protein from Gonyaulax polyedra. J. comp. Physiol. 128, SWEENEY, B. M. (1987). Rhythmic Phenomena in Plants, 2nd edn. Academic Press, N.Y. SWEENEY, B. M. AND HASTINGS, J. W. (1958). Rhythmic cell division in populations of Gonyaulax polvedra. J. Protozool. 5, (Received 25 September 1989-Accepted 27 October 1989) 328 L. Fritz et al.

Close Window Preview a Different Module

1 of 21 2/12/2006 3:02 PM Close Window Preview a Different Module Dinoflagellate Bioluminescence and its Circadian Regulation by J. Woodland Hastings 1, Liyun Liu 1 and Wayne Schultz 2 1 Department of