Name: observation of photobehavior in chlamydomonas reinhardtii
Uploaded: 2022-05-06T15:00:00
Description: Watch this Scientific Journal Video about Observation of Photobehavior in Chlamydomonas reinhardtii at JoVE.com

这项研究得到了日本科学促进会KAKENHI（https://www.jsps.go.jp/english/index.html）到NU（19K23758，21K06295），TH（16H06556）和KW（19H03242，20K21420，21H00420），大隅前沿科学财团（https://www.ofsf.or.jp/en/）到KW的资助，以及从人类，环境和材料桥接人类，环境和材料动态联盟（http://alliance.tagen.tohoku.ac.jp/english/）到NU，TH和KW的资助。

在本研究中，使用了杂交CC-124 x CC-125与agg1 + mt-的杂交的生殖细胞 衣原体的野生型菌株21。从衣原体资源中心获得CC-124和CC-125（见 材料表），并在20-25°C下维持在三醋酸磷酸酯（TAP）22，1.5%琼脂糖培养基上。 任何运动菌株都可用于该方案。
1. 细胞培养
<ol><li>在TAP液体培养基中培养感兴趣的 衣原体 菌?...

<ol>
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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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+the+deflagellation+pathway+in+Chlamydomonas.">Genetics of the deflagellation pathway in Chlamydomonas.</a> Genetics. 149 (2), 927-936 (1998).</li><li>Morel-Laurens, N. <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+phototactic+orientation+in+Chlamydomonas+reinhardtii:+sign+and+strength+of+response.">Calcium control of phototactic orientation in Chlamydomonas reinhardtii: sign and strength of response.</a> Photochemistry and Photobiology. 45 (1), 119-128 (1987).</li><li>Wakabayashi, K., King, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Chlamydomonas+reinhardtii+flagellar+motility+by+redox+poise.">Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.</a> Journal of Cell Biology. 173 (5), 743-754 (2006).</li><li>Wakabayashi, K., Misawa, Y., Mochiji, S., Kamiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction-oxidation+poise+regulates+the+sign+of+phototaxis+in+Chlamydomonas+reinhardtii.">Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), 11280-11284 (2011).</li><li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in The Chlamydomonas Sourcebook Second Edition. 1, 25-64 (2009).</li><li>Mergenhagen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clock:+genetic+characterization+of+a+short+period+mutant+of+Chlamydomonas+reinhardii.">Circadian clock: genetic characterization of a short period mutant of Chlamydomonas reinhardii.</a> European Journal of Cell Biology. 33 (1), 13-18 (1984).</li><li>Ozasa, K., Lee, J., Song, S., Hara, M., Maeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+optical+feedback+control+of+Euglena+confined+in+closed-type+microfluidic+channels.">Two-dimensional optical feedback control of Euglena confined in closed-type microfluidic channels.</a> Lab on a Chip. 11 (11), 1933-1940 (2011).</li><li>Tanno, A., 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=The+four-celled+Volvocales+green+alga+Tetrabaena+socialis+exhibits+weak+photobehavior+and+high-photoprotection+ability.">The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.</a> PLoS One. 16 (10), 0259138 (2021).</li><li>Ueno, Y., Aikawa, S., Kondo, A., Akimoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+light-harvesting+functions+of+unicellular+green+algae+to+different+light+qualities.">Adaptation of light-harvesting functions of unicellular green algae to different light qualities.</a> Photosynthesis Research. 139 (1-3), 145-154 (2019).</li><li>Takahashi, T., Watanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthesis+modulates+the+sign+of+phototaxis+of+wild-type+Chlamydomonas+reinhardtii.+Effects+of+red+background+illumination+and+3-(3',4'-dichlorophenyl)-1,1-dimethylurea.">Photosynthesis modulates the sign of phototaxis of wild-type Chlamydomonas reinhardtii. 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>

目前的协议简单且不耗时。如果怀疑 莱纳迪 猪笼草突变体存在光感受或睫状体运动缺陷，则该方法可用作主要表型分析。
但是，存在一些关键步骤。一种是在对数生长阶段的早期到中期在实验中使用细胞。长时间培养后，细胞变得不那么活跃，对光敏感，甚至形成棕榈类化合物（细胞团块）27。另一个重要步骤是在实验前将细胞暴露在红光下（步骤2...

光是光合生物不可缺少的能量来源，但过多的光可能会造成光氧化损伤。因此，光养生物需要在中等强度的光下生存，在那里它们可以光合作用而不会遭受光氧化损伤1。在陆地植物中，叶绿体不能从叶子中移出并在细胞中显示照片运动;叶绿体在高光下移动到细胞的外围，在低光下移动到细胞表面2，而许多运动藻类显示出光行为，使它们能够找到合适的光合作用光条件，从而促进它们的生存3。
衣原体是 一种单细胞绿藻，在纤毛（又名鞭毛）、光合作用和光行为等研究领域被视为模型生物。 莱因哈蒂猪笼草 每个细胞有一个眼斑和两个纤毛，分别用于光感受和游泳。眼斑有两个组成部分：通道视紫红质（ChRs），质膜中的光门控离子通道以及位于ChR后面的富含类胡萝卜素的颗粒层。眼斑充当定向光受体，因为富含类胡萝卜素的颗粒层起到光反射器的作用4，5。
ChRs最初被确定为在莱恩哈迪衣原体6，7，8，9中引起光行为的光感受器。虽然在眼斑中发现了两种亚型ChR1和ChR2，但敲低实验表明，ChR1是光行为10的主要光感受器。尽管如此，ChR2受到了更多的关注，并在光遗传学的发展中发挥了核心作用，光遗传学是一种通过光11控制细胞激发的技术。因此，研究控制雷恩哈迪光行为的调控机制将进一步了解ChR功能并改善光遗传学。
在光接收后， 莱恩哈蒂猪笼草 细胞显示出两种类型的光行为：光出租车和光锤反应12。光出租车是细胞在光源方向或相反方向上游动的行为，分别称为正光出租车或负光出租车。光晕反应是细胞在感知到光强度的突然变化后表现出的一种行为，例如在闪光灯照射时。细胞停止游泳或向后游（即，随着细胞体向前游动）一小段时间，通常为<1 s。
莱因哈迪猪笼草的纤毛运动参与了其摄影行为。两个纤毛通常像人类的蛙泳一样跳动，这是针对光行为调制的。对于光出租车，由两个纤毛产生的力通过调节跳频和每个纤毛13的波形幅度而不平衡。最靠近眼斑的纤毛称为顺式纤毛，另一种称为反式纤毛。这两个纤毛在各个方面都有不同之处。例如，反式纤毛在体外的睫状跳动频率高出30%-40%14。此外，它们的Ca2 +灵敏度是不同的。去膜细胞模型15的再激活表明，对于Ca2+ <1 x 10−8 M，顺式纤毛的跳动比反式纤毛更强，而对于Ca2+ >1 x 10−7 M则相反。这种Ca2+灵敏度的不对称性对于光定向转弯可能很重要，因为缺乏这种不对称性的突变体不会表现出正常的光出租车16，17。相反，波形转换对于光震是必要的。睫状波形从前向游泳中的非对称波形转换为向后游泳中的对称波形。该波形转换也由Ca2+调节，阈值为1 x 10−4 M18，19。由于调节睫状体运动的缺陷导致人类原发性睫状体运动障碍，因此研究莱因哈迪猪笼草的光行为可能有助于更好地了解这些疾病和治疗发展20.
本文演示了四种观察 雷恩哈蒂猪笼 草中光行为的简单方法。首先，显示了使用培养皿的光出租车测定，其次，显示了针对细胞悬浮液滴的光出租车测定。在这两种情况下观察到的现象都不是严格的光出租车，而是光积累，其中细胞倾向于在光源侧或另一侧附近积聚。在 C. reinhardtii 中，光累积主要由光出租车引起，其方式可用作光出租车的近似值。第三，显示了在显微镜下对光氧化的更严格的测定，最后是在显微镜下进行光锤测定。

<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

对于运动光养微生物的生存，在适当的光照条件下至关重要。因此，它们显示出光诱导的行为（或光行为），并改变它们的运动方向以响应光。典型的光行为包括光震（或光恐惧症）响应和光出租车。光晕是对光强度突然变化（例如，闪光照明）的反应，其中生物体短暂地停止移动或向后移动。在光氧化期间，生物体向光源或相反方向移动（分别称为正光共济或负光氧化）。单细胞绿藻 衣原体是 研究光行为的极好生物体，因为它通过在光感受后调节纤毛（又名鞭毛）的跳动来迅速改变其游泳模式。在这里，展示了各种简单的方法来观察 莱因哈迪猪笼草中的光行为。对 C. reinhardtii的光行为的研究导致了真核纤毛和通道视紫红质之间的共同调节机制的发现，这可能有助于更好地了解纤毛病和开发新的光遗传学方法。

这里显示了典型的 莱因哈迪 猪笼草光出租车和光冲击反应测定。细胞密度估计后，用光行为实验溶液洗涤野生型细胞培养物（交叉CC-124×CC-125的后代，agg1 + mt -）23 用于光出租车培养皿测定。将细胞悬浮液置于昏暗的红光下约1小时。将2mL细胞悬浮液置于3.5cm培养皿中。培养皿被轻轻摇晃，放在灯箱上，并用固定在支架上的相机拍照。在这种情况下，细胞显示出明显的负光?...

Watch this Scientific Journal Video about Observation of Photobehavior in Chlamydomonas reinhardtii at JoVE.com

Observation of Photobehavior in Chlamydomonas reinhardtii

大多数游泳的光自养生物表现出光诱导的行为改变（光行为）。本方案在模式生物 衣原体中观察到所述光行为。

对衣原体视网膜的光行为观察

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

这种方法解决的关键问题是，一株有趣的衣原体是否保留了照片行为的能力。这种方法简单，快速且易于测试，如果一个有趣的菌株表现出照片行为。该方法有助于纤毛运动的光生物学和细胞生物学。演示该过程的将是我实验室的实验室技术人员Atsuko Isu。首先，在3.5厘米的培养皿中加入2至3毫升的细胞悬浮液，并将培养皿放在灯箱上。轻轻摇动培养皿以均匀分布细胞，并在照明前获得图像。在桌面暗室中用绿色发光二极管或LED从一侧照亮盘子。在获取图像之前，将盘子放置约五分钟。对于使用细胞培养液滴的光出租车测定，使用微量移液管将25微升的细胞悬浮液滴直接置于白色塑料板上。在桌面暗室中用绿色LED从一侧照亮液滴，并在获取图像之前将其放置三分钟。将30微升的细胞悬浮液放在载玻片上，并在侧面放置一个带有垫片的18×18毫米盖玻片。在没有带有绿色LED的垫片的情况下从盖玻片的一侧照亮样品，并在暗淡的红光下用10X物镜在暗场显微镜下观察细胞。光照后，使用配备相机的显微镜记录细胞照度20秒。对于光锤反应测定，将30微升细胞悬浮液置于载玻片上，并在载玻片顶部放置带有垫片的盖玻片。在具有暗红色光的显微镜下观察细胞。在记录细胞运动时使用相机闪光灯应用闪光灯照明。此处显示了在侧面照明五分钟后，在衣原体中出现负光出租车的代表性示例。大多数细胞积聚在光源的另一侧。在光冲击反应测定中，几乎所有细胞都表现出光冲击反应。细胞向后游了一小段时间，并恢复了向前游泳。要执行此过程，请使用干旱二咪结相细胞，并在测定前将其暴露在红光下。如果不暴露在红光下，细胞将对光变得不敏感。该技术分离出具有异常光锤反应的各种突变体。此外，还发现了影响照片行为的各种细胞内和细胞外因素。

Research

JoVE Journal

Biology

Observation of Photobehavior in Chlamydomonas reinhardtii

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

科研

生物学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

解剖和蜜蜂背血管的观察心功能的研究

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

莱因哈蒂衣原体中去膜化细胞模型的再激活

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in ...