Circadian rhythmicity of protein synthesis in the dinoflagellate, gonyaulax polyedra: a biochemical and radioautographic investigation

Comp. Biochem. Physiol. Vol. 77B, No. 3, pp. 493-500, 1984

0305-0491/84 $3.00 + 0.00 ,~-~'1984 Pergamon Press Ltd

Printed in Great Britain

CIRCADIAN RHYTHMICITY OF PROTEIN SYNTHESIS IN THE DINOFLAGELLATE, G O N Y A U L A X P O L Y E D R A " A BIOCHEMICAL AND RADIOAUTOGRAPHIC INVESTIGATION W. VOLKNANDT and R. HARDELAND I. Zoologisches Institut, Universit~it G6ttingen, Berliner Str. 28, D-3400 G6ttingen, FRG

(Received 11 August 1983) Abstract--1. In constant light and temperature, Gonyaulax polyedra exhibited a free-running circadian rhythm of [3H]leucine incorporation, with a broad maximum occurring between circadian time (CT) 6 and 18. 2. The relative amounts of 70S and 80S protein syntheses were determined in the presence of cycloheximide and anisomycin or chloramphenicol, respectively. 3. With regard to the circadian pattern, the maximum of 70S translation tended to precede that of 80S translation. 4. Circadian changes of incorporation were confirmed by both light and electron microscopic radioautography. 5. Labeled proteins were separated by SDS-polyacrylamide gel electrophoresis. Several individual protein bands showed rhythms of synthesis and/or concentration.

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INTRODUCTION Inhibition o f protein synthesis has been d e m o n s t r a t e d to provoke considerable phase shifts in circadian rhythms o f various organisms [Karakashian and Schweiger, 1976a,b; R o t h m a n and Strumwasser, 1976; Jacklet, 1980a,b; E h r h a r d t et al., 1980 (review); G o o d e n o u g h et al., 1981 ], including Gonyaulax polyedra (Dunlap et al., 1980; Rensing et al., 1980; Taylor et a[., 1982a,b). These findings, suggesting a central role o f protein synthesis in the circadian clock mechanism, led to a study o f the circadian behaviour o f translational activity in the respective species. In this context, it should be noted that particularly the unicellular model organisms of chronobiology are poorly studied with regard to protein synthesis. This holds especially true for Gonyaulax, on which even the basic data on translation are missing in the literature. MATERIALS AND METHODS

Cells Gonyaulax polyedra was obtained in 1975 from Dr J. W. Hastings, Cambridge, MA. Cells were grown in Falcon flasks at 20 + 0.2cC using a modified I"/2 medium [Guillard and Ryther, 1962; Stahr et al., 1980 (modification)] and were subjected to an artificial illumination cycle of LD 12:12 (1200:0 lx). Experiments were carried out in constant light (LL). Cells were harvested at or slightly above a density of 10,000/ml. Cultures of Gonyaulax were unialgal, but could not be efficiently grown in axenic conditions. Therefore contaminating bacteria had to be removed prior to measuring protein synthesis. For this purpose, cells of 80ml suspensions were sedimented at 30g for 5 min and washed thrice in 80 ml of sterile sea water which was diluted to the same extent as the medium. In order to correct for residual bacterial contamination, [3H]leucine incorporation was determined in the last supernatant. Cells of the last sediment were resuspended in ca. 2.5 ml. 493

Aliquots of 1 ml were removed from the concentrated cell suspensions and preincubated for 30min at 20C, in the presence or absence of antibiotics. Anisomycin and cycloheximide were dissolved in sterile medium, chloramphenicol in DMSO; the antibiotics were added in a volume of 10pl. Controls received the same volume of sterile medium or DMSO. After pre-treatment, cells were incubated with lOpl of [3H]leucine for 1 hr at 20~C. Incorporation was stopped by adding 2 ml of 20~o perchloric acid containing 0.1 M leucine. After addition of 2 mg of bovine serum albumin as a carrier (dissolved in l ml of distilled water), precipitation was completed for 15min on ice. After a centrifugation of 20 min at 7000g and 4c'C, the pellets were washed twice in 3 ml of 5~o perchloric acid containing 0.1 M leucine, dissolved in 1 ml 0.15 N NaOH for 4 hr at 60"C and transferred into 10 ml of scintillation cocktail (Quickszint 212, Zinsser, Frankfurt/Main). As a sensitive measure of protein content suitable for low concentrations, fluorescence of tryptophan was determined by means of an Aminco Bowman ratio spectrophotofluorometer with an ellipsoidal condensing system. Cell suspensions were diluted 1:4 with distilled water and homogenized by 25 strokes in a Potte~Elvehjem homogenizer with a tightly fitting Teflon pestle. After removal of cell armours by a 20-min centrifugation at 7000g, tryptophan fluorescence was determined at an excitation wave length of 290nm and an emission of 350nm. Calibration measurements with tryptophan were carried out for each experiment. Within the working range, tryptophan fluorescence of the supernatant showed a linear dependence on extract concentration.

Radioautography Radioautographic procedures were carried out basically according to Williams (1973, 1977a,b). Cells were washed, concentrated and preincubated as described, and subsequently incubated with 20pl of [3H]leucine for 2hr at 2ff~C. To remove the major quantity of extracellular leucine, cells were sedimented twice at 50g for 5 rain. The following steps were performed at 4°C in the dark: fixation was carried

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Fig. 1. Incorporation of [3H]leucine into Gonyaulax protein. (a) Time dependence (10 #l [3H]lcuc ne/'m cell suspension); (b) dependence on [3H]leucine concentration (incubation I hr).

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out using 2~o glutaraldehyde in 0.15 M phosphate buffer, pH 7.3, 0.2 M glucose (l hr); to eliminate the low-molecularweight material, cells were washed 4 times within 24 hr in 10 ml phosphate buffer, pH 7.3, 0.2 M glucose; this step was followed by a final fixation for l hr in 1'~{,OsO4, 0.05 M phosphate buffer, pH 7.3. After washing in 0.05 M phosphate buffer, pH 7.3, the material was dehydrated (ascending ethanol concentrations, 2 x propylene oxide) and embedded in araldite. For the purpose of light microscopic radioautography, sections of 1 pm were layered with Kodak AR-10 stripping film, and after an exposure of 16 days at 4~C the films were developed at 2 0 C for 5 min in Kodak D 19. For electron microscopic radioautography, sections of 50 nm were cut by means of an ultramicrotome Reichert Om U 2, mounted on copper grids, contrasted with uranyl

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Fig. 2. Effects of translational inhibitors on protein synthesis in Gonyaulax. (a) Dependence on duration of preincubation with antibiotic (ehloramphenicol 1 mg/ml, cycloheximide 10 #g/ml, anisomycin 2.5 #g/ml); (b) dependence on dosage of inhibitor (preincubation 30 rain). • • Chloramphenicol, 0 - - 0 cycloheximide, x x anisomycin.

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ation, 0 - - - 0 incorporation in the presence of chloramphenicol (1 mg/ml); (b) incorporation in the presence of • • cycloheximide (lO#g/ml) and of U I - - [ ] anisomycin (2.5 #g/ml). Vertical lines: three-fold SEM.

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Fig. 4. Light microscopic radioautography of [3H]leucine incorporation at two circadian phases. (a) Central section from CT 22-24; (b) central section from CT 16-18; (c) peripheral section from CT 22-24; (d) peripheral section from CT 16-18. c = chloroplasts, n = nucleus, p = P A S body. Magnification, x 939.25.

acetate and lead citrate, and coated with carbon. Ilford L 4 emulsion was applied using a platinum wire loop and developed after 60 days of exposure with Kodak D 19 (3 min, 20°C). Radioautographs were examined in a Zeiss electron microscope 10 B operated at 60 kV.

Gel electrophoresis Cell suspensions concentrated as described were homogenized by 25 strokes in the two-fold volume of 75mM Tris-HC1 buffer, pH7.6, 50mM KCI, 10mM MgCI2, 8%

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sucrose, 2 mM 2-mercaptoethanol and 0.44 mM phenylmethylsulfonylfluoride using a tightly fitting Potter-Elvehjem homogenizer. Homogenates were centrifuged for 1 hr at 40,000 g. Proteins of the supernatant were precipitated by adding 5 #1 of 10°/0sodium deoxycholate/ml and trichloroacetic acid ad 10°J~,and keeping the mixture for 20 min on ice. The sediment was centrifuged for 20 rain at 7000g and the pellet was washed twice with -20~C-cold acetone. The pellets were dissolved in 6~ sodium dodecyl sulphate (SDS), 10~ glycerol, 75 mM Tris HCI, pH 6.8 and 2 mM 2-mercaptoethanol by sonication using a KNL ultrasound generator 281/101 with sonatrode TU 175/1 (intensity 5, 10 sec). In order to inactivate residual protease activity, samples were exposed to 95°C for 4 min. SDS-polyacrylamide gel electrophoresis was carried out in l-mm slab gels according to Laemmli (1970): stacking gel 3~o, separation gel 10~o acrylamide; 100 pg protein per slot; separation 8 hr at 6°C, controlled by a Spinco Duostat RD-2 (Beckman Instruments). Proteins were precipitated in trichloroacetic acid (20 min on ice), stained with Serva Blau G 250, diffusion destained with 20~ glycerol, 20~0 ethanol and 8~o acetic acid, and fixed in 7% acetic acid. Molecular weights were determined using 12 protein standards of 13.7-140kD (Weber and Osborn, 1969, 1975). Gels were cut into l-mm slices and burnt in an Intertechnik Oximat. Radioactivity was measured by means of a PPO MSD toluene scintillation cocktail. Gels were also examined by autofluorography, using DMSO as dehydrating agent, PPO as scintillator, Siemens enhancer foil and Kodak X-Omat R-film [exposure 2 weeks at -70°C; for other details see Bonner and Laskey (1974)]. In this case, cells had been incubated with 10/~1 [3SS]methionine instead of [3H]leucine. Protein concentrations were measured, prior to electrophoresis, by the method of Zaman and Verwilghen (1979). Stained gels were evaluated by means of a Joyce and Loebl microdensitometer 3 CS. Chemicals

Anisomycin and cycloheximide were obtained from Sigma (Munich) and chloramphenicol from Serva (Heidelberg); [3H]leucine > ll0Ci/mmol, 1 mCi/ml) and

[35S]methionine (> 800 Ci/mmol, 10mCi/ml) were purchased from New England Nuclear (Dreieich).

RESULTS With regard to the lack of data on protein synthesis in Gonyaulax, incorporation kinetics and efficiency of translational inhibitors had to be investigated prior to the chronobiological experiments. Under the conditions chosen the incorporation rate showed a linear dependence on both time and [3H]leucine concentration (Fig. 1). Figure 2 demonstrates the effects of the 70S inhibitor, chloramphenicol, and of the 80S inhibitors, cycloheximide and anisomycin. All three antibiotics reached the respective maximal inhibition rate after a preincubation o f ca. 20 min (Fig. 2a). Full suppression of 70S synthesis was achieved at 1 mg/ml chloramphenicol, and of 80S synthesis at 5/~g/ml cycloheximide or 1.25/lg/ml anisomycin (Fig. 2b). A further reduction of incorporation seen at concentrations of 50/~g/ml anisomycin should be due to additional effects on 70S translation, which can be neglected at the dosages normally applied. In constant light, overall incorporation of [3H]leucine exhibits a highly significant circadian rhythm characterized by a broad maximum between CT 6 and 18 (Fig. 3a). In the presence of chloramphenicol, the remaining 80S synthesis reveals a likewise pronounced rhythmicity, however, with maximal values at CT 18. Inhibition of 80S translation by cycloheximide and anisomycin resulted in two very similar curves; the remaining 70S synthesis tended to highest values at CT 6 9 (Fig. 3b). The amounts of 70S and 80S syntheses, as determined with the antibiotics applied, are additive within the range of statistical precision. Light microscopic radioautography of cells from two circadian phases (CT 22 24, CT 16--18) confirms

Fig. 5. Effect of chloramphenicol (1 mg/ml) at CT 1(~18, as demonstrated by light microscopic radioautography. Abbreviations as in Fig. 4. Magnification, x 867.

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Fig. 6. Electron microscopic radioautography of central sections. (a) Incorporation at CT 22-24; (b) incorporation at CT 16-18. Magnification, × 3395.75.

the biochemical results (Fig. 4). At the phase of the minimum, higher grain densities are only seen at or in the vicinity of the nucleus or in the PAS body. The maximum is characterized by a much higher labelling of the other parts of the cell, especially in the periphery. Chloramphenicol, given at the time of the maximum (Fig. 5), exerted a reduction of incorporation, as expected from the data of Fig. 3a.

Electron microscopic radioautography shows, apart from circadian variations in overall density of silver corn filaments, distinct changes in intracellular distribution of labelling (Fig. 6). At the minimum, silver corn filaments are concentrated in typical strandqike structures--besides those found over the nucleus. At the maximum of incorporation, the strands are less pronounced, and labelling is more

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Fig. 7. Electron microscopic radioautography of peripheral sections. (a) Incorporation at CT 22 24; (b) incorporation at CT 16-18. Magnification, x 2890.

isodisperse and much higher in the periphery (cf. also Fig. 7). Gel electrophoretic separation of proteins from the 40,000g supernatant revealed that this fraction contains several constituents with circadian behaviour. Figure 8 shows (a) the gel electrophoretic pattern, (b) the corresponding autofluorograph, (c-g) five examples of protein bands with significant circadian rhythmicity in [3H]leucine incorporation and indications for changes in concentration and (h) an example of an arrhythmic protein band, standing for many others. Certain other protein bands apparently varied with regard to concentration, although rhythms of incorporation could not be detected (Fig. 9).

DISCUSSION

Our data demonstrate circadian rhythmicity in free-running conditions of both 70S and 80S protein syntheses. The high amount of 70S translation, as seen in this investigation, is supported by several findings. Throughout the whole circadian cycle the two types of translation are additive, to give the overall incorporation values. Two structurally completely different blockers of 80S synthesis, cycloheximide and anisomycin, result in the same plateau of inhibition over a wide range of concentrations. The curves of circadian rhythmicity obtained in the presence of these two antibiotics are almost identical. Moreover, the data correspond to the relatively large size of the plastidary compartment within the Gonyaulax cell, as seen by light, electron and autofluorescence microscopy (Volknandt, 1983). Changes in peripheral labelling of the cell parallel

circadian variations observed in intracellular distribution and shape of chloroplasts (cf. Rensing et al., 1980). Furthermore, peripheral labelling is reduced after administration of chloramphenicol (Fig. 5). Although the radioautographs demonstrate changes in grain density and distribution, they do not represent a visualization of protein synthesis sites. In particular, this becomes obvious by the high label over nucleus and PAS body, which must be due to intracellular translocation. The latter organelle has been presumed to possess autophagic digestive functions (Schmitter, 1971; Schmitter and Jurkiewicz, 1981). In this context, also, the exact identification of the strand-like structures seen in the electron microscopic radioautographs is at least problematic; it would require a more detailed study to clarify in which way these strands are related to substructures, surface or vicinity of chloroplasts. Gel electrophoretic separation demonstrates considerable differences in the chronobiological behaviour of the various proteins. Clear-cut rhythms with distinct temporal patterns were seen in several protein bands, whereas many others turned out to be arrhythmic. Moreover, a comparison of incorporation and protein density within the bands revealed both similarities and deviations, indicating differences in synthesis and turnover rates of the individual proteins. Rhythmicity was detected in several, but by no means all, highly labelled and, hence, presumably short-lived proteins. Circadian changes in protein concentration were in general less well expressed. A few examples of presumably existing rhythms in this function are shown in Figs 8 and 9. In the latter, these variations were not accompanied by rhythms of incorporation. This

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might reflect circadian changes of protein turnover, at more or less constant rates of synthesis. However, it also cannot be excluded that this finding resulted from overlapping of proteins with same molecular weight. This reservation in fact has to be made more

generally in this investigation. Remarkably low circadian variations of electrophoretically separated proteins were also seen in particulate preparations from Gonyaulax, especially plastidary and microsomal membranes (Wunram, 1982).

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Acknowledgements--This work was supported by a research grant of the Deutsche Forschungsgemeinschaft to R.H. and a personal grant of the Friedrich-Ebert-Stiftung to W.V. We are thankful to Drs S. and E. Buchner, Max-Planck-lnstitut f/Jr biologische Kybernetik, Tiibingen, for valuable advice in the methodology of electron microscopic radioautography.

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R. HARDELAND

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