Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii

Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]–ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms. Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]–ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms. Measurement of ATP in live cells is important for understanding cellular activities. Various gene-encoded ATP sensors, including the fluorescence-based sensors ATeam (1Imamura H. Nhat K.P. Togawa H. Saito K. Iino R. Kato-Yamada Y. Nagai T. Noji H. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 15651-15656Crossref PubMed Scopus (604) Google Scholar), B-Queen (2Yaginuma H. Kawai S. Tabata K.V. Tomiyama K. Kakizuka A. Komatsuzaki T. Noji H. Imamura H. Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.Sci. Rep. 2014; 4: 6522Crossref PubMed Scopus (161) Google Scholar), and MaLionB/G/R (3Arai S. Kriszt R. Harada K. Looi L.S. Matsuda S. Wongso D. Suo S. Ishiura S. Tseng Y.H. Raghunath M. Ito T. Tsuboi T. Kitaguchi T. RGB-color intensiometric indicators to visualize spatiotemporal dynamics of ATP in single cells.Angew. Chem. Int. Ed. Engl. 2018; 57: 10873-10878Crossref PubMed Scopus (28) Google Scholar) and the bioluminescence-based sensor BTeam (4Yoshida T. Kakizuka A. Imamura H. BTeam, a novel BRET-based biosensor for the accurate quantification of ATP concentration within living cells.Sci. Rep. 2016; 6: 39618Crossref PubMed Scopus (43) Google Scholar), have been developed for this purpose. These sensors can be expressed in specific cellular compartments, such as the endoplasmic reticulum and mitochondria, and quantitatively monitor local ATP concentrations, [ATP], based on ratiometric analyses. These sensors, however, are not easy to use in some kinds of cells because they must be expressed as recombinant proteins in the target cells, they could perturb the cellular metabolism and the cellular ATP level when overexpressed (5Magidson V. Khodjakov A. Circumventing photodamage in live-cell microscopy.Methods Cell Biol. 2013; 114: 545-560Crossref PubMed Scopus (123) Google Scholar), and in the case of phototrophic organisms, they are susceptible to chloroplast autofluorescence. In this study, we explored the possibility of easily estimating cytosolic [ATP] in the unicellular green alga Chlamydomonas reinhardtii from the beat frequency of its cilia (also called flagella). C. reinhardtii is an excellent model organism in various research fields, including photosynthesis, respiration, reproduction, and ciliary function. Because it can be cultured either photoautotrophically or heterotrophically, numerous mutants with defects in photosynthesis or respiration pathways have been isolated (6Cardol P. Remacle C. The mitochondrial genome.in: Stern D.B. Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 445-467Crossref Scopus (7) Google Scholar, 7Cardol P. Figueroa F. Remacle C. Franzén L.-G. González-Halphen D. Oxidative phosphorylation: building blocks and related components.in: Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 469-502Crossref Scopus (9) Google Scholar, 8de Vitry C. Kuras R. The cytochrome b 6 f complex.in: Stern D.B. Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 603-637Crossref Scopus (7) Google Scholar, 9Finazzi G. Drapier D. Rappaport F. The CF 0F1 ATP synthase complex of photosynthesis.in: Stern D.B. Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 639-670Crossref Scopus (5) Google Scholar, 10Gokhale Z. Sayre R.T. Photosystem II, a structural perspective.in: Stern D.B. Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 573-602Crossref Scopus (12) Google Scholar, 11Redding K.E. Photosystem I.in: Stern D.B. Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA2009: 541-572Crossref Scopus (4) Google Scholar, 12Wostrikoff K. Stern D.B. Rubisco.in: 2nd Ed. Chlamydomonas Sourcebook. Academic Press, San Diego, CA2009: 303-332Google Scholar). Some of these mutant cells swim slower than WT cells (13Wakabayashi K. King S.M. Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.J. Cell Biol. 2006; 173: 743-754Crossref PubMed Scopus (65) Google Scholar). Low motility of such mutants may reflect a decrease in their intracellular [ATP]. Eukaryotic cilia are motile organelles driven by microtubule-based motor proteins: dyneins. Ciliary dyneins belong to the protein superfamily containing ATPases associated with diverse cellular activities (AAA+ proteins) and generate force between adjacent doublet microtubules through ATP hydrolysis (14Erzberger J.P. Berger J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins.Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 93-114Crossref PubMed Scopus (535) Google Scholar). The inner structure of the cilia, called the axoneme, is detergent-insoluble, and the motility of such a cytoskeleton-based structure has been traditionally studied in vitro by detergent extraction followed by the addition of ATP. This kind of experiments is originated from the in vitro contraction of glycerinated muscle by Szent-Györgyi (15Szent-Györgyi A. Free energy relations and contraction of actomyosin.Biol. Bull. 1949; 94: 140-161Crossref Scopus (103) Google Scholar), and the method was applied for sperm flagella (16Bishop D.W. Hoffman-Berling H. Extracted mammalian sperm models. I. Preparation and reactivation with adenosine triphosphate.J. Cell Comp. Physiol. 1959; 53: 445-466Crossref PubMed Scopus (21) Google Scholar), Paramecium cilia (17Naitoh Y. Kaneko H. Reactivated triton-extracted models of paramecium: modification of ciliary movement by calcium ions.Science. 1972; 176: 523-524Crossref PubMed Scopus (288) Google Scholar), and then C. reinhardtii cilia (18Kamiya R. Witman G.B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.J. Cell Biol. 1984; 98: 97-107Crossref PubMed Scopus (246) Google Scholar). In each system, after detergent-extraction, motor proteins can be activated by the addition of ATP to show sliding motion against cytoskeletons, and the cell motility can be reproduced in vitro. This system enables in vitro assessment for the effects of various factors such as ions and nucleotides on cell motility. The detergent-extracted cilia or whole cells (cell models) of C. reinhardtii display motility in the presence of ATP such that demembranated cilia beat with almost the same pattern as that in live cells (Fig. 1A) (18Kamiya R. Witman G.B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.J. Cell Biol. 1984; 98: 97-107Crossref PubMed Scopus (246) Google Scholar). The ciliary beat frequency (CBF) of C. reinhardtii increases with [ATP] in a Michaelis–Menten pattern (19Kamiya R. Okamoto M. A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim.J. Cell Sci. 1985; 74: 181-191Crossref PubMed Google Scholar), as found originally with sea urchin sperm flagella (20Brokaw C.J. Adenosine triphosphate usage by flagella.Science. 1967; 156: 76-78Crossref PubMed Scopus (45) Google Scholar). This ATP-dependence of CBF conforming to Michaelis–Menten kinetics is an empirical observation that cannot be theoretically explained as representing the function of single or multiple enzymes. Nevertheless, we have experienced that the [ATP]–CBF curve is reproducible when the same cilia or flagella are tested under constant conditions. We thus hypothesized that cytosolic [ATP] could be estimated by interpolating the CBF of live cells if we can draw a reliable [ATP]–CBF curve from in vitro experiments. The methods for preparing detergent-extracted cell models of C. reinhardtii and reactivating their motility with the addition of ATP are well established and have been used in various studies (13Wakabayashi K. King S.M. Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.J. Cell Biol. 2006; 173: 743-754Crossref PubMed Scopus (65) Google Scholar, 18Kamiya R. Witman G.B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.J. Cell Biol. 1984; 98: 97-107Crossref PubMed Scopus (246) Google Scholar, 21Takada S. Kamiya R. Beat frequency difference between the two flagella of Chlamydomonas depends on the attachment site of outer dynein arms on the outer- doublet microtubules.Cell Motil. Cytoskeleton. 1997; 36: 68-75Crossref PubMed Scopus (36) Google Scholar, 22Zhang H. Mitchell D.R. Cpc1, a Chlamydomonas central pair protein with an adenylate kinase domain.J. Cell Sci. 2004; 117: 4179-4188Crossref PubMed Scopus (63) Google Scholar). However, all of these methods are flawed in that the maximal CBF of the cell models under physiological [ATP] is lower than the CBF of live cells. Thus, an improved method has been awaited. In this study, we improved the conditions for motility reactivation of the cell models so that they display higher CBF comparable to the in vivo CBF over a more extensive [ATP] range. Using the improved [ATP]–CBF curve for calibration, we estimated intracellular [ATP] in cells under various conditions. The values thus obtained showed a reasonable agreement with those measured based on luciferin/luciferase phosphorescence. The CBF-based method is entirely noninvasive and rapid, and it is useful for monitoring the dynamics of cellular [ATP] in live cells. To draw a better [ATP]–CBF curve and estimate the cellular [ATP] from CBF, we first improved the buffer conditions for motility reactivation in detergent-extracted cell models. In previous studies, a reactivation buffer was used that contained 30 mM Hepes (pH 7.4), 5 mM MgSO4, 1 mM dithiothreitol, 1 mM EGTA, 50 mM potassium acetate, and 1% polyethylene glycol (Mw: 20,000) (18Kamiya R. Witman G.B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.J. Cell Biol. 1984; 98: 97-107Crossref PubMed Scopus (246) Google Scholar, 21Takada S. Kamiya R. Beat frequency difference between the two flagella of Chlamydomonas depends on the attachment site of outer dynein arms on the outer- doublet microtubules.Cell Motil. Cytoskeleton. 1997; 36: 68-75Crossref PubMed Scopus (36) Google Scholar). The motility of the cell models is readily reactivated by adding ATP to the cell models in this buffer. However, in these conventional experimental conditions, at [ATP] > 1 mM, CBF is lower than projected from the Michaelis–Menten curve (Fig. 1B). Because the physiological [ATP] is suggested to be 1 to 2 mM in plant cells (23De Col V. Fuchs P. Nietzel T. Elsasser M. Voon C.P. Candeo A. Seeliger I. Fricker M.D. Grefen C. Moller I.M. Bassi A. Lim B.L. Zancani M. Meyer A.J. Costa A. et al.ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology.eLife. 2017; 6e26770Crossref PubMed Scopus (53) Google Scholar, 24Voon C.P. Guan X. Sun Y. Sahu A. Chan M.N. Gardestrom P. Wagner S. Fuchs P. Nietzel T. Versaw W.K. Schwarzlander M. Lim B.L. ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E10778-E10787Crossref PubMed Scopus (25) Google Scholar) and C. reinhardtii WT cells swim with a CBF higher than the maximal CBF (Vmax) calculated from Michaelis–Menten kinetics, this problem might be caused by nonoptimal experimental conditions. Assuming that ATP uncoordinated with Mg2+ increases when [ATP] is increased with limited [Mg2+] (Fig. S1) and that such free ATP may inhibit dynein ATPase (25Hayashi M. Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP.Biochim. Biophys. Acta. 1976; 422: 225-230Crossref PubMed Scopus (4) Google Scholar), we changed [Mg2+] in the buffer to 5 to 20 mM. The CBF, measured from the cell body vibration (26Kamiya R. Analysis of cell vibration for assessing axonemal motility in Chlamydomonas.Methods. 2000; 22: 383-387Crossref PubMed Scopus (38) Google Scholar), showed that 15 mM MgSO4 conditions yielded a curve well fitted to the Michaelis–Menten equation and gave the highest Vmax value, which was higher than the CBF in live cells (Table 1). The increase in Vmax with [Mg2+] may mean that Mg2+-free ATP indeed inhibits the mechanochemical cycle of dyneins.Table 1Km, Vmax, and |R| values for Figure 1BMg2+ATP only[ATP]:[ADP] = 20:1Km (mM)Vmax (Hz)|R|Km (mM)Vmax (Hz)|R|5 mM0.14 ± 0.04456.00 ± 3.6690.98370.10 ± 0.01652.33 ± 2.1110.866510 mM0.22 ± 0.04562.11 ± 2.9200.99630.21 ± 0.03463.47 ± 1.3660.994515 mM0.49 ± 0.08172.85 ± 1.1560.99730.52 ± 0.06676.90 ± 1.6400.998920 mM0.52 ± 0.01059.70 ± 5.0270.98880.58 ± 0.10859.57 ± 4.9920.9735 Open table in a new tab As an additional improvement, we added ADP together with ATP. Several studies have suggested that ADP enhances dynein motor activity (27Furuta A. Yagi T. Yanagisawa H.A. Higuchi H. Kamiya R. Systematic comparison of in vitro motile properties between Chlamydomonas wild-type and mutant outer arm dyneins each lacking one of the three heavy chains.J. Biol. Chem. 2009; 284: 5927-5935Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Kikushima K. Yagi T. Kamiya R. Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein.FEBS Lett. 2004; 563: 119-122Crossref PubMed Scopus (39) Google Scholar). A previous study showed that in C. reinhardtii cells, the ATP: ADP ratio is almost always ∼20:1 (29Forti G. Furia A. Bombelli P. Finazzi G. In vivo changes of the oxidation-reduction state of NADP and of the ATP/ADP cellular ratio linked to the photosynthetic activity in Chlamydomonas reinhardtii.Plant Physiol. 2003; 132: 1464-1474Crossref PubMed Scopus (46) Google Scholar). We thus added ADP at 1/20 concentration of ATP for reactivation of the motility of the cell models. The addition of ADP resulted in a curve showing an excellent fit to the Michaelis–Menten equation and a higher Vmax (Fig. 1B, Table 1). Based on the excellent in vitro motility attained, we decided to use the [ATP]–CBF curve obtained in the buffer containing 15 mM MgSO4 and ADP, in addition to other ordinary components, to estimate the cellular ATP concentration from the CBF of live cells (Videos S1–S4). The mean CBF of live WT cells (see Table 2 for the strains used in this study) grown in the light or the dark was measured as 61.0 ± 0.2 or 56.8 ± 0.8 Hz, respectively (mean ± SEM, n = 4) (Fig. 2A). These values corresponded with 2.0 ± 0.1 and 1.5 ± 0.1 mM [ATP], respectively, in the 15 mM Mg2+ curve of Figure 1B (and Fig. S1). It is reasonable that dark-adapted cells, not undergoing photosynthesis, showed lower CBF and cytosolic [ATP] than light-adapted cells.Table 2Strains used in this studyStrainDescriptionReferenceWild type (WT)A progeny of the cross CC-124 × CC125, agg1+, mt−This studyCC-124Wild-type strain, nit1−, nit2−, agg1−, mt−(44Harris E.H. Chlamydomonas in the laboratory.in: Harris E.H. The Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA, 2009: 241-302Google Scholar)CC-125Wild-type strain, nit1−, nit2−, mt+(44Harris E.H. Chlamydomonas in the laboratory.in: Harris E.H. The Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA, 2009: 241-302Google Scholar)FUD50Deletion in the atpB gene encoding β subunit of chloroplast F1ATPase in the chloroplast genome. Lacking chloroplast FoF1 ATPase.(30Woessner J.P. Masson A. Harris E.H. Bennoun P. Gillham N.W. Boynton J.E. Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas.Plant Mol. Biol. 1984; 3: 177-190Crossref PubMed Scopus (63) Google Scholar)FUD50PA progeny of the cross FUD50 (mt+) × WT strain (mt−)This studydum11Deletion of 0.7 kb in the cob gene and adjacent end of the mitochondrial genome. Lacking complex III. Respiration deficient.(31Dorthu M.P. Remy S. Michel-Wolwertz M.R. Colleaux L. Breyer D. Beckers M.C. Englebert S. Duyckaerts C. Sluse F.E. Matagne R.F. Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii.Plant Mol. Biol. 1992; 18: 759-772Crossref PubMed Scopus (53) Google Scholar)dum22Deletion in the cob gene and the 3′ end of the nd4 gene. Lacking complexes I and III. Respiration deficient.(32Cardol P. Matagne R.F. Remacle C. Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme.J. Mol. Biol. 2002; 319: 1211-1221Crossref PubMed Scopus (103) Google Scholar)oda1Mutation in the ODA1 locus corresponds to the gene encoding DC2 of the outer dynein arm docking complex (ODA-DC). Lacking outer arm dynein and the ODA-DC.(45Takada S. Wilkerson C.G. Wakabayashi K. Kamiya R. Witman G.B. The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly.Mol. Biol. Cell. 2002; 13: 1015-1029Crossref PubMed Scopus (90) Google Scholar)ida9Mutation in the IDA9 locus corresponds to the gene encoding DHC9, a dynein heavy chain for inner arm dynein subspecies c.(46Yagi T. Minoura I. Fujiwara A. Saito R. Yasunaga T. Hirono M. Kamiya R. An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9.J. Biol. Chem. 2005; 280: 41412-41420Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) Open table in a new tab Next, we assessed the cytosolic [ATP] of several photosynthetic or respiratory mutants listed in Table 2. Before measuring live-cell CBF in these mutants, we examined the motility of their demembranated cells reactivated at different ATP concentrations to verify that the ciliary properties are unchanged. [ATP]–CBF curves (Fig. S3) were used to extrapolate and compare the Vmax values (Table S1). Mutants used were FUD50P lacking the beta subunit of chloroplast FoF1ATP synthase (CF1β) as a photosynthesis-deficient mutant (30Woessner J.P. Masson A. Harris E.H. Bennoun P. Gillham N.W. Boynton J.E. Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas.Plant Mol. Biol. 1984; 3: 177-190Crossref PubMed Scopus (63) Google Scholar) and dum11 and dum22 as respiratory mutants (31Dorthu M.P. Remy S. Michel-Wolwertz M.R. Colleaux L. Breyer D. Beckers M.C. Englebert S. Duyckaerts C. Sluse F.E. Matagne R.F. Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii.Plant Mol. Biol. 1992; 18: 759-772Crossref PubMed Scopus (53) Google Scholar, 32Cardol P. Matagne R.F. Remacle C. Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme.J. Mol. Biol. 2002; 319: 1211-1221Crossref PubMed Scopus (103) Google Scholar). Because the original FUD50 cilia contain less of an outer-arm dynein component probably caused by unintentional mutation, FUD50P, a progeny of the cross WT × FUD50 was used (see Experimental procedures and Fig. S2). The dum11 strain lacks complex III, and the dum22 strain lacks complexes I and III in the respiration chain in mitochondria. The cytosolic [ATP] of these stains were calculated from each strain's [ATP]–CBF curve (Fig. S3). The cytosolic [ATP] for each mutant was calculated from the respective CBF values of live mutant cells (Fig. 2). Because FUD50P does not grow in the light and dum11 and dum22 do not grow in the dark, these strains were kept only in their preferred dark or light conditions, respectively. The cytosolic [ATP] of FUD50P was 1.3 ± 0.1 mM, which was close to the value of dark-grown WT (1.5 ± 0.1 mM). Consistently, dark-grown WT and the photosynthesis-deficient mutant showed similar cytosolic [ATP] values (Fig. 2). By contrast, the respiration mutants showed significantly lower [ATP] values (0.9 ± 0.1 mM in dum11 and 0.5 ± 0.1 mM in dum22) than the other strains, suggesting that respiration contributes to the cytosolic [ATP] more significantly than does photosynthesis under heterotrophic culture conditions (Fig. 2). For a more straightforward estimation of the cytosolic [ATP] in a mutant, its CBF can be extrapolated into the WT calibration curve instead of its own. The [ATP] values estimated from the WT calibration curve were not significantly different from those from the respective strain's curve, except for FUD50P (Table 3). This difference may be caused by the remaining unintentional mutations in FUD50P, suggested from a slightly lower Vmax value of the calibration curve (Table S1, Fig. S3). Therefore, when estimating the cytosolic [ATP] from CBF, the calibration curve should be changed depending on the purpose: the respective strain's calibration curve for better estimation and the WT calibration curve ([ATP] =0.52 mM ∗CBF/(76.9 Hz-CBF)) for easier estimation. We employed the former in this study below.Table 3Comparison of [ATP] estimated by different calibration curvesStrain and light conditionEstimated [ATP] (mM)p valueaStudent's t test.By WT calibration curveBy each strain's calibration curvedum11, light0.96 ± 0.0960.91 ± 0.0980.3120dum22, light0.51 ± 0.0390.47 ± 0.0380.1700FUD50P, dark1.07 ± 0.0151.29 ± 0.0210.0001a Student's t test. Open table in a new tab To validate the [ATP] values estimated from CBF, we next measured the intracellular ATP concentration by a bioluminescence-based method. Whole-cell extracts were prepared from each strain after TCA fixation, and the ATP amounts in cell lysates were measured by an ATP detection system based on the luciferin-luciferase reaction. To convert the ATP amount to its intracellular concentration, we approximated cells to spheres and calculated the total cell volume from the cell number and the mean cell radius (Fig. S4). The bioluminescence-based method yielded significantly higher [ATP] values than those estimated by the CBF method. However, both methods reported the same patterns in the [ATP] difference among cells under different physiological or genetic conditions (Fig. 3). The difference in the [ATP] values assessed by the two methods may not be surprising because the CBF reflects the [ATP] in cilia, whereas the bioluminescence method measures the total quantity of [ATP] contained in both the cytoplasm and chloroplasts. If we consider that the chloroplast occupies ∼51% of the total cell volume (33Gaffal K. Arnold C.-G. Friedrichs G. Gemple W. Morphodynamical changes of the chloroplast of Chlamydomonas reinhardtii during the 1st round of division.Arch. Protistenkd. 1995; 145: 10-23Crossref Scopus (11) Google Scholar), whereas the cytoplasm occupies ∼40% (34Harris E.H. Cell architecture.in: The Chlamydomonas Sourcebook. 2nd Ed. Academic Press, San Diego, CA, 2009: 25-64Google Scholar), we may consider that [ATP] measured by the bioluminescence-based assay is greatly affected by the [ATP] in the chloroplasts. Taking advantage of the rapidity of the CBF-based method, we assessed the effects of two kinds of inhibitors on ATP production. First, we tested rotenone, a respiration inhibitor that targets complex I. WT, dum11, and dum22 cells were treated with 100 μM rotenone, and the CBF of each strain was measured for 30 min. The CBFs of dum11 and dum22 were lower than those of the WT before rotenone treatment (Fig. 4A). After the treatment, the CBFs of WT and dum11 (lacking complex III) cells decreased within 1 min, whereas the CBF of dum22 (lacking complexes I and III) cells assumed a low level at time zero that did not decrease further with time (Fig. 4A). These data suggest that rotenone inhibited complex I in the respiratory chain. These changes in CBF were converted to the change in the cytosolic [ATP] from each strain's calibration curve (Fig. S3). Rotenone decreased cytosolic [ATP] in WT cells from 2.0 ± 0.2 to 1.6 ± 0.3 mM within 1 min (Fig. 4B), suggesting that respiration contributes to the cytosolic [ATP] by ∼0.4 mM under normal conditions. Next, we tested 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthesis inhibitor that targets photosystem II. CBF decreased within 1 min after treatment with DCMU and recovered almost completely by 30 min (Fig. 5A). Conversion of CBF to [ATP] showed that, similarly, the cytosolic [ATP] dropped from 2.1 ± 0.2 to 1.0 ± 0.1 mM immediately after the DCMU treatment and spontaneously recovered to 2.1 ± 0.1 mM within 30 min (Fig. 5B). These data suggest that photosynthesis contributes to cytosolic [ATP] by ∼1.1 mM under heterotrophic culture conditions. We surmised that the activation of respiration is primarily responsible for this recovery. To test this idea, we treated the cells with both DCMU and rotenone. After the treatment, [ATP] decreased from 1.6 ± 0.1 to 0.5 ± 0.1 mM within 1 min and partially recovered to 1.0 ± 0.2 mM by 30 min (Fig. 5B). This modest recovery supports the concept that respiration activation is indeed crucial for recovery but simultaneously suggests that other metabolic pathways, such as glycolysis, also play a role. We estimated the intracellular [ATP] in C. reinhardtii from CBF in vivo, using in vitro data on the [ATP]-dependence of ciliary movement in demembranated cells (cell models). Measurements of CBF in several metabolic mutants or cells treated with metabolic inhibitors allowed us to quantify the contribution of photosynthesis and respiration to the cytosolic [ATP]. CBF must reflect [ATP] in the cilia, but whether or how much this level differs from the cytosolic [ATP] is unclear. Because ATP molecules are produced in the cell body and diffuse into the cilia, where they are consumed by axonemal dyneins and other ATPases such as intraflagellar transport motors, the ATP concentration along each cilium could form a steep gradient (35Rosenbaum J.L. Cole D.G. Diener D.R. Intraflagellar transport: the eyes have it.J. Cell Biol. 1999; 144: 385-388Crossref PubMed Scopus (146) Google Scholar). 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