搜索

Phototaxis Assay for Chlamydomonas reinhardtii
莱茵衣藻的趋光性分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Phototaxis is a behavior in which organisms move toward or away from the light source (positive or negative phototaxis, respectively). It is crucial for phototrophic microorganisms to inhabit under proper light conditions for phototaxis. The unicellular green alga Chlamydomonas reinhardtii rapidly changes its swimming direction upon light illumination, and thus is a nice model organism for phototaxis research. Here we show two methods to assay Chlamydomonas phototaxis; one is a quick, easy and qualitative analysis, so-called the dish assay; and the other is a quantitative single-cell analysis.

Keywords: Phototaxis(趋光性), Green algae(绿藻), Flagella(鞭毛), Channelrhodopsin(光敏感通道蛋白), Photoreception(感光)

Background

The unicellular green alga Chlamydomonas reinhardtii is used as a model organism in various research fields including phototaxis of microorganisms, photosynthesis, and ciliary/flagellar motility (Hegemann and Berthold, 2009). A Chlamydomonas cell perceives light at its eyespot, the photoreceptive organelle observed as an orange spot located near the cell equator. The eyespot contains the photoreceptor proteins channelrhodopsins localized in the cellular membrane and the carotenoid-rich granule layers right behind the channelrhodopsins which function as a light reflector. Because of their relative position, the eyespot undergoes highly directional photoreception, and the cell can accurately detect the direction of light illumination (Foster and Smyth, 1980; Ueki et al., 2016). Upon photoreception, two flagella change their beating balance, and the cell changes its swimming direction either toward or away from the light source.

The Chlamydomonas phototactic direction (or ‘sign’) is regulated by cellular reduction-oxidation state, which is affected by cellular metabolism such as photosynthetic and respiratory activities (Wakabayashi et al., 2011). The phototactic sign thus indirectly reflects those activities in vivo. For instance, a mutant showing fast phototactic response has been shown to have high photosynthetic activity (Kim et al., 2016). In addition, for the regulation of flagellar beating for phototactic turning of the cell, flagellar dyneins should be strictly regulated (Kamiya and Witman, 1984; Okita et al., 2005; Hegemann and Berthold, 2009). Therefore, phototaxis assay contributes to a wide variety of biological researches, such as photoreception, photosynthesis, respiration, and motor proteins.

Various methods have been developed to quantify Chlamydomonas phototaxis. Mergenhagen developed an automatic assay system for phototaxis (photoaccumulation), which detects the density of cells in the light path by a photocell (Mergenhagen, 1984). Takahashi et al. developed a computer-assisted system that automatically detects the direction of cellular movement using an infrared-sensitive video camera (Takahashi et al., 1991). Comparing to those sophisticated systems with hand-made equipment, our protocol is rather simple, and can be carried out with equipment that is commercially or freely available.

Materials and Reagents

  1. 50 ml tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  2. 4 cm Petri dish (AS ONE, catalog number: 1-8549-01 )
  3. Chlamydomonas strain of interest
  4. Tris-acetate-phosphate medium
  5. (Optional) Tertiary-butyl hydroperoxide (t-BOOH: final concentration, 0.2 mM) (WAKO Pure Chemical Industries, catalog number: 026-13451 )
  6. (Optional) N,N’-dimethylthiourea (DMTU: final concentration, 75 mM) (Sigma-Aldrich, catalog number: D188700 )
  7. HEPES (pH 7.4) (NACALAI TESQUE, catalog number: 17514-15 )
  8. EGTA (DOJINDO, catalog number: 346-01312 )
  9. Potassium chloride (KCl) (NACALAI TESQUE, catalog number: 28514-75 )
  10. Calcium chloride dihydrate (CaCl2·2H2O) (NACALAI TESQUE, catalog number: 06731-05 )
  11. Phototaxis assay solution (Okita et al., 2005) (see Recipes)

Equipment

  1. Centrifuge (Hitachi Koki, model: CR20GIII )
  2. Swing rotor (Hitachi Koki, model: R4SS )
  3. Green light-emitting diode (LED) (λ = 525 nm) (OptoSupply, model: OSPG5111A-VW )
  4. Red light (or white light with a red filter [λ > 600 nm])
  5. White sheet of paper/plastic
  6. Dark room or dark box
  7. Digital still camera (SONY, model: RX100II )
  8. Imaging table with camera mount (AS ONE, model: NS-CPS360N )
  9. Inverted microscope equipped with video camera (Olympus, model: IX70 ; Wraymer, model: 1129HMN1/3)
  10. Red filter (630 nm long-pass filter) (SCHOTT, model: RG630 )
  11. (Optional) Photometer (Apogee Instrument, model: MQ-200 )
  12. (Optional) Neutral density filters (HOYA, models: ND10AH and ND30AH )

Software

  1. Image Hyper (Science Eye, Japan) or any particle-tracking software (e.g., ImageJ with MTrack2 plugin)
  2. Microsoft Excel or any spreadsheet software

Procedure

  1. Culture cells in Tris-acetate-phosphate medium (Gorman and Levine, 1965) with aeration at 22 °C under a 12 h/12 h light/dark cycle (light: ~30 µmol photons m-2 sec-1 white light) to the mid-log phase (~3 x 106 cells/ml).
    Note: Change the medium and the other culture conditions when necessary.
  2. Harvest cells by centrifugation at 600 x g for 5 min at room temperature.
  3. Suspend cells in a phototaxis assay buffer at ~1 x 107 cells/ml (for dish assay) or ~1 x 106 cells/ml for quantitative assay in a 50 ml tube.
  4. Place the cells under red light (~40 µmol photons m-2 sec-1) for 30-60 min.
    Note: This step makes cells swim actively (Sineshchekov et al., 2000). In addition, red light (λ) does not stimulate channelrhodopsin 1 (ChR1), the main photoreceptor protein for phototaxis, and thus makes cells sensitive to following green-light illumination (Berthold et al., 2008).
  5. (For dish assay) Put 2 ml cell suspensions in a Petri dish, place it on a white sheet and take a picture before illumination (Figure 1A).
    Note: When control data for positive or negative phototaxis are necessary, add final 0.2 mM t-BOOH or 75 mM DMTU to the cell suspensions, respectively. These reagents can be added either to the harvested cell suspensions in a test tube or directly the cell suspensions in a dish. t-BOOH is a kind of reactive oxygen species (ROS) which has similar effects to H2O2, and can be substituted with 25 μM H2O2. DMTU is a H2O2 scavenger, and can be substituted with 50 mM 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) (Wakabayashi et al., 2011). Another option to derive negative phototaxis control is to use the strain CC-124 (agg1). It is regarded as a ‘wild type’, but its mutation in the agg1 locus causes strong negative phototaxis (Ide et al., 2016).


    Figure 1. Dish phototaxis assay. A. A 4 cm Petri dish is set on a white plastic sheet. A green LED is set on the side of the dish and fixed. B. During and C. after illumination for 5 min. Positive phototaxis is observed. D. For dish assay, the Petri dish and the LED is covered with a box to block the room light. The inside of the box is masked with pieces of black cloth for antireflection. E. During the dish assay. Before and after 5 min (or longer) illumination, pictures are taken from the top with a camera.

  6. Illuminate the dish from one side with green LED and cover the dish and the LED with a box (Figures 1B, 1D and 1E). Leave them for 5 min (or longer). Check the light intensity by a photometer. Typically, wild-type cells show positive phototaxis at low intensity (< 1 μmol photons m-2 sec-1) and negative phototaxis at higher intensity (> 5 μmol photons m-2 sec-1). To change the light intensity, either change the distance from the LED to the dish and/or set ND filters in front of the LED.
    Note: The action spectra of phototaxis peak at 495 to 505 nm (blue-green light) (Foster et al., 1984). We use green light because blue light activates photosynthesis, which changes cellular redox state as well as the phototactic sign (Takahashi and Watanabe, 1993; Wakabayashi et al., 2011).
  7. Take pictures (Figure 1C).
    Note: Please note that this ‘dish assay’ is not an accurate method for phototaxis assay, because accumulation of cells caused by light illumination (called photo-accumulation) could occur also by the photoshock response, in that cells either stop or swim backward for a short period upon sensing the sudden change in the light intensity. The purpose of this method is a quick test for phototactic capability. Phototactic movement is defined as the movement along the light beam, which should be examined by the following single-cell analysis.
  8. (For the single-cell analysis) Place a Petri dish on the stage of an inverted microscope and illuminate it with a green LED from the side (Figure 2). Observe the cells in the area near the light source (to estimate the light intensity when necessary) with dim red light (λ > 600 nm, ~5 μmol photons m-2 sec-1) and video-record using a CCD camera.


    Figure 2. Setting for cell-level phototaxis analysis. A 4 cm Petri dish is set on a stage of the inverted microscope. Arrow indicates the red filter (630 nm long-pass filter). Arrowhead indicates the green LED.

Data analysis

  1. Track the swimming cells using a particle-tracking software. The Image Hyper software can be used in a semi-automatic manner. For tracking cells using Image J software, save the video in the uncompressed Audio Video Interleave (AVI) format. Playback the AVI file using ImageJ. Binarize the images (cells appear in black and background in white). Run MTrack2 plugin.
  2. Export the data of the trajectories (i.e., positions of cells at each frame) as the Comma-Separated Values (CSV) or any format for spreadsheet softwares.
  3. Measure the angle (θ) between the light direction and the swimming direction from the trajectories during 1.5 sec following illumination with a green LED for 15 sec (Figure 3A). The angle can be calculated by a spreadsheet software. (If Microsoft Excel is used, it can be calculated as following: ‘= degrees(atan2((x2 - x1),(y2 - y1)))’, where (x1, y1) represents the columns for the primary position of a cell and (x2, y2) represents those for the position after 1.5 sec.)
    Note: For a few seconds after illumination, the photoshock response could occur and the phototactic behavior should be recorded after that period


    Figure 3. Cell-level phototaxis analysis. A. Swimming direction of each cell is measured for 1.5 sec following 15 sec illumination with a green LED to avoid the effects of photoshock response. B. An example of cell-level analysis (wild-type cells, random swimming). Well-focused images of swimming cells were auto-tracked using the Image Hyper software. Circles represent the starting points of cells, and lines indicate their swimming trajectories for 1.5 sec. (see Video 1). C and D. Polar histograms depicting the percentage of cells moving in a particular direction relative to light illuminated from the right (0°) (12 bins of 30°; n = 50 cells per condition). C. Representative data for random swimming when not illuminated; D. Representative data for positive phototaxis. ~80% of cells swim to 0°. Modified from (Wakabayashi et al., 2011).

    Video 1. Tracking cells using Image Hyper software. Cells showing phototactic swimming were auto-tracked under a dark-field microscope. Four cells were tracked in this movie, and the former three cells show positive, and the last cell show negative phototaxis. (This software is a Japanese product and some letters in the screen are written in Japanese.)

  4. For drawing polar histograms, typically, set 12 bins of 30° and draw a histogram (Figures 3C and 3D). (If necessary, more bins can be set [such as 24 bins of 15°].) If Microsoft Excel is used, draw graphs with ‘radar’ (Figure 4).


    Figure 4. Drawing polar histogram. A screen shot of Microsoft Excel is shown. Calculate the first histogram (columns A and B) by the swimming angle against the light illumination axis. The numbers of cells are then pooled to different angular categories (columns C and D) as followings: D2 = B11, D4 = B10, D6 = B9, D8 = B8, D10 = B7, D12 = B6, D14 = B5, D16 = B4, D18 = B3, D20 = B2 + B14, D22 = B13, and D24 = B12. Insert an empty bin between each two categories (e.g., 75 between categories 90 and 60) so that the values are drawn as bars, not as the points of a polygon in the radar graph. In this example, most cells show negative phototaxis (the light is illuminated from 0°).

  5. For estimation of phototactic index, calculate cosθ for each cell. When the sign of  phototaxis is examined, average the value of cosθ. When cells are not illuminated and swim in random directions, the phototactic index should be ~0. When 100% of cells show clear positive or negative phototaxis, the phototactic index is 1 or -1, respectively. When the phototactic capability (i.e., swimming parallel to the light beam) is examined and the phototactic sign can be disregarded, average the value of |cosθ|. When 100% of cells swim parallel to the light beam, this index is 1. When cells swim in random directions, index is ~2/π (Okita et al., 2005).

Recipes

  1. Phototaxis assay solution (Okita et al., 2005)
    5 mM HEPES (pH 7.4)
    0.2 mM EGTA
    1 mM KCl
    0.3 mM CaCl2

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers 15H01206, 15H01314, and 16K14752 to KW. This protocol was used in Wakabayashi et al., 2011 and Ueki et al., 2016.

References

  1. Berthold, P., Tsunoda, S. P., Ernst, O. P., Mages, W., Gradmann, D. and Hegemann, P. (2008). Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization. Plant Cell 20(6): 1665-1677.
  2. Foster, K. W., Saranak, J., Patel, N., Zarilli, G., Okabe, M., Kline, T. and Nakanishi, K. (1984). A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature 311(5988):756-759.
  3. Foster, K. W. and Smyth, R. D. (1980). Light Antennas in phototactic algae. Microbiol Rev 44(4): 572-630.
  4. Gorman, D. S. and Levine R. P. (1965). Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 54: 1665-1669.
  5. Hegemann, P. and Berthold, P. (2009). Sensory photoreceptors and light control of flagellar activity. In: George, B. W. (Ed). The Chlamydomonas Sourcebook Second Edition Volume 3. Academic Press pp: 395-430.
  6. Ide, T., Mochiji, S., Ueki, N., Yamaguchi, K., Shigenobu, S., Hirono, M. and Wakabayashi, K. (2016). Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii. Biochem Biophys Reports 7: 379-385.
  7. Kamiya, R. and Witman, G. B. (1984). Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J Cell Biol 98(1): 97-107.
  8. Kim, J. Y., Kwak, H. S., Sung, Y. J., Choi, H. I., Hong, M. E., Lim, H. S., Lee, J. H., Lee, S. Y. and Sim, S. J. (2016). Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis. Sci Rep 6: 21155.
  9. Mergenhagen, D. (1984). Circadian clock: genetic characterization of a short period mutant of Chlamydomonas reinhardii. Eur J Cell Biol 33(1): 13-18.
  10. Okita, N., Isogai, N., Hirono, M., Kamiya, R. and Yoshimura, K. (2005). Phototactic activity in Chlamydomonas 'non-phototactic' mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein. J Cell Sci 118(Pt 3): 529-537.
  11. Sineshchekov, O., Lebert, M. and Hader, D. P. (2000). Effects of light on gravitaxis and velocity in Chlamydomonas reinhardtii. J Plant Physiol 157(3): 247-254.
  12. Takahashi, T. and Watanabe, M. (1993). Photosynthesis modulates the sign of phototaxis of wild-type Chlamydomonas reinhardtii. Effects of red background illumination and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. FEBS Lett 336(3): 516-520.
  13. Takahashi, T., Yoshihara, K., Watanabe, M., Kubota, M., Johnson, R., Derguini, F. and Nakanishi, K. (1991). Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: simultaneous measurements of phototactic and photophobic responses. Biochem Biophys Res Commun 178(3): 1273-1279.
  14. Ueki, N., Ide, T., Mochiji, S., Kobayashi, Y., Tokutsu, R., Ohnishi, N., Yamaguchi, K., Shigenobu, S., Tanaka, K., Minagawa, J., Hisabori, T., Hirono, M. and Wakabayashi, K. (2016). Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 113(19): 5299-5304.
  15. Wakabayashi, K., Misawa, Y., Mochiji, S. and Kamiya, R. (2011). Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 108(27): 11280-11284.

简介

光趋向性是一种生物朝向或远离光源(分别为正或负光趋势)移动的行为。 光合微生物在光照条件适宜的条件下居住是至关重要的。 单细胞藻类莱茵衣藻在光照下迅速改变其游泳方向,因此是光线研究的一个很好的模型生物。 我们在这里展示了两种测定衣藻感染的方法; 一个是快速,容易和定性的分析,所谓的盘测定; 另一个是定量单细胞分析
【背景】单细胞藻类莱茵衣藻被用作各种研究领域的示范生物,包括微生物光合作用,光合作用和睫毛/鞭毛运动(Hegemann and Berthold,2009)。衣藻细胞在其眼窝感觉到光,观察细胞器作为位于细胞赤道附近的橙色斑点。眼窝包含位于细胞膜中的光感受器蛋白质通道视紫质和富含类胡萝卜素的颗粒层,其作用于光反射器之后的通道视紫质。由于它们的相对位置,眼睛受到高度定向的光感受,细胞可以准确地检测光照的方向(Foster和Smyth,1980; Ueki等,2016)。在光接收时,两个鞭毛改变它们的跳动平衡,并且细胞改变其游泳方向朝向或远离光源。
衣原体光镜方向(或“符号”)由细胞还原氧化态调节,其受细胞代谢如光合作用和呼吸活动的影响(Wakabayashi等,2011)。因此,光照标记间接地反映了体内的这些活性。例如,显示快速光敏反应的突变体已被证明具有高的光合活性(Kim等,2016)。此外,为了调节鞭毛打击细胞的光动力转动,鞭毛动力蛋白应严格调节(Kamiya和Witman,1984; Okita等,2005; Hegemann和Berthold,2009)。因此,光滴测定有助于广泛的生物学研究,如光接收,光合作用,呼吸和运动蛋白。
已经开发了各种各样的方法来定量衣藻光合速率。 Mergenhagen开发了一种用于光照(光累积)的自动测定系统,其通过光电池检测光路中细胞的密度(Mergenhagen,1984)。高桥等开发了一种使用红外敏感摄像机自动检测细胞运动方向的计算机辅助系统(Takahashi等,1991)。与手工设备相比,我们的协议相当简单,可以使用商业上可用的设备进行。

关键字:趋光性, 绿藻, 鞭毛, 光敏感通道蛋白, 感光

材料和试剂

  1. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339652)
  2. 4厘米培养皿(AS ONE,目录号:1-8549-01)
  3. 感染菌株
  4. Tris-乙酸盐 - 磷酸盐培养基
  5. (任选的)叔丁基过氧化氢(t-BOHOH:终浓度,0.2mM)(WAKO Pure Chemical Industries,目录号:026-13451)
  6. (可选)N,N' - 二甲基硫脲(DMTU:终浓度,75mM)(Sigma-Aldrich,目录号:D188700)
  7. HEPES(pH 7.4)(NACALAI TESQUE,目录号:17514-15)
  8. EGTA(DOJINDO,目录号:346-01312)
  9. 氯化钾(KCl)(NACALAI TESQUE,目录号:28514-75)
  10. 氯化钙二水合物(CaCl 2·2H 2 O)(NACALAI TESQUE,目录号:06731-05)
  11. Phototaxis测定溶液(Okita等人,2005)(参见食谱)
    1. 设备

      1. 离心机(日立Koki,型号:CR20GIII)
      2. 旋转转子(日立Koki,型号:R4SS)
      3. 绿色发光二极管(LED)(λ= 525nm)(OptoSupply,型号:OSPG5111A-VW)
      4. 红光(或具有红色滤光片的白光[λ> 600nm])
      5. 白纸/塑料
      6. 暗室或黑匣子
      7. 数码相机(SONY,型号:RX100II)
      8. 带相机安装的成像台(AS ONE,型号:NS-CPS360N)
      9. 带有摄像机的倒置显微镜(Olympus,型号:IX70; Wraymer,型号:1129HMN1/3)
      10. 红色滤光片(630 nm长通滤光片)(肖特,型号:RG630)
      11. (可选)光度计(Apogee Instrument,型号:MQ-200)
      12. (可选)中性密度滤光片(HOYA,型号:ND10AH和ND30AH)
        1. 软件

          1. Image Hyper(Science Eye,Japan)或任何粒子跟踪软件(例如,,具有MTrack2插件的ImageJ)
          2. Microsoft Excel或任何电子表格软件
            1. 程序

              1. 培养细胞在Tris-乙酸盐 - 磷酸盐培养基(Gorman和Levine,1965)中,在22℃,12小时/12小时光照/暗循环(光:〜30μmol光子, sec -1 白光)至对数中期(〜3×10 6个细胞/ml)。
                注意:必要时更改中等和其他文化条件。
              2. 通过在室温下以600×g离心5分钟收获细胞。
              3. 将细胞以〜1×10 7个细胞/ml(用于培养皿测定)或〜1×10 6个细胞/ml悬浮在光趋向性测定缓冲液中,用于在50 ml管
              4. 将细胞置于红光下(约40μmol光子m -2 sec -1 )30-60分钟。
                注意:这一步使细胞积极游泳(Sineshchekov et al。,2000)。另外,红光(λ)不刺激光诱导的主要感光蛋白1(ChR1),从而使细胞对后续的绿光照明敏感(Berthold等,2008)。 />
              5. (用于餐具测定)将2ml细胞悬浮液放入陪替氏培养皿中,将其置于白片上并照射前拍照(图1A)。
                注意:当需要正或负光趋向性的控制数据时,分别将最终的0.2mM t-BOOH或75mM DMTU加入到细胞悬浮液中。这些试剂可以添加到试管中的收获的细胞悬液中,或直接加入盘中的细胞悬液。 t-BOOH是一种对H 2 O 2 O 2具有相似作用的活性氧(ROS),可以用25μMH 2 O取代, /子> 0 <子> 2 。 DMTU是H 2 O 2 O 2清除剂,可以用50mM的4-羟基-2,2,6,6-四甲基哌啶1-氧基(TEMPOL)(TEMPOL) Wakabayashi等人,2011)。导出阴性光趋势控制的另一个选择是使用菌株CC-124(agg1)。它被认为是"野生型",但其在agg1基因座中的突变导致强负的光合速率(Ide et al。,2016)。


                图1.碟形光谱测定。 A.将4cm培养皿放置在白色塑料片上。一个绿色的LED被放在盘的侧面并固定。 B.在照明期间和C.照射5分钟。观察到正光趋势。 D.对于碟形测定,培养皿和LED被一个盒子覆盖以阻挡房间的光线。盒子的内部被黑色的布料遮掩,用于防反射。 E.在盘测定期间。在5分钟(或更长时间)照明之前和之后,照相机从顶部拍摄照片。

              6. 用绿色LED从一侧照亮菜,并用一个盒子盖住菜肴和LED(图1B,1D和1E)。离开他们5分钟(或更长时间)。用光度计检查光强度。通常,野生型细胞在低强度(<1μmol光子> sec -1)处显示正光趋势,在较高强度(>5μmol光子m -2 sec -1 )。要改变光强度,可以改变从LED到盘的距离和/或设置LED前面的ND滤镜。
                注意:光吸收峰在495〜505nm(蓝 - 绿光)的作用光谱(Foster等,1984)。我们使用绿光,因为蓝光激活光合作用,这改变了细胞氧化还原状态以及光照标志(Takahashi和Watanabe,1993; Wakabayashi等,2011)。
              7. 拍照(图1C)。
                注意:请注意,这种"盘测定"不是光趋化测定的准确方法,因为光照会引起的细胞积累(称为光累积)也可能由于光休克反应而发生,因为细胞停止或停止感觉到光线强度的突然变化,会在短时间内向后游泳。该方法的目的是快速测试光触媒能力。光照运动被定义为沿着光束的运动,应该通过以下单细胞分析来检查。
              8. (对于单细胞分析)将培养皿放置在倒置显微镜的平台上,并从侧面用绿色LED照明(图2)。观察光源附近区域的细胞(以必要时估计光强度)(λ> 600nm,〜5μmol光子,m sec -1) )和使用CCD相机的视频记录。


                图2.细胞级光趋势分析的设置。将4厘米培养皿放置在倒置显微镜的一个阶段。箭头表示红色滤光片(630 nm长通滤光片)。箭头表示绿色LED。
                1. 数据分析

                  1. 使用粒子跟踪软件跟踪游泳池。 Image Hyper软件可以半自动的方式使用。对于使用Image J软件跟踪单元格,请将视频保存在未压缩的音频视频交错(AVI)格式中。使用ImageJ播放AVI文件。对图像进行二值化(单元格以黑色和背景显示为白色)。运行MTrack2插件。
                  2. 将逗号分隔值(CSV)或电子表格软件的任何格式导出轨迹的数据(即,每个帧的单元格位置)。
                  3. 在用绿色LED照射15秒后的1.5秒内,从轨迹测量光线方向和游泳方向之间的角度(θ)(图3A)。角度可以通过电子表格软件来计算。 (如果使用Microsoft Excel,则可以计算如下:'= degrees(atan2((x 2 - x 1 ),(y 2 <其中(x 1,...,y 1)表示单元的主要位置的列,并且(x 2,y 2)代表1.5秒后的位置)。
                    注意:照射后几秒钟,可能会发生拍摄反应,并在该时间段后记录光照行为。


                    图3.细胞级光趋势分析。 A.用绿色LED照射15秒后测量每个细胞的游泳方向1.5秒,以避免拍摄反应的影响。 B.细胞水平分析(野生型细胞,随机游泳)的一个例子。使用Image Hyper软件自动跟踪游泳细胞的精心集中的图像。圆圈表示细胞的起点,线条表示其游泳轨迹1.5秒。 (见视频1)。 C和D.极坐标直方图描述了相对于从右侧(0°)照射的光(特定方向为12°,30°; n =每个条件50个细胞)在特定方向移动细胞的百分比。 C.不亮时随机游泳的代表性数据; D.正面趋光性的代表性数据。 〜80%的细胞游泳到0°。 (Wakabayashi等人,2011)。

                    Video 1. Tracking cells using Image Hyper software. Cells showing phototactic swimming were auto-tracked under a dark-field microscope. Four cells were tracked in this movie, and the former three cells show positive, and the last cell show negative phototaxis. (This software is a Japanese product and some letters in the screen are written in Japanese.)

                    To play the video, you need to install a newer version of Adobe Flash Player.

                    Get Adobe Flash Player


                  4. 为了绘制极坐标直方图,通常设定为12°的30°并绘制直方图(图3C和3D)。 (如果需要,可以设置更多的箱子[例如24个箱子的15°]。)如果使用Microsoft Excel,使用"雷达"绘制图形(图4)。


                    图4.绘制极坐标直方图。显示Microsoft Excel的屏幕截图。通过相对于光照度轴的游泳角度计算第一直方图(列A和B)。然后将单元数合并到不同的角度类别(列C和D),如下:D2 = B11,D4 = B10,D6 = B9,D8 = B8,D10 = B7,D12 = B6,D14 = B5,D16 = B4,D18 = B3,D20 = B2 + B14,D22 = B13,D24 = B12。在每个类别(例如,类别90和60之间的每个类别(例如,75)之间插入空箱,以便将值绘制为条,而不是作为雷达图中多边形的点。在这个例子中,大多数细胞显示出负光照(光从0°照亮)
                  5. 为了估计光照指数,计算每个细胞的cosθ。当 检查光照,平均值cosθ。当细胞不被照射并沿随机方向游泳时,光照指数应为〜0。当100%的细胞显示明显的正或负光趋势时,光敏指数分别为1或-1。当检查光触媒能力(即平行于光束游动)时,可以忽略光触觉标志,平均|cosθ|的值。当100%的细胞与光束平行游动时,该指数为1.当细胞沿随机方向游泳时,指数为〜2 /π(Okita等人,2005)。
                    1. 食谱

                      1. Phototaxis测定溶液(Okita等人,2005)
                        5mM HEPES(pH 7.4)
                        0.2 mM EGTA
                        1 mM KCl
                        0.3mM CaCl 2
                        1. 致谢

                          这项工作得到了JSS KAKENHI Grant号15H01206,15H01314和KKK的16K14752的支持。该协议在2011年的Wakabayashi等人,2011和Ueki等人,2016中使用。

                          参考

                          1. Berthold,P.,Tsunoda,SP,Ernst,OP,Mages,W.,Gradmann,D.and Hegemann,P。(2008)。视紫质是单细胞真核细胞衣藻中光合作用的功能感光体。 自然 311(5988):756-759。
                          2. Foster,KW和Smyth,RD(1980)。  Light天线在光触媒藻类中。微生物修订版44(4):572-630。
                          3. Gorman,DS和Levine RP(1965)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC300531/"target ="_ blank" >细胞色素f和质体蛋白:它们在莱茵衣藻光合电子传递链中的序列。 Proc Natl Acad Sci USA 54:1665-1669。 >
                          4. Hegemann,P.和Berthold,P。(2009)。感光感受器和鞭毛活动的光控。在:George,B.W。(Ed)。 "衣原体资料手册"第二版第3卷。 学术出版社 pp:395-430。
                          5. Ide,T.,Mochiji,S.,Ueki,N.,Yamaguchi,K.,Shigenobu,S.,Hirono,M。和Wakabayashi,K。(2016)。< a class ="ke-insertfile"href ="http://www.sciencedirect.com/science/article/pii/S2405580816301248"target ="_ blank">鉴定在"野生型"菌株中负责负光趋向性的'agg1 突变的衣藻衣原体。生物化学生物物理报告 7:379-385。
                          6. Kamiya,R。和Witman,GB(1984)。  亚微摩尔钙水平控制了衣藻模型中两种鞭毛之间跳动的平衡。 98细胞生物 98(1):97-107。 br />
                          7. Kim,JY,Kwak,HS,Sung,YJ,Choi,HI,Hong,ME,Lim,HS,Lee,JH,Lee,SY and Sim,SJ(2016)。  微流控高通量选择具有优异光合效率的微藻菌株,使用竞争性光照。 Sci Rep 6:21155.
                          8. Mergenhagen,D。(1984)。  昼夜节律钟:遗传表征衣藻的短期突变体。 Eur J Cell Biol 33(1):13-18。
                          9. Okita,N.,Isogai,N.,Hirono,M.,Kamiya,R。和Yoshimura,K。(2005)。在衣原体中的光敏作用缺乏Ca 2 + - 鞭毛的依赖性控制的非光学突变体优势或内臂动力蛋白。细胞科学 118(Pt 3):529-537。
                          10. Sineshchekov,O.,Lebert,M.and Hader,DP(2000)。  光对莱茵衣藻的重力和速度的影响 植物生理157(3):247-254。
                          11. Takahashi,T.和Watanabe,M.(1993)。光合作用调节野生型莱茵衣藻的光趋向性的迹象。红色背景照明和3-(3',4'-二氯苯基)-1,1-二甲基脲的影响。 FEBS Lett 336(3):516-520。
                          12. Takahashi,T.,Yoshihara,K.,Watanabe,M.,Kubota,M.,Johnson,R.,Derguini,F.and Nakanishi,K。(1991)。 13-ene视网膜的光异构化对于莱茵衣藻的光趋向性是重要的:同时测量光化学和光致反应。生物化学生物学通讯 178(3):1273-1279。
                          13. Ueki,N.,Ide,T.,Mochiji,S.,Kobayashi,Y.,Tokutsu,R.,Ohnishi,N.,Yamaguchi,K.,Shigenobu,S.,Tanaka,K.,Minagawa, Hisabori,T.,Hirono,M。和Wakabayashi,K。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27122315"目标="_ blank">眼药水依赖性确定衣原体中的光敏征标志。 Proc Natl Acad Sci USA 113(19):5299-5304。
                          14. Wakabayashi,K.,Misawa,Y.,Mochiji,S.和Kamiya,R。(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21690384"target ="_ blank">还原氧化泊松调节了莱茵衣藻的光趋势标志。美国Proc Natl Acad Sci USA 108(27 ):11280-11284。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ueki, N. and Wakabayashi, K. (2017). Phototaxis Assay for Chlamydomonas reinhardtii. Bio-protocol 7(12): e2356. DOI: 10.21769/BioProtoc.2356.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。