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NOTES
Humphreys-Beher, M.G., Schneyer, C.A., Kidd, V.J., and Marchase, R.B. 1987. Isoproterenol-mediated parotid gland hypertrophy is inhibited by effectors of 40-galactosyltransferase. J. Biol. Chem. 262: 11 706 - 11 713. Humphreys-Beher, M.G., Bunnell, B., Van Tuinen, P., et al. 1989. Correction: Molecular cloning and chromosomal localization of human 40-galactosyltransferase.Proc. Natl. Acad. Sci. U.S.A. 86: 8747. Humphreys-Beher, M.G., Zelles, T., Maeda, N., et al. 1990. Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation. Mol. Cell. Biochem. 95: 1-1 1. Hunter, T., and Cooper, J.A. 1986. Viral oncogenes and tyrosine phosphorylation. In The enzymes. Vol. 17. Edited by P.D. Boyer and E.G. Krebs. Academic Press, Orlando. pp. 191-246. Magee, S.C., Mawal, R., and Ebner, K.E. 1974. Multiple forms of galactosyltransferase from bovine milk. Biochemistry, 23: 99-107. Ottaviano, Y., and Gerace, L. 1985. Phosphorylation of the nuclear lamins during interphase and mitosis. J. Biol. Chem. 260: 624-632. Pugsley, A.D., and Schnaitman, C.A. 1979. Factors affecting the electrophoretic mobility of the major outer membrane proteins
of Escherichia coli in polyacrylamide gels. Biochim. Biophys. Acta, 581: 163-178. Purushotham, K.R., and Humphreys-Beher, M.G. 1991. Use of calmodulin agarose ALD in the purification of calcium dependent calmodulin kinase. Focus, 13: 63-65. Purushotham, K.R., Zelles, T., and Humphreys-Beher, M.G. 1991. Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation. Mol. Cell. Biochem. 102: 19-33. Sato, H., Yamauchi, T., and Fujisawa, H. 1990. Purification and characterization of calmodulin dependent protein kinase I1 from rat spleen: a new type of calmodulin dependent protein kinase 11. J. Biochem. (Tokyo), 197: 802-809. Strous, G.J., Van Kerkhof, P., Fullon, R.J., and Schwartz, A.L. 1987. Golgi galactosyltransferase contains serine-linked phosphate. Eur. J. Biochem. 169: 307-31 1. Yamauchi, T., Ohsako, S., and Deguchi, T. 1989. Expression and characterization of calmodulin dependent protein kinase I1 from cloned cDNAs in Chinese hamster ovary cells. J. Biol. Chem. 264: 19 108 - 19 116.
Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants BRADFORD S. HAMILTON Institute of Medical Science and Department of Medicine, Sunnybrook Health Science Centre University of Toronto, Toronto, Ont., Canada M4N 3M5
K ~ z u oNAKAMURA Department of Biological Sciences, University of Lethbridge, Lethbridge, Alta., Canada TIK 3M4 AND
DANIELA. K.
RONCARI'
Institute of Medical Science and Department of Medicine, Sunnybrook Health Science Centre University of Toronto, Toronto, Ont., Canada M4N 3M5 Received July 23, 1991 HAMILTON, B. S., NAKAMLTRA, K., and RONCARI,D. A. K. 1992. Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants. Biochem. Cell Biol. 70: 255-258. Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c + . The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonasreinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms. Key words: Chlamydomonas, cytoskeleton, paralyzed flagella, starch, bioenergetics. K., et RONCARI, D. A. K. 1992. Accumulation of starch in Chlamydomonasreinhardtii HAMILTON,B. S., NAKAMURA, flagellar mutants. Biochem. Cell Biol. 70 : 255-258. Les mutants flagellaires paralysCs pf-1, pf-2, pf-7 et pf-18 de l'algue verte, Chlamydomonasreinhardtii (Dangeard) emmagasinent une quantitt beaucoup plus grande d'amidon que le type sauvage mobile 137c + . Cette augmentation d'emmagasinage de l'amidon est relike de facon importante aux protCines, B la chlorophylle et au nombre de cellules. L'analyse des dimensions moyennes des cellules rCvtle que celles des mutants paralysCs sont plus grandes que celles de type sauvage. Cet accroissement de l'accumulation des mol&ules de storage supporte une relation inverse entre l'emmagasinage de 1'Cnergie chimique et l'utilisation CnergCtique pour les fonctions cefiulaires biomCcaniques/motiles. Chlam~domonasreinhardtii fournit un modtle utile Dour les ttudes du rSle de I'activitC cvtosauelettiaue dans la relation - et lYCq-uilibreCnergCtiques des organismes. Mots elks : Chlamydomonas, cytosquelette, flagelles paralysts, amidon, biotnergttique. [Traduit par la rCdaction] I
Author to whom all correspondence should be sent at the following address: Sunnybrook Health Science Centre, 2075 Bayview Ave., North York, Ont., Canada M4N 3M5. Printed in Canada / Imprime au Canada
BIOCHEM. CELL BIOL. VOL. 70,
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Introduction The precise determinants of energy balance or its perturbations still require elucidation. A particularly important objective has been to learn the mechanisms mediating adaptive thermogenesis. We have postulated that energy utilized for cellular biomechanical functions, which are mediated by the cytoskeleton, constitutes an appreciable quantity of the energy expended by the organism (Roncari 1987). According to our hypothesis, moreover, after energy utilization for life-sustaining processes such as maintenance of electrochemical gradients across membranes, a significant portion of the extra energy that is not consumed by biomechanical processes is converted to chemical energy storage. The unicellular green alga Chlamydomonas reinhardtii provides a valuable model for studies of energy fluxes and transductions. This organism is both photoautotrophic and heterotrophic, and under each condition, the amount of nutrient energy as well as the details of the nutritional requirements are known (Harris 1989). Starch is the main form of chemical energy storage in Chlamydomonas.Some information is available regarding the amounts stored under different conditions, including the important interrelationship between starch content and degree of cell division (Cohen and Parnas 1976; Craigie and Cavalier-Smith 1982; Voight and Munzner 1987; Ball et al. 1990). During darkness, the energy required for cell division is derived from stored energy and is thus associated with diminishing amounts of starch (Harris 1989). In these energy relationships, the potentially large contribution of the energy utilized for biomechanical functions has been largely ignored. In this context, C. reinhardtii is useful because several mutants have been characterized in detail at the genetic level (Ebersold et al. 1962; McVittie 1972). Flagella, particularly in unicellular organisms, must consume a large proportion of the available energy through the dynein ATPase of the microtubules which power these organelles (Alberts et al. 1989). Thus, the Chlamydomonas mutants may provide a useful model for studies of the role of biomechanical functions in overall bioenergetics, as described in this study. Materials and methods Strains Chlamydomonas reinhardtii (Dangeard) wild-type strain 137c + (obtained from Dr. N. Gillham, Duke University, Durham, N.C.) and flagellar mutants pf-1 (mt+), pf-2 (mt'), pf-7 (mt-), and pf-18 (mt') (Ebersold et al. 1962; McVittie 1972) (obtained from Dr. E. Harris. Chlamydomonas Genetics Centre, Duke University, Durham. N.C.) were used. Growth The cells were grown with continuous stirring at 2S°C, in 500-mL Erlenmeyer flasks containing a modified Gowans' (1960)minimal medium, made by substituting NH4NOSwith NH,Cl (0.1 g/L). Constant illumination (28.1 pE .m -'s - ') was provided by General Electric Daylight fluorescent bulbs. Fourteen days after inoculation, similar growing log-phase cultures were harvested for experiments by centrifugation. Analysis Starch was determined by a modification of the method of Gfeller and Gibbs (1984). After centrifugation (6000 x g for 5 min) of the cell suspensions, the pellets were resuspended in 0.1 mL of methanol, washed with 2 mL of methanol, washed with
-
1992
FIG. 1. Starch per cell in Chlamydomonas reinhardtii wild type (137c +) and flagellar mutants (pf-1, pf-2, pf-7, and pf-18). Means + SE are shown (n = 3). The difference in storage of starch between the wild type and each of the murants was significant at P < 0.001. 2 mL of sodium acetate (100 mM, pH 4.9, and resuspended in
1.7 mL of the acetate buffer. The samples were then sonicated and boiled for 10 min to solubilize the starch. Amyloglucosidase(2.2 U, pH 4.5; 1 U liberates 1.0 mg glucose from starch in 3 min at 55°C) was added and the samples were incubated at 55°C for 14 h. After clarification by centrifugation, glucose in the supernatant was determined with a Sigma assay kit (510-A). Chlorophyll was extracted with 80% acetone and its content was measured by the method of Arnon (1949). Protein was determined according to Bradford (1976). Cell number was enumerated with a hemacytometer (Ultra Plane Spot lite Counting Chamber). Cell size was determined with a Becton/Dickinson cell sorter. Standard curves were made with latex beads of known diameter and the mean cell size was obtained using regression curves. Statistical analysis was carried out by the Student's t-test, performed with Statworks (Version 1.2) on a Macintosh IIx computer. Results Starch content in the mutants and wild type was examined relative to cell number, protein, and chlorophyll. Figure 1 indicates that, irrespective of the mutation, the nonmotile cells stored significantly ( P < 0.001) greater amounts of starch per cell number, ranging from 1.6-2.5 times that stored in the wild type (137c +). As there might be some problem with determining accurate cell number because of the possibility of mutant daughter cells remaining inside the mother cell wall (Nakamura et al. 1978), additional criteria were examined. The content of starch relative to protein was also significantly ( P < 0.001) increased in the pf mutants, on the average 1.5 times that in 137c + (Fig. 2). Relative to chlorophyll, moreover, mutant cells stored significantly ( P < 0.001, or P < 0.01 for pf-2) larger quantities of starch (Fig. 3). Representative size curves of 1 x lo4 cells from each strain are shown in Fig. 4. The curve for each of the mutants turned out to lie to the right of the wild type, indicating that the majority of each strain's cells were larger than those of 137c+. Discussion This study has shown that flagellar impairment in Chlamydomonasreinhardtii results in augmented storage of starch, the major form of macromolecular chemical energy
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FIG. 2. Starch relative to total protein in Chlamydomonas reinhardtii wild type (137c+) and flagellar mutants (pf-1, pf-2, pf-7 and pf-18). Means SE are shown (n = 3). The difference in storage of starch between the wild type and each of the mutants was significant at P < 0.001.
*
storage in C. reinhardtii. This finding may represent a general principle in bioenergetic interactions. In fact, the extra energy made available by decreased flagellar work could conceivably be utilized and (or) released through increased chemical oxidation, futile biochemical (substrate) cycles, and thermogenesis. But this study has indicated that at least part of the energy is retained for anabolic processes, particularly the formation of starch. Thus, these findings suggest an inverse relationship between energy utilized for cellular motility, at least in the form of flagellar function, and chemical energy storage. More specifically, genetically determined flagellar dysfunctions of the unicellular green alga C. reinhardtii are associated with accumulation of starch. Seemingly, this organism does not have a mechanism for consuming energy beyond that used for enhanced growth, or dissipating the energy that would have been utilized for normal flagellar function. As an example of lack of stringency in regulating the content of starch, Ball et al. (1990) have shown that when growth is restricted by limiting specific nutrients, large amounts of starch accumulate, irrespective of whether the cells are grown as photoautotrophs or heterotrophs. Similarly, de Hostos et al. (1988) have reported that cells deprived of sulfate cease to divide, enlarge, and accumulate starch. These findings suggest that limitation of specific nutrients coupled with provision of adequate energy, leads to accumulation of macromolecular stores. Duration and quality of light influence cell size and replicative rate of the alga. Jones (1970) has reported that at high light intensities, cell size after 6 h is equal to the cell size after 12 h at lower intensity. In Chlamydomonas, moreover, characteristic cell size is maintained by the number of divisions, the mother cell continuing to divide until a minimal volume of daughter cells is reached. In general, there is a positive correlation between intensity/ duration of the photoperiod and cell size/extent of division (Cohen and Parnas 1976; Craigie and Cavalier-Smith 1982; Voight and Munzner 1987; Harris 1989). In addition to the light energy requirements, starch accumulation in Chlamydomonas is dependent on carbon supply. Indeed, Kuchitsu et al. (1988) have found that a high ambient C 0 2 content and acetate supplementation result in increased
FIG. 3. Starch relative to total chlorophyll in Chlamydomonas reinhardtii wild type (137c +) and flagellar mutants @f-1, pf-2, pf-7 and pf-18). Means + SE are shown (n = 3). The difference in storage of starch between the wild type and the mutants was significant at P < 0.001, except pf-2 where the difference was significant at P < 0.01.
u ?me+
-
ROO
m
pr-7 pr-1%
4(KI
200
0
0
5
10
15
20
25
DIAMETER (elm)
FIG.4. Representative size distribution of Chlamydomonas reinhardtii wild type (137c+) and flagellar mutants (pf-1, pf-2, pf-7, and pf-18). The mean diameter for the wild type was 8.06 pm, while pf-1, pf-2, pf-7, and pf-18 diameters were 8.88,9.71, 10.78, and 9.96 am, respectively.
storage. These observations would suggest that under conditions of optimal growth, an increase in energy supply results in greater storage of chemical energy. Thus, both selective restriction of cell division and (or) increase of available energy result in an expanded starch content in Chlamydomonas. These findings indicate that the extent of macromolecular storage is not regulated stringently, at least under certain conditions. In general, cell differentiation, with its associated anabolism, including storage of chemical energy, onsets at or after cessation of replication (Umek et al. 1991). However, the postreplicative synthetic processes are modulated by different regulatory mechanisms including the degree of cytoskeletal activity, as suggested by our findings. The degree of chemical energy storage could vary through a wide range, as an inverse function of the energy utilized by the cytoskeleton. While it is tempting to generalize the inverse relationship between cellular motility and chemical energy storage to include all living forms, experimental verification is
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obviously required. However, it may well be relevant that agents that interfere with cytoskeletal functions (i.e., cytochalasin B, Colcemid, and trifluoperazine) lead to accumulation of triacylglycerol in cultured human preadipocytes (Healy 1987). While there has been increasing recognition of the critical role of the cytoskeleton in numerous cell functions, the contribution of these activities to the energy interactions of an organism has not been appreciated. It is nonetheless highly probable that the various motile functions consume relatively large quantities of energy (Roncari 1987; Alberts et al. 1989).
Acknowledgements The authors thank Christine A. Goertzen for her technical assistance. This research was supported by grant MT-8460 of the Medical Research Council of Canada, a grant from the Heart and Stroke Foundation of Canada (Ontario), and an operating grant from the Natural Sciences and Engineering Research Council of Canada (A-6224). AIberts, B., Bray, D., Lewis, J., et al. 1989. Molecular biology of the cell. 2nd ed. Garland Publishing Inc., New York. pp. 41-86. Arnon, D. 1949. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol. 24: 1-15. Ball, S.G., Dirick, L., Decq, A., et al. 1990. Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii. Plant Sci. (Limerick, Irel.), 66: 1-9. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Cohen, D.. and Parnas, H. 1976. An optimal policy for the metabolism of storage materials in unicellular algae. J. Theor. Biol. 56: 1-18. Craigie, R.A., and Cavalier-Smith, T. 1982. Cell volume and the control of the Chlamydomonas cell cycle. J. Cell Sci. 54: 173-191.
1992
De Hostos, E.L., Togasaki, R.K., and Grossman, A. 1988. Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonasreinhardtii. J. Cell Biol. 106: 29-37.
Ebersold, W.T., Levine, R.P., Levine, E.E., and Olmsted, M.A. 1962. Linkage maps in Chlamydomonas reinhardtii. Genetics, 47: 531-543. Gfeller, R.P., and Gibbs, M. 1984. Fermentative metabolism of Chlamydomonas reinhardtii. Plant Physiol. 75: 212-21 8. Gowans, C.S. 1960. Some genetic investigations on Chlamydomonas eugametos. Z . Vererbungsl. 91: 63-73. Harris, E.H. 1989. The Chlarnydomonas sourcebook. Academic Press, Toronto. Healy, I.A. 1987. Influence of inhibitors of cytoskeletal function on triacylglycerol accretion in cultured adipocyte precursors. M.Sc. thesis, University of Calgary, Calgary, Alta. Jones, R.F. 1970. Physiological and biochemical aspects of growth and gametogenesis in Chlamydomonas reinhardtii. Ann. N.Y. Acad. Sci. 175: 648-659. Kuchitsu, K., Tsuzuki, M., and Miyachi, S. 1988. Changes of starch localization within the chloroplast induced by changes in COz concentration during growth of Chlamydomonas reinhardtii: independent regulation of pyrenoid starch and stroma starch. Plant Cell Physiol. 29: 1269-1278. McVittie, A. 1972. Genetic studies on flagellum mutants of Chlamydomonas reinhardtii. Genet. Res. 19: 157-164. Nakamura, K., Bray, D.F., and Wagenaar, E.B. 1978. Ultrastructure of palmelloid-forming strain of Chlamydomonas eugametos. Can. J . Bot. 56: 2348-2356. Roncari, D.A.K. 1987. Individual variations in energy utilized biomechanical processes and molecular mobility account for diverse susceptibility to obesity. Med. Hypotheses, 23: 11-18. Umek, R.M., Friedman, A.D., and McKnight, S.L. 1991. CCAATenhancer binding protein: a component of a differentiation switch. Science (Washington, D.C.), 251: 288-292. Voight, J., and Munzner, P. 1987. The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch. Planta, 172: 463-472.
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NOTES
Humphreys-Beher, M.G., Schneyer, C.A., Kidd, V.J., and Marchase, R.B. 1987. Isoproterenol-mediated parotid gland hypertrophy is inhibited by effectors of 40-galactosyltransferase. J. Biol. Chem. 262: 11 706 - 11 713. Humphreys-Beher, M.G., Bunnell, B., Van Tuinen, P., et al. 1989. Correction: Molecular cloning and chromosomal localization of human 40-galactosyltransferase.Proc. Natl. Acad. Sci. U.S.A. 86: 8747. Humphreys-Beher, M.G., Zelles, T., Maeda, N., et al. 1990. Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation. Mol. Cell. Biochem. 95: 1-1 1. Hunter, T., and Cooper, J.A. 1986. Viral oncogenes and tyrosine phosphorylation. In The enzymes. Vol. 17. Edited by P.D. Boyer and E.G. Krebs. Academic Press, Orlando. pp. 191-246. Magee, S.C., Mawal, R., and Ebner, K.E. 1974. Multiple forms of galactosyltransferase from bovine milk. Biochemistry, 23: 99-107. Ottaviano, Y., and Gerace, L. 1985. Phosphorylation of the nuclear lamins during interphase and mitosis. J. Biol. Chem. 260: 624-632. Pugsley, A.D., and Schnaitman, C.A. 1979. Factors affecting the electrophoretic mobility of the major outer membrane proteins
of Escherichia coli in polyacrylamide gels. Biochim. Biophys. Acta, 581: 163-178. Purushotham, K.R., and Humphreys-Beher, M.G. 1991. Use of calmodulin agarose ALD in the purification of calcium dependent calmodulin kinase. Focus, 13: 63-65. Purushotham, K.R., Zelles, T., and Humphreys-Beher, M.G. 1991. Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation. Mol. Cell. Biochem. 102: 19-33. Sato, H., Yamauchi, T., and Fujisawa, H. 1990. Purification and characterization of calmodulin dependent protein kinase I1 from rat spleen: a new type of calmodulin dependent protein kinase 11. J. Biochem. (Tokyo), 197: 802-809. Strous, G.J., Van Kerkhof, P., Fullon, R.J., and Schwartz, A.L. 1987. Golgi galactosyltransferase contains serine-linked phosphate. Eur. J. Biochem. 169: 307-31 1. Yamauchi, T., Ohsako, S., and Deguchi, T. 1989. Expression and characterization of calmodulin dependent protein kinase I1 from cloned cDNAs in Chinese hamster ovary cells. J. Biol. Chem. 264: 19 108 - 19 116.
Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants BRADFORD S. HAMILTON Institute of Medical Science and Department of Medicine, Sunnybrook Health Science Centre University of Toronto, Toronto, Ont., Canada M4N 3M5
K ~ z u oNAKAMURA Department of Biological Sciences, University of Lethbridge, Lethbridge, Alta., Canada TIK 3M4 AND
DANIELA. K.
RONCARI'
Institute of Medical Science and Department of Medicine, Sunnybrook Health Science Centre University of Toronto, Toronto, Ont., Canada M4N 3M5 Received July 23, 1991 HAMILTON, B. S., NAKAMLTRA, K., and RONCARI,D. A. K. 1992. Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants. Biochem. Cell Biol. 70: 255-258. Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c + . The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonasreinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms. Key words: Chlamydomonas, cytoskeleton, paralyzed flagella, starch, bioenergetics. K., et RONCARI, D. A. K. 1992. Accumulation of starch in Chlamydomonasreinhardtii HAMILTON,B. S., NAKAMURA, flagellar mutants. Biochem. Cell Biol. 70 : 255-258. Les mutants flagellaires paralysCs pf-1, pf-2, pf-7 et pf-18 de l'algue verte, Chlamydomonasreinhardtii (Dangeard) emmagasinent une quantitt beaucoup plus grande d'amidon que le type sauvage mobile 137c + . Cette augmentation d'emmagasinage de l'amidon est relike de facon importante aux protCines, B la chlorophylle et au nombre de cellules. L'analyse des dimensions moyennes des cellules rCvtle que celles des mutants paralysCs sont plus grandes que celles de type sauvage. Cet accroissement de l'accumulation des mol&ules de storage supporte une relation inverse entre l'emmagasinage de 1'Cnergie chimique et l'utilisation CnergCtique pour les fonctions cefiulaires biomCcaniques/motiles. Chlam~domonasreinhardtii fournit un modtle utile Dour les ttudes du rSle de I'activitC cvtosauelettiaue dans la relation - et lYCq-uilibreCnergCtiques des organismes. Mots elks : Chlamydomonas, cytosquelette, flagelles paralysts, amidon, biotnergttique. [Traduit par la rCdaction] I
Author to whom all correspondence should be sent at the following address: Sunnybrook Health Science Centre, 2075 Bayview Ave., North York, Ont., Canada M4N 3M5. Printed in Canada / Imprime au Canada
BIOCHEM. CELL BIOL. VOL. 70,
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256
Introduction The precise determinants of energy balance or its perturbations still require elucidation. A particularly important objective has been to learn the mechanisms mediating adaptive thermogenesis. We have postulated that energy utilized for cellular biomechanical functions, which are mediated by the cytoskeleton, constitutes an appreciable quantity of the energy expended by the organism (Roncari 1987). According to our hypothesis, moreover, after energy utilization for life-sustaining processes such as maintenance of electrochemical gradients across membranes, a significant portion of the extra energy that is not consumed by biomechanical processes is converted to chemical energy storage. The unicellular green alga Chlamydomonas reinhardtii provides a valuable model for studies of energy fluxes and transductions. This organism is both photoautotrophic and heterotrophic, and under each condition, the amount of nutrient energy as well as the details of the nutritional requirements are known (Harris 1989). Starch is the main form of chemical energy storage in Chlamydomonas.Some information is available regarding the amounts stored under different conditions, including the important interrelationship between starch content and degree of cell division (Cohen and Parnas 1976; Craigie and Cavalier-Smith 1982; Voight and Munzner 1987; Ball et al. 1990). During darkness, the energy required for cell division is derived from stored energy and is thus associated with diminishing amounts of starch (Harris 1989). In these energy relationships, the potentially large contribution of the energy utilized for biomechanical functions has been largely ignored. In this context, C. reinhardtii is useful because several mutants have been characterized in detail at the genetic level (Ebersold et al. 1962; McVittie 1972). Flagella, particularly in unicellular organisms, must consume a large proportion of the available energy through the dynein ATPase of the microtubules which power these organelles (Alberts et al. 1989). Thus, the Chlamydomonas mutants may provide a useful model for studies of the role of biomechanical functions in overall bioenergetics, as described in this study. Materials and methods Strains Chlamydomonas reinhardtii (Dangeard) wild-type strain 137c + (obtained from Dr. N. Gillham, Duke University, Durham, N.C.) and flagellar mutants pf-1 (mt+), pf-2 (mt'), pf-7 (mt-), and pf-18 (mt') (Ebersold et al. 1962; McVittie 1972) (obtained from Dr. E. Harris. Chlamydomonas Genetics Centre, Duke University, Durham. N.C.) were used. Growth The cells were grown with continuous stirring at 2S°C, in 500-mL Erlenmeyer flasks containing a modified Gowans' (1960)minimal medium, made by substituting NH4NOSwith NH,Cl (0.1 g/L). Constant illumination (28.1 pE .m -'s - ') was provided by General Electric Daylight fluorescent bulbs. Fourteen days after inoculation, similar growing log-phase cultures were harvested for experiments by centrifugation. Analysis Starch was determined by a modification of the method of Gfeller and Gibbs (1984). After centrifugation (6000 x g for 5 min) of the cell suspensions, the pellets were resuspended in 0.1 mL of methanol, washed with 2 mL of methanol, washed with
-
1992
FIG. 1. Starch per cell in Chlamydomonas reinhardtii wild type (137c +) and flagellar mutants (pf-1, pf-2, pf-7, and pf-18). Means + SE are shown (n = 3). The difference in storage of starch between the wild type and each of the murants was significant at P < 0.001. 2 mL of sodium acetate (100 mM, pH 4.9, and resuspended in
1.7 mL of the acetate buffer. The samples were then sonicated and boiled for 10 min to solubilize the starch. Amyloglucosidase(2.2 U, pH 4.5; 1 U liberates 1.0 mg glucose from starch in 3 min at 55°C) was added and the samples were incubated at 55°C for 14 h. After clarification by centrifugation, glucose in the supernatant was determined with a Sigma assay kit (510-A). Chlorophyll was extracted with 80% acetone and its content was measured by the method of Arnon (1949). Protein was determined according to Bradford (1976). Cell number was enumerated with a hemacytometer (Ultra Plane Spot lite Counting Chamber). Cell size was determined with a Becton/Dickinson cell sorter. Standard curves were made with latex beads of known diameter and the mean cell size was obtained using regression curves. Statistical analysis was carried out by the Student's t-test, performed with Statworks (Version 1.2) on a Macintosh IIx computer. Results Starch content in the mutants and wild type was examined relative to cell number, protein, and chlorophyll. Figure 1 indicates that, irrespective of the mutation, the nonmotile cells stored significantly ( P < 0.001) greater amounts of starch per cell number, ranging from 1.6-2.5 times that stored in the wild type (137c +). As there might be some problem with determining accurate cell number because of the possibility of mutant daughter cells remaining inside the mother cell wall (Nakamura et al. 1978), additional criteria were examined. The content of starch relative to protein was also significantly ( P < 0.001) increased in the pf mutants, on the average 1.5 times that in 137c + (Fig. 2). Relative to chlorophyll, moreover, mutant cells stored significantly ( P < 0.001, or P < 0.01 for pf-2) larger quantities of starch (Fig. 3). Representative size curves of 1 x lo4 cells from each strain are shown in Fig. 4. The curve for each of the mutants turned out to lie to the right of the wild type, indicating that the majority of each strain's cells were larger than those of 137c+. Discussion This study has shown that flagellar impairment in Chlamydomonasreinhardtii results in augmented storage of starch, the major form of macromolecular chemical energy
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FIG. 2. Starch relative to total protein in Chlamydomonas reinhardtii wild type (137c+) and flagellar mutants (pf-1, pf-2, pf-7 and pf-18). Means SE are shown (n = 3). The difference in storage of starch between the wild type and each of the mutants was significant at P < 0.001.
*
storage in C. reinhardtii. This finding may represent a general principle in bioenergetic interactions. In fact, the extra energy made available by decreased flagellar work could conceivably be utilized and (or) released through increased chemical oxidation, futile biochemical (substrate) cycles, and thermogenesis. But this study has indicated that at least part of the energy is retained for anabolic processes, particularly the formation of starch. Thus, these findings suggest an inverse relationship between energy utilized for cellular motility, at least in the form of flagellar function, and chemical energy storage. More specifically, genetically determined flagellar dysfunctions of the unicellular green alga C. reinhardtii are associated with accumulation of starch. Seemingly, this organism does not have a mechanism for consuming energy beyond that used for enhanced growth, or dissipating the energy that would have been utilized for normal flagellar function. As an example of lack of stringency in regulating the content of starch, Ball et al. (1990) have shown that when growth is restricted by limiting specific nutrients, large amounts of starch accumulate, irrespective of whether the cells are grown as photoautotrophs or heterotrophs. Similarly, de Hostos et al. (1988) have reported that cells deprived of sulfate cease to divide, enlarge, and accumulate starch. These findings suggest that limitation of specific nutrients coupled with provision of adequate energy, leads to accumulation of macromolecular stores. Duration and quality of light influence cell size and replicative rate of the alga. Jones (1970) has reported that at high light intensities, cell size after 6 h is equal to the cell size after 12 h at lower intensity. In Chlamydomonas, moreover, characteristic cell size is maintained by the number of divisions, the mother cell continuing to divide until a minimal volume of daughter cells is reached. In general, there is a positive correlation between intensity/ duration of the photoperiod and cell size/extent of division (Cohen and Parnas 1976; Craigie and Cavalier-Smith 1982; Voight and Munzner 1987; Harris 1989). In addition to the light energy requirements, starch accumulation in Chlamydomonas is dependent on carbon supply. Indeed, Kuchitsu et al. (1988) have found that a high ambient C 0 2 content and acetate supplementation result in increased
FIG. 3. Starch relative to total chlorophyll in Chlamydomonas reinhardtii wild type (137c +) and flagellar mutants @f-1, pf-2, pf-7 and pf-18). Means + SE are shown (n = 3). The difference in storage of starch between the wild type and the mutants was significant at P < 0.001, except pf-2 where the difference was significant at P < 0.01.
u ?me+
-
ROO
m
pr-7 pr-1%
4(KI
200
0
0
5
10
15
20
25
DIAMETER (elm)
FIG.4. Representative size distribution of Chlamydomonas reinhardtii wild type (137c+) and flagellar mutants (pf-1, pf-2, pf-7, and pf-18). The mean diameter for the wild type was 8.06 pm, while pf-1, pf-2, pf-7, and pf-18 diameters were 8.88,9.71, 10.78, and 9.96 am, respectively.
storage. These observations would suggest that under conditions of optimal growth, an increase in energy supply results in greater storage of chemical energy. Thus, both selective restriction of cell division and (or) increase of available energy result in an expanded starch content in Chlamydomonas. These findings indicate that the extent of macromolecular storage is not regulated stringently, at least under certain conditions. In general, cell differentiation, with its associated anabolism, including storage of chemical energy, onsets at or after cessation of replication (Umek et al. 1991). However, the postreplicative synthetic processes are modulated by different regulatory mechanisms including the degree of cytoskeletal activity, as suggested by our findings. The degree of chemical energy storage could vary through a wide range, as an inverse function of the energy utilized by the cytoskeleton. While it is tempting to generalize the inverse relationship between cellular motility and chemical energy storage to include all living forms, experimental verification is
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obviously required. However, it may well be relevant that agents that interfere with cytoskeletal functions (i.e., cytochalasin B, Colcemid, and trifluoperazine) lead to accumulation of triacylglycerol in cultured human preadipocytes (Healy 1987). While there has been increasing recognition of the critical role of the cytoskeleton in numerous cell functions, the contribution of these activities to the energy interactions of an organism has not been appreciated. It is nonetheless highly probable that the various motile functions consume relatively large quantities of energy (Roncari 1987; Alberts et al. 1989).
Acknowledgements The authors thank Christine A. Goertzen for her technical assistance. This research was supported by grant MT-8460 of the Medical Research Council of Canada, a grant from the Heart and Stroke Foundation of Canada (Ontario), and an operating grant from the Natural Sciences and Engineering Research Council of Canada (A-6224). AIberts, B., Bray, D., Lewis, J., et al. 1989. Molecular biology of the cell. 2nd ed. Garland Publishing Inc., New York. pp. 41-86. Arnon, D. 1949. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol. 24: 1-15. Ball, S.G., Dirick, L., Decq, A., et al. 1990. Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii. Plant Sci. (Limerick, Irel.), 66: 1-9. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Cohen, D.. and Parnas, H. 1976. An optimal policy for the metabolism of storage materials in unicellular algae. J. Theor. Biol. 56: 1-18. Craigie, R.A., and Cavalier-Smith, T. 1982. Cell volume and the control of the Chlamydomonas cell cycle. J. Cell Sci. 54: 173-191.
1992
De Hostos, E.L., Togasaki, R.K., and Grossman, A. 1988. Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonasreinhardtii. J. Cell Biol. 106: 29-37.
Ebersold, W.T., Levine, R.P., Levine, E.E., and Olmsted, M.A. 1962. Linkage maps in Chlamydomonas reinhardtii. Genetics, 47: 531-543. Gfeller, R.P., and Gibbs, M. 1984. Fermentative metabolism of Chlamydomonas reinhardtii. Plant Physiol. 75: 212-21 8. Gowans, C.S. 1960. Some genetic investigations on Chlamydomonas eugametos. Z . Vererbungsl. 91: 63-73. Harris, E.H. 1989. The Chlarnydomonas sourcebook. Academic Press, Toronto. Healy, I.A. 1987. Influence of inhibitors of cytoskeletal function on triacylglycerol accretion in cultured adipocyte precursors. M.Sc. thesis, University of Calgary, Calgary, Alta. Jones, R.F. 1970. Physiological and biochemical aspects of growth and gametogenesis in Chlamydomonas reinhardtii. Ann. N.Y. Acad. Sci. 175: 648-659. Kuchitsu, K., Tsuzuki, M., and Miyachi, S. 1988. Changes of starch localization within the chloroplast induced by changes in COz concentration during growth of Chlamydomonas reinhardtii: independent regulation of pyrenoid starch and stroma starch. Plant Cell Physiol. 29: 1269-1278. McVittie, A. 1972. Genetic studies on flagellum mutants of Chlamydomonas reinhardtii. Genet. Res. 19: 157-164. Nakamura, K., Bray, D.F., and Wagenaar, E.B. 1978. Ultrastructure of palmelloid-forming strain of Chlamydomonas eugametos. Can. J . Bot. 56: 2348-2356. Roncari, D.A.K. 1987. Individual variations in energy utilized biomechanical processes and molecular mobility account for diverse susceptibility to obesity. Med. Hypotheses, 23: 11-18. Umek, R.M., Friedman, A.D., and McKnight, S.L. 1991. CCAATenhancer binding protein: a component of a differentiation switch. Science (Washington, D.C.), 251: 288-292. Voight, J., and Munzner, P. 1987. The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch. Planta, 172: 463-472.
