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. 2006 Jun 5;173(5):743-54.
doi: 10.1083/jcb.200603019.

Modulation of Chlamydomonas Reinhardtii Flagellar Motility by Redox Poise

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Free PMC article

Modulation of Chlamydomonas Reinhardtii Flagellar Motility by Redox Poise

Ken-ichi Wakabayashi et al. J Cell Biol. .
Free PMC article

Abstract

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+ -binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.

Figures

Figure 1.
Figure 1.
Light/dark sampling regime and analysis of the photophobic response. (a) Light/dark conditions. A C. reinhardtii preculture was grown under a 15/9 h light/dark cycle for 2–3 d. This culture was then inoculated into several bottles and grown for 1–2 d more (total 3–4 d). On the day before the experiment, half of the bottles were kept under the regular illumination regime, and the other half were wrapped with aluminum foil and kept completely in the dark. Experiments were initiated ∼6 h after the beginning of the light phase. We prepared samples and/or observed cell behavior for each treatment group, and then switched the light conditions; i.e., light-adapted cells were placed in total darkness and vice-versa. Samples were taken 30 and 60 min after the light switch (arrows); for assessment of the in vivo redox state (Fig. 6), additional samples were taken at 1 and 120 min. For chloroplast mutants, precultures were grown in complete darkness for 6–7 d. 1 h before the experiment, half of the culture was transferred to the light. (b) The PPR. When C. reinhardtii cells perceive a sudden change in light intensity, they exhibit the PPR. Cells alter their flagellar waveform from an asymmetric (or ciliary) beat to a symmetric (or flagellar) wave (Phase I) and swim backward or simply stop. After a short period, the cell body rotates to randomize swimming direction (Phase II), and, subsequently, the cell returns to forward swimming. We measured the duration of Phase I, II, and total PPR of single cells by video microscopy.
Figure 2.
Figure 2.
Light/dark transitions alter C. reinhardtii swimming behavior. (a) Beat frequency (Hz) of cells adapted to different light conditions. (left) Transition of light-adapted cells to the dark. (right) Transition of dark-adapted cells to the light. Peaks in the FFT power spectra represent the mean beat frequency of 30–50 cells. Reference lines are added at 60 (left) and 50 Hz (right). (b) Duration of the PPR of cells under different light conditions. Phase I represents the duration of backward swimming, and phase II the duration of rotation before recovery of forward swimming. Data represent the mean ± SD for 30 cells. In each graph, data indicated by white and black bars are significantly different. P < 0.05; two-tailed t test.
Figure 3.
Figure 3.
Exogenous oxidants/antioxidants modify C. reinhardtii photobehavior. (a) Beat frequency of light-adapted cells before and after treatment with low levels of hydrogen peroxide (left), and of dark-adapted cells before and after treatment with DMTU (middle) and TEMPOL (right) is shown. Peaks in the FFT power spectra represent the mean flagellar beat frequency of 30–50 cells. Reference lines are added at the peak value of the untreated samples shown in the top plots. (b) Duration of the PPR of cells treated as in a. In each histogram, data indicated by black and white bars are significantly different (P < 0.05; two-tailed t test), except for phase II of the DMTU treatment series. (c) Beat frequency power spectra for dark-adapted cells treated with H2O2 (left) and light-adapted cells treated with DMTU (middle) and TEMPOL (right). The solid line in each plot represents the peak beat frequency obtained in the absence of oxidizing/reducing agents; see power spectra in top row of a. (d) PPR duration of cells treated as in c. Data represent the mean ± SD for 30 cells.
Figure 4.
Figure 4.
Photosystem I is required for the photokinetic effect. (a) Simplified diagram illustrating the chloroplast light reactions. Electrons are obtained from water by photosystem II (PS-II), passed to photosystem I (PS-I), and ultimately used to reduce NADP+ to NADPH. Consequently, the cellular NADP/NADPH ratio changes in response to light/dark conditions (Forti et al., 2003). Mutants defective in either of these systems require acetate for growth. (b) Beat frequency analysis of light- and dark-adapted mutants defective for photosystem II (left) and I (right). Both mutants have a beat frequency of 40–45 Hz in the light. However, only the photosystem II mutant exhibits a decrease in flagellar beat frequency upon dark adaption, suggesting that a functional photosystem I is required for this response.
Figure 5.
Figure 5.
Redox poise modulates flagellar beat frequency in reactivated cell models. Detergent-extracted cell models were prepared from wild-type cells and mutants lacking the entire ODA (oda1), the ODA α HC and LC5 (oda11), and the β HC motor domain (oda4-s7). Models were reactivated by addition of ATP and an ATP-regenerating system under fully reducing (10 mM DTT and GSH) and oxidizing (10 mM DTNB and GSSG) conditions, and in the presence of various redox buffers. Flagellar beat frequency was determined, and the data plotted as the mean ± SEM for three independent experiments. Note that in the presence of 10 mM DTNB or GSSG, all cell models lost their flagella and were therefore immotile; visual inspection determined that these detached axonemal structures did not reactivate in the presence of ATP.
Figure 6.
Figure 6.
Assessment of the in vivo redox state of LC3, LC5, and DC3. (a) Structure of AMS. (b) Scheme detailing the AMS method to distinguish oxidized and reduced dithiols. The maleimide group of AMS reacts only with reduced thiols and, thus, a protein with a reduced vicinal dithiol will incorporate two AMS moieties (980 D). In contrast, oxidized dithiols are protected from modification. Consequently, reduced proteins may be readily separated from the oxidized forms by nonreducing SDS-PAGE. (c) Axonemes were incubated with redox buffers containing different GSH/GSSG ratios, alkylated with AMS, electrophoresed in the absence of reducing agents, and probed with anti-LC3, -LC5, and -DC3 antibodies. Axonemes alkylated after incubation with 10 mM DTNB or DTT were used as fully oxidized and reduced controls, respectively. The lane marked Cont was only treated with HMEK buffer before AMS modification. The redox state of the three dynein/ODA-DC proteins is altered depending on the GSH/GSSG ratio. The location of the Mr markers (× 103) is at right. (d) Flagella from cells grown under different light conditions (Fig. 1) were fixed with TCA immediately after deflagellation and treated with AMS. Samples were separated in a 15% acrylamide gel without reducing reagents, transferred to nitrocellulose membranes and probed with anti-LC3, -LC5, and -DC3 antibodies. Although LC5 is essentially always reduced, small amounts of LC3 (∼10%) and DC3 (∼30%) are present in the oxidized form. (e) Long exposure of a flagella sample obtained 1 min after a light → dark transition and probed for LC3. Three distinct bands are evident, suggesting that this protein contains two redox-sensitive vicinal dithiols. The fully oxidized and reduced markers are at left.
Figure 7.
Figure 7.
Identification of redox-sensitive flagellar components. (a) Schematic of the redox 2D gel method. Samples were initially alkylated with IAA in the absence of reducing reagents and electrophoresed in the first dimension (intermolecular disulfides are stable under these conditions). The gel lane was excised, reduced, and alkylated, and then electrophoresed again. Any proteins initially cross-linked by intermolecular disulfide bonds focus under the diagonal. (b) Redox 2D gel analysis of C. reinhardtii flagella from light-adapted cells treated with 0.5 mM H2O2 and stained with SYPRO Ruby; multiple spots are observed below the diagonal. The inset shows a similar 2D gel that used reducing reagents in both dimensions; note that no spots were detected below the diagonal. The arrows indicate protein spots identified by mass spectrometry (Table I). (c) Redox 2D gel analysis of flagella from light- and dark-adapted cells stained with SYPRO Ruby. The insets show the boxed region of similar gels stained with silver. Arrowheads indicate a spot (corresponds to spot 5 in b) that is more prominent in light-adapted cells. (d) Immunoblots of redox-2D gels probed with antibodies against LC3, LC5, and DC3. Flagellar samples were obtained from cells adapted to the light (L) and the dark (D), and cells treated with 0.5 mM H2O2 in the light (H). R represents the control reducing–reducing 2D gel. (bottom right) An immunoblot probed with antibody against LC7a, which does not contain any Cys residues. In each immunoblot, off-diagonal spots which derive from binding other proteins are indicated with arrowheads. Note that additional spots appear in the dark versus light for all three redox-active proteins. H2O2 treatment also gave a distinct pattern for LC3, suggesting that enhanced oxidative stress results in the preferential formation of certain mixed disulfides containing this protein. Samples lacking the entire ODA (including LC3 and LC5), but retaining the ODA-DC (from oda2) and missing only the α HC and LC5 (from oda11) were probed for DC3 and LC3, respectively (bottom right). Asterisks indicate minor cracks in the original gel and dotted lines mark the diagonal on which most proteins align. The location of Mr markers are indicated (× 103).

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References

    1. Aitken, R.J., D. Harkiss, W. Knox, M. Paterson, and S. Irvine. 1998. On the cellular mechanisms by which the bicarbonate ion mediates the extragenomic action of progesterone on human spermatozoa. Biol. Reprod. 58:186–196. - PubMed
    1. Aksenov, M.Y., M.V. Aksenova, D.A. Butterfield, J.W. Geddes, and W.R. Markesbery. 2001. Protein oxidation in the brain in Alzheimer's disease. Neurosci. Lett. 103:373–383. - PubMed
    1. Baker, M.A., and R.J. Aitken. 2004. The importance of redox regulated pathways in sperm cell biology. Mol. Cell. Endocrinol. 216:47–54. - PubMed
    1. Bessen, M., R.B. Fay, and G.B. Witman. 1980. Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas. J. Cell Biol. 86:446–455. - PMC - PubMed
    1. Bowman, A.B., R.S. Patel-King, S.E. Benashski, J.M. McCaffery, L.S. Goldstein, and S.M. King. 1999. Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis. J. Cell Biol. 146:165–180. - PMC - PubMed

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