This study was supported by grants from the Japan Society for the Promotion of Science KAKENHI (https://www.jsps.go.jp/english/index.html) to NU (19K23758, 21K06295), TH (16H06556), and KW (19H03242, 20K21420, 21H00420), from the Ohsumi Frontier Science Foundation (https://www.ofsf.or.jp/en/) to KW, and from the Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials (http://alliance.tagen.tohoku.ac.jp/english/) to NU, TH, and KW.&#160;

In the present study, a wild-type strain of Chlamydomonas reinhardtii, a progeny of the cross CC-124 x CC-125 with agg1+mt-, was used21. CC-124 and CC-125 were obtained from the Chlamydomonas Resource Center (see Table of Materials) and maintained on a Tris-acetate-phosphate (TAP)22, 1.5% agarose medium at 20-25 &#176;C. Any motile strain can be used for this protocol.
1. Cell culture
<ol>
	<li>Culture...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoprotection+and+other+responses+of+plants+to+high+light+stress.">Photoprotection and other responses of plants to high light stress.</a> Annual Reviews Plant Physiology and Plant Molecular Biology. 43, 599-626 (1992).</li><li>Wada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement.">Chloroplast movement.</a> Plant Science. 210, 177-182 (2013).</li><li>Sgarbossa, A., Checcucci, G., Lenci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreception+and+photomovements+of+microorganisms.">Photoreception and photomovements of microorganisms.</a> Photochemical &amp; Photobiological Sciences. 1 (7), 459-467 (2002).</li><li>Ueki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyespot-dependent+determination+of+the+phototactic+sign+in+Chlamydomonas+reinhardtii.">Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (19), 5299-5304 (2016).</li><li>Foster, K. 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A., Jung, K. -. H., Spudich, J. 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J., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ptx1,+a+nonphototactic+mutant+of+Chlamydomonas,+lacks+control+of+flagellar+dominance.">ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.</a> Journal of Cell Biology. 120 (3), 733-741 (1993).</li><li>Hyams, J. S., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+flagellar+apparatus+of+Chlamydomonas:+characterization+of+forward+swimming+and+alteration+of+waveform+and+reversal+of+motion+by+calcium+ions+in+vitro.">Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro.</a> Journal of Cell Science. 33, 235-253 (1978).</li><li>Bessen, M., Fay, R. B., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+control+of+waveform+in+isolated+flagellar+axonemes+of+Chlamydomonas.">Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas.</a> Journal of Cell Biology. 86 (2), 446-455 (1980).</li><li>Reiter, J. F., Leroux, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+f+and+plastocyanin:+their+sequence+in+the+photosynthetic+electron+transport+chain+of+Chlamydomonas+reinhardi.">Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.</a> Proceedings of the National Academy of Sciences of the United States of America. 54 (6), 1665-1669 (1965).</li><li>Finst, R. J., Kim, P. J., Quarmby, L. 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Effects of red background illumination and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea.</a> FEBS Letters. 336 (3), 516-520 (1993).</li><li>Morishita, J., Tokutsu, R., Minagawa, J., Hisabori, T., Wakabayashi, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Chlamydomonas+reinhardtii+mutants+that+exhibit+strong+positive+phototaxis.">Characterization of Chlamydomonas reinhardtii mutants that exhibit strong positive phototaxis.</a> Plants (Basel). 10 (7), (2021).</li><li>Fujiu, K., Nakayama, Y., Yanagisawa, A., Sokabe, M., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+CAV2+encodes+a+voltage-+dependent+calcium+channel+required+for+the+flagellar+waveform+conversion.">Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.</a> Current Biology. 19 (2), 133-139 (2009).</li><li>Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensors+of+ciliary+outer+arm+dynein:+functions+and+phylogenetic+considerations+for+eukaryotic+evolution.">Calcium sensors of ciliary outer arm dynein: functions and phylogenetic considerations for eukaryotic evolution.</a> Cilia. 4 (1), 6 (2015).</li></ol>

The authors have nothing to disclose.

The present protocol is easy and not time-consuming. If a C. reinhardtii mutant is suspected of presenting with defects in photoreception or ciliary motion, this method could serve as primary phenotypic analysis.
However, some critical steps exist. One is to use cells in the experiment in the early to mid-log growth phase. After culturing for long periods, cells become less motile, less light-sensitive, and even form palmelloids (cell clumps)27. Another importa...

Light is an indispensable energy source for photosynthetic organisms, but too much light may cause photo-oxidative damage. Thus, phototrophic organisms need to survive under moderate intensity light, where they can photosynthesize but not suffer photo-oxidative damage1. In land plants, chloroplasts cannot move out from the leaf and show photo movements in the cell; chloroplasts move to the periphery of the cell under high light and the cell surface under low light2, whereas many motile algae show photobehaviors that allow them to find proper light conditions for photosynthesis and, thus, facilitate their survival3.
Chlamydomonas reinhardtii is a unicellular green alga regarded as a model organism in research fields like cilia (a.k.a., flagella), photosynthesis, and photobehavior. C. reinhardtii presents with one eyespot and two cilia per cell, used for photoreception and swimming, respectively. The eyespot has two components: channelrhodopsins (ChRs), light-gated ion channels in the plasma membrane, and the carotenoid-rich granule layers located right behind the ChRs. The eyespot acts as a directional light receptor since the carotenoid-rich granule layers function as a light reflector4,5.
ChRs were initially identified as photoreceptors causing photobehaviors in C. reinhardtii6,7,8,9. Although two isoforms, ChR1 and ChR2, are found in the eyespot, knock-down experiments showed that ChR1 is the primary photoreceptor for photobehaviors10. Despite this, ChR2 has received more attention and played a central role in developing optogenetics, a technique to control cell excitation by light11. Therefore, studying the regulatory mechanisms governing photobehaviors in C. reinhardtii will further the understanding of ChR function and improve optogenetics.
After photoreception, C. reinhardtii cells show two types of photobehaviors: phototaxis and photoshock response12. Phototaxis is the behavior of cells swimming in the direction of the light source or the opposite direction, called positive or negative phototaxis, respectively. Photoshock response is a behavior that cells show after sensing a sudden change in light intensity, such as when illuminated by a flash. Cells stop swimming or swim backward (i.e., swimming with the cell body forward) for a short period, typically &#60;1 s.
Ciliary movements in C. reinhardtii are involved in its photobehaviors. Two cilia usually beat like a human&#39;s breaststroke swimming, and this is modulated for photobehaviors. For phototaxis, the forces generated by the two cilia are imbalanced by the modulation of the beating frequency and the waveform amplitude of each cilium13. The cilium closest to the eyespot is called cis cilium, and the other is called trans cilium. These two cilia differ on various points. For example, the ciliary beating frequency of trans cilium in vitro is 30%-40% higher14. In addition, their Ca2+ sensitivity is different. Reactivation of demembranated cell models15 showed that the cis cilium beats more strongly than the trans cilium for Ca2+ &#60;1 x 10&#8722;8 M, while the opposite is true for Ca2+ &#62;1 x 10&#8722;7 M. This asymmetry in Ca2+ sensitivity is possibly important for phototactic turns since mutants lacking this asymmetry do not exhibit normal phototaxis16,17. Conversely, waveform conversion is necessary for photoshock. The ciliary waveform transforms from the asymmetrical waveform in forward swimming to the symmetrical waveform in backward swimming. This waveform conversion is also regulated by Ca2+, at a threshold of 1 x 10&#8722;4 M18,19. Since defects in regulating ciliary movements cause primary ciliary dyskinesia in humans, studying photobehaviors in C. reinhardtii might help in better understanding of these diseases and therapeutic developments20.
Herein, four simple methods to observe photobehaviors in C. reinhardtii are demonstrated. First, a phototaxis assay using Petri dishes is shown, and second, a phototaxis assay against cell suspension droplets. The phenomenon observed in both cases is not strictly phototaxis but photo accumulation, where the cells tend to accumulate close to the light source side or the opposite side. In C. reinhardtii, photo accumulation is mainly caused by phototaxis in a way that can be used as an approximation to phototaxis. Third, a more rigorous assay for phototaxis under a microscope is shown, and last is a photoshock assay under a microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm Cannonball green LED</td>
			<td>Optosupply</td>
			<td>OSPG5161P</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>AC adaptor for the light box</td>
			<td>ATTO</td>
			<td>2196161</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto cell counter</td>
			<td>DeNovix</td>
			<td>CellDrop BF</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Nakalai tesque</td>
			<td>06731-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera flash</td>
			<td>NEWWER</td>
			<td>TT560</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlamydomonas strains CC-124 and CC-125</td>
			<td>Chlamydomonas Resource Center</td>
			<td></td>
			<td>https://www.chlamycollection.org/</td>
		</tr>
		<tr>
			<td>C-mout CCD camera</td>
			<td>Wraymer</td>
			<td>1129HMN1/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop darkroom</td>
			<td>Scientex</td>
			<td>B-S8</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital still camera</td>
			<td>SONY</td>
			<td>RX100II</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dojindo</td>
			<td>G002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Green LED plate</td>
			<td>CCS</td>
			<td>ISLM-150X150-GG</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fujifilm WAKO</td>
			<td>080-01066</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dojindo</td>
			<td>GB70</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nakalai tesque</td>
			<td>238514-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightbox (Flat viewer)</td>
			<td>ATTO</td>
			<td>2196160</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (&#966;3.5 cm)</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium acetate</td>
			<td>Nakalai tesque</td>
			<td>28434-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply for the green LED plate</td>
			<td>CCS</td>
			<td>ISC-201-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Shibuya Optical</td>
			<td>S-RG630</td>
			<td></td>
		</tr>
	</tbody>
</table>

observation of photobehavior in chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Typical C. reinhardtii phototaxis and photoshock response assays are shown here. After cell density estimation, wild-type cell culture (a progeny of the cross CC-124 &#215; CC-125 with agg1+ mt -)23 was washed with photobehavior experimental solution for the phototaxis dish assay. The cell suspension was placed under dim red light for ~1 h. A 2 mL cell suspension was placed in a 3.5 cm Petri dish. The Petri dish was shaken gently, put on a lightbox, and photographed with a camera fixed on...

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Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced ...

The key question this method addresses is, if a strain of interesting Chlamydomonas retains the ability for photo behaviors or not. This method is simple, quick and easy to test if a strain of interesting exhibits photo behaviors. This method can contribute to the photobiology and cell biology of ciliary motility.Demonstrating the procedure will be Atsuko Isu, a lab technician from my laboratory. To begin, add two to three milliliters of cell suspension in a 3.5 centimeter Petri dish and place the dish on a light box. Shake the dish gently to uniformly distribute the cells and acquire a picture before illumination.Illuminate the dish from one side with a green light-emitting diode or LED in a desktop dark room. Leave the dish for approximately five minutes before acquiring images. For phototaxis assay using cell culture droplets, place 25 microliters of cell suspension droplets directly onto a white plastic plate using a micro pipette.Illuminate the droplets from one side with a green LED in a desktop dark room and leave them for three minutes before acquiring images. Place 30 microliters of cell suspension onto a glass slide and place an 18 by 18 millimeter cover slip with spacers on the side. Illuminate the samples from one side of the cover slip without a spacer with a green LED, and observe the cells under a darkfield microscope with a 10X objective lens under dim red light.After light illumination, record the cell illumination for 20 seconds using a camera-equipped microscope. For the photoshock response assay, place 30 microliters of cell suspension onto a glass slide, and place a cover slip with spacers on top of the slide. Observe the cell under a microscope with dim red light.Apply flash illumination using a camera flash while recording the cell movement. A representative example of negative phototaxis in Chlamydomonas reinhardtii after five minutes of side illumination is shown here. Most of the cells accumulated on the opposite side of the light source.In the photoshock response assay, almost all cells exhibited the photoshock response. The cells swam backward for a short period and recovered forward swimming. To perform this procedure, use aridimigudolphase cells and expose them to the red light before the assay.If not exposed to the red light, cells will become insensitive to the light. This technique isolates various mutants with abnormal phototaxis of photoshock responses. In addition, various intracellular and extracellular factors that influence photo behaviors have been identified.

observation-of-photobehavior-in-chlamydomonas-reinhardtii

Research

JoVE Journal

Biology

3.5K vistas.  Hosei University. La pregunta clave que aborda este método es, si una cepa de Chlamydomonas interesantes conserva la capacidad de comportamientos fotográficos o no. Este método es simple, rápido y fácil de probar si una cepa de interesante exhibe comportamientos fotográficos. Este método puede contribuir a la fotobiología y biología celular de la motilidad ciliar.Demostrando el procedimiento estará Atsuko Isu, un técnico de laboratorio de mi laboratorio. Para comenzar, agregue de dos a tres m...