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本实验方案简略版
May 2019

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Visualizing Filamentous Actin in Chlamydomonas reinhardtii
在莱茵衣藻中利用鬼笔环肽进行纤维状肌动蛋白可视化分析   

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Abstract

This protocol aims to visualize the filamentous actin network in Chlamydomonas reinhardtii. We improved fixed-cell labeling conditions using the F-actin probe, phalloidin. We created a Chlamydomonas-optimized protocol by halving the phalloidin incubation time, electing for optimal fixation conditions, and selecting for a healthy cell population. This phalloidin protocol is quick, effective, and is the only labeling method to date that allows for reliable actin filament detection in fixed vegetative Chlamydomonas cells. This method reveals previously unidentified actin structures in Chlamydomonas and novel insights into cytoskeletal dynamics.

Keywords: Chlamydomonas reinhardtii (莱茵衣藻), Actin (肌动蛋白), Phalloidin (鬼笔环肽), Cytoskeleton (细胞骨架), Filaments (纤维)

Background

Chlamydomonas reinhardtii is a single-celled green alga widely used in studying photosynthesis, cilia, chloroplast biology, and the synthesis of biofuels. It is a leading model system in these fields due to the organism’s well-characterized two apical flagella, inexpensive and simple culture conditions, and a fully sequenced haploid genome allowing for excellent genetic studies. Understanding the Chlamydomonas cytoskeleton will play an important role in advancing these areas of research. However, the filamentous actin network of Chlamydomonas reinhardtii remains uncharacterized, partly due to the difficulties in visualizing filaments in this eukaryotic alga.

Recently, there has been some success in detecting filamentous actin in Chlamydomonas. Through the expression of the fluorescently-tagged actin binding peptide, LifeAct, live-cell imaging revealed an actin-based perinuclear structure (Avasthi et al., 2014; Onishi et al., 2016). However, there is increasing evidence that LifeAct alters cell morphology, actin organization, actin-dependent functions, and preferentially labels a subset of actin structures (Courtemanche et al., 2016, Flores et al., 2019). Further, achieving stable expression is difficult in Chlamydomonas due to epigenetic silencing of exogenous genes (Cerutti et al., 1997) and the need to colocalize actin probes with fixed cell organelle markers is essential for understanding the revitalized field of Chlamydomonas actin biology. Thus, an efficient and specific fixed-cell method of actin visualization would lead to significant advancement in the field and offer novel insights into basic actin biology.

Available actin antibodies do not discriminate between monomeric and filamentous actin. It was previously shown that the widely used fluorescent actin probe phalloidin was ineffective in labeling filamentous actin in vegetative Chlamydomonas cells (Harper et al., 1992). However, phalloidin does label an actin-dense structure in gametic cells known as the fertilization tubule (Detmers et al., 1985). By optimizing phalloidin staining conditions, we are now able to visualize the Chlamydomonas actin network with exquisite detail in vegetative cells (Figure 1). Specifically, reducing the staining incubation time from 30 min to 16 min was essential in achieving optimal signal to noise which allowed for detection above Chlamydomonas’s auto-fluorescent chloroplast. Usage of a brighter and more photostable fluorophore, Atto 488, was also critical in obtaining a high signal to noise ratio and uniform labeling. This protocol allows us to understand how cell body actin filaments are distributed in gametic and vegetative cells for the first time. The previously unidentified filamentous actin structures will be crucial in exploring actin-dependent behaviors and how co-expressed actin genes in Chlamydomonas function to regulate cytoskeletal dynamics. Development of these strategies may prove useful for the study of actin filaments in other protists for which actin visualization has also been challenging.


Figure 1. Phalloidin staining in wild-type vegetative Chlamydomonas reinhardtii. A. Representative raw single widefield image of specific filamentous actin staining using our optimized protocol. B. The deconvolved maximum intensity projection of a Z-stack (0.3 μm steps) of the field visualized in A. C. Overlay of brightfield and fluorescence channels with phalloidin signal indicated in green. D. Deconvolved maximum intensity projection of a Z-stack (0.3 μm steps) of a single cell demonstrating effective phalloidin staining. Scale bars = 5 μm.

Materials and Reagents

  1. Kimwipes
  2. Parafilm
  3. Hydrophobic Marker (Elite PAP Pen, Diagnostic Biosystems Ref: K039)
  4. 1.5 ml Microcentrifuge Tubes
  5. Columbia Jars (DWK Life Sciences WheatonTM Columbia Jars for Coverslips, catalog number: 02-912-637)
  6. 10 μl inoculating loop
  7. Culture tubes (Borosilicate Glass Tubes 13 x 100 mm item) (Globe Scientific, catalog number: 1510)
  8. Micropipette tips
  9. Microscope slides
  10. Coverglass (22 x 22 mm square, 0.13 mm to 0.16 mm thick) (Corning, catalog number: 2845-22)
  11. Disposable Petri Dishes 60 x 15 mm (VWR®, catalog number: 25384-164)
  12. Whatman® Qualitative Filter Paper, Grade 1 (Whatman, catalog number: 1001 125)
  13. Chlamydomonas Strains
    Note: The wild-type strain CC-125 mating type + was obtained from the Chlamydomonas Resource Center (University of Minnesota).
  14. Atto 488 Phalloidin (Sigma, catalog number: 49409), store at -20 °C, shield from light
  15. Poly-L-lysine solution (Sigma, catalog number: P8920)
  16. PBS Tablet (Research Products International PBS 100 ml Tablets SKU P32080-100T)
  17. PFA aqueous solution 16% (Electron Microscopy Sciences, catalog number: 15710)
  18. HEPES (Corning, catalog number: 61-034-RM)
  19. NaOH pellets (J.T. Baker, catalog number: 3722)
  20. HCl (Fischer Chemical, catalog number: A481-212)
  21. Acetone (Histological) (Fisher Chemical, catalog number: A16P-4)
  22. Fluoromount-GTM (Invitrogen, Ref: 00-4958-02)
  23. DI Water
  24. BD Biosciences DifcoTM Agar, Granulated (Ref: 214530)
  25. Hunter’s Trace Elements (Can be ordered from the Chlamydomonas Resource Center)
  26. Acetic Acid, Glacial (Certified ACS) (Fisher Chemical, catalog number: A35S-500)
  27. Methanol
  28. Tris Base (C4H11NO3), Ultra Pure
  29. Ammonium Chloride (NH4Cl)
  30. Magnesium Sulfate Heptahydrate (MgSO4·7H2O)
  31. Calcium Chloride Dihydrate (CaCl2·2H2O)
  32. Potassium Phosphate Dibasic Anhydrous (K2HPO4)
  33. Potassium Phosphate Monobasic (KH2PO4)
  34. 1x PBS (see Recipes)
  35. 4% PFA in 7.5 mM HEPES Buffer (see Recipes)
  36. Phalloidin Stock Preparation (see Recipes)
  37. TAP liquid media (Tris Acetate Phosphate Media) (see Recipes)
    1. 100x Tris buffer (see Recipes)
    2. 100x TAP Salts (see Recipes)
    3. 1000x Phosphate Solution (see Recipes)
  38. 1.5% TAP Agar Plates (see Recipes)

Equipment

  1. Chemical spatula
  2. Graduated cylinder
  3. Beaker
  4. Stir plate
  5. Stir rod
  6. pH meter
  7. Scale
  8. Tweezers
  9. Pipettes
  10. Gusto® High-Speed Mini Centrifuge (Heathrow Scientific, catalog number: HEA10050)
  11. Empty glove boxes, cover to shield samples from light
  12. Microscope for image acquisition (We use a Nikon Eclipse Ti-S equipped with a QiIMAGING QICAM)
  13. Roller Drum (Cel Gro Tissue Culture Rotator) (Thermo Scientific, catalog number: 1640Q)
  14. -20 °C freezer

Software

  1. Huygens Essential (Scientific Volume Imaging)
  2. ImageJ (Schneider et al., 2012)

Procedure

  1. Cell culture and experimental setup
    1. Grow two inoculating loops of CC-125 mating type + cells, taken from a Tris Acetate Phosphate (TAP) agar plate, in 2 ml of tris acetate phosphate (TAP) liquid media on a roller drum (40 rpm) overnight (~16 h) in growth lighting at room temperature.
    2. The next day, set up a parafilm workspace (Figure 2). Use a hydrophobic marker to draw a circle on the coverslip where you will eventually pipette the cells and all future media used in the experiment. Make sure to label coverslips appropriately for each trial, we find it helpful to write “F” in the corner of each coverslip to help denote front from back. Add 200 μl of Poly-L-Lysine at room temperature to the hydrophobic circle and wash after 10 min by quickly dipping in distilled water. The Poly-L-Lysine will immediately fall off the coverslip once it is submerged in water, quickly place coverslip back onto parafilm and use a Kimwipe to remove any excess moisture around the edges of the coverslip. Cover your workspace to shield from debris that might adhere to the Poly-L-Lysine coated coverslips. Unused Poly-L-Lysine coverslips can be stored at room temperature for future use.


      Figure 2. Parafilm Workspace

    3. It is essential to select healthy cells by centrifuging 1 ml of cell culture at 1,800 rpm (203 x g) for 1.5 min. Discard the supernatant and resuspend cells in 600 μl fresh TAP media. Slowly pipette media up and down a few times then let culture sit. Healthy, swimming cells should migrate to the top of the culture after about 10 min. Then pipette (from the top of the culture) 200 μl of resuspended cells to Poly-L-Lysine-coated coverslips for 5 min and shield samples from light. The cells are shielded from the light because they phototax and swim upward towards the light, reducing the number of cells that adhere to the coverslip.

  2. Cell fixation and permeabilization
    1. After 5 min, tilt off liquid from the coverslips and replace with 200 μl 4% fresh paraformaldehyde (PFA) in 7.5 mM HEPES pH 7.4 to the middle of the hydrophobic circle. Allow cells to incubate in fixative for 15 min at room temperature.
      Note: If non-specific chloroplast or bright pyrenoid signal is present (Figures 3A and 3B), dilute PFA solution fresh. We do not make up PFA from powder, but find by simply diluting fixative to 4% from an unopened ampule of 16% PFA solution improves labeling. We find after 2-3 weeks of PFA use, labeling efficiency decreases and makes pyrenoid signal very prominent. Diluting fixative from a fresh ampule alleviates this issue.


      Figure 3. Phalloidin staining using non-optimal conditions. A. If cells incubate too long in staining solution or PFA has expired, cells will show strong pyrenoid signal (yellow arrow) and non-specific cytoplasmic staining. This type of signal can be easily distinguished from Atto 488 signal as there will be no filamentous perinuclear or apical staining, instead a dim hazy signal found throughout the entire cell body as pictured in A and B above. Scale bars = 5 μm. C. A general diagram of a Chlamydomonas cell highlighting filamentous actin in pink, the pyrenoid in blue, and the chloroplast in green.

    2. Tilt of PFA solution into a Kimwipe and place coverslips in a Columbia jar containing 1x PBS to wash for 3 min. Use enough PBS so that the coverslip is completely submerged in liquid. 
    3. For cell permeabilization, submerge coverslips in a Columbia jar containing 80% pre-cooled acetone (diluted in water and stored at -20 °C) and then incubate for 5 min at -20 °C.
    4. Quickly transfer coverslips into a second Columbia jar containing 100% pre-cooled acetone and incubate for another 5 min at -20 °C.
    5. Place coverslips back on parafilm and allow them to air dry for a minimum of 2 min or longer if needed.

  3. Phalloidin Stain
    1. Rehydrate cells by transferring coverslips to a Columbia jar containing 1x PBS and incubate for 5 min.
    2. Place coverslips back on parafilm. Stain coverslips with Atto 488 Phalloidin (Sigma) for 16 min in the dark. This shorter than recommended staining time significantly reduces background and increases the signal to noise ratio.
      Note: The Atto 488 Phalloidin reagent greatly enhances photostability and brightness compared to Alexa-488, which allows for more uniform and reproducible filament labeling. Due to the photostability, Atto 488-labeled slides can be reimaged.
    3. Quickly tilt of staining solution and wash cells by transferring coverslips to a Columbia jar containing 1x PBS. Wash once for 5 min.
    4. Remove excess liquid from the coverslip with a Kimwipe, but be careful to not disturb the inside of the hydrophobic circle (where the cells are adhered). Mount coverslips with self-sealing Fluoromount-GTM (Invitrogen) as quickly as possible. This sealant is designed for staining experiments where the final step is aqueous.

  4. Image acquisition
    Capture 0.3 μm step Z-stacks in brightfield and widefield fluorescence channels (GFP filter set) using a Nikon Eclipse Ti-S equipped with a QiIMAGING QICAM. Deconvolve fluorescence images using Huygens Essential deconvolution software and format in ImageJ.

Data analysis

Hyugens Essential deconvolution software (Scientific Volume Imaging) was used for post-image analysis. Images were deconvolved using the following parameters:

  1. Algorithm: Classic MLE
  2. PSF
    Mode: Theoretical
    Max Iterations: 10
  3. Iteration Mode: Optimized 
  4. Quality Change Thresh. (%): 0.001
  5. Signal to Noise Ratio: 20
  6. Background Mode: Auto
  7. Background Estimation Radius: 0.7
  8. Relative Background: 0.0
  9. Background Correction: If Possible
  10. Brick Mode: Auto
  11. PSFs per Brick Mode: Auto
  12. PSFs per Brick, Manual Mode: 1

Recipes

  1. 1x PBS
    1. Dissolve 1 tablet of PBS in 80 ml of purified water and add up to 100 ml for a 1x solution. Stir until dissolved
    2. 1 Tablet contains 137 mM Sodium Chloride, 2.7 mM Potassium Chloride, and 11.9 mM Phosphate Buffer
  2. 4% PFA in 7.5 mM HEPES buffer pH 7.4
    1. PFA aqueous solution 16% (Electron Microscopy Sciences)
    2. HEPES Stock Solution 10x (0.1 M, pH 7.4) (Farhat, 2013)
      1. For 100 ml of 0.1 M HEPES, pH 7.4, add 2.38 g of HEPES to an appropriate beaker (100-200 ml in this case)
      2. Add 80 ml of deionized water to the beaker
      3. Add a stir bar to the beaker and leave it on a stir plate until completely dissolved (~1 min)
      4. Add one NaOH pellet to raise pH towards 7.4. Adding one pellet will bring the pH to around 7
      5. Once the first pellet is fully dissolved, add a second NaOH pellet if necessary, to raise the pH to 7.4. Monitor carefully, and if the pH approaches 7.3/7.4 before the pellet is fully dissolved, carefully remove the NaOH pellet with a clean spatula
        Note: In our experience, about 1.5 pellets are just the right amount to raise the pH to 7.4, so retrieving the second pellet is necessary for achieving the right pH.
      6. If the pH goes too high, lower it back to a pH of 7.4 by carefully adding a little HCl, while monitoring the pH
        Caution: Wear gloves, eye protection and exercise extreme caution with this acid solution.
      7. Once the pH of the solution is 7.4, add enough deionized water to raise the volume to 100 ml
      8. Filter (if Whatman filter paper is used here), if possible, and store in the refrigerator for up to 4 months or aliquot and freeze at -20 °C for future use
      9. For 50 ml, add 37.5 ml of 1x HEPES solution to 12.5 ml fresh 16% PFA for final concentration of 4% PFA in 7.5 mM HEPES buffer. Vortex lightly and store at 4 °C
  3. Phalloidin stock preparation
    1. Follow the manufacturer’s instructions and resuspend lyophilized phalloidin reagent in 0.5 ml pre-cooled methanol and store at -20 °C. Shield from light
    2. To prepare working stock, add 5 μl of phalloidin stock to 200 μl of 1x PBS per coverslip. For example, if you need to stain 4 coverslips, add 20 μl of the stock to 800 μl of 1x PBS. Keep phalloidin stocks shielded from light at all times
  4. TAP liquid media (1 L)
    1. Put ~800 ml deionized water into a beaker, flask, or bottle
    2. Add the following solutions:
      1. 10 ml 100x tris buffer
        Dissolve 24.2 g tris (free base, not tris HCl) in a final volume of 100 ml H2O
      2. 10 ml 100x TAP salts
        Dissolve 18.75 g NH4Cl, 5 g MgSO4·7H2O (heptahydrate), and 2.5 g CaCl2·2H2O (dihydrate) in a final volume of 500 ml dH2O
      3. 1 ml 1,000x Phosphate Solution
        Dissolve 21.6 g K2HPO4 and 10.8 g KH2PO4 in a final volume of 200 ml dH2O
      4. 1 ml Hunter trace elements
      5. 1 ml glacial acetic acid
    3. Add deionized water to a total volume of 1 L
    4. Test the pH of the solution. It should be between 7 and 7.3. If required, adjust pH using acetic acid. If pH is below 7 initially, start the process over from scratch
    5. Autoclave and let cool before use
  5. 1.5% TAP Agar Plates
    1. Follow Recipe 4 a-d for preparing TAP liquid media
    2. For 1.5% TAP plates, add 15 g of DifcoTM Agar, granulated to the 1 L TAP solution
    3. Add a stir bar and autoclave
    4. Remove agar solution from autoclave and allow solution to cool. Stir the agar so it is evenly distributed throughout the container
    5. Pour 30-40 ml of agar into each Petri dish. 1 L of agar should make ~2 sleeves of 1.5% TAP agar plates which can be stored in a 4 °C refrigerator or at room temperature for short term use

Acknowledgments

Thank you to members of the Avasthi Lab for troubleshooting and manuscript feedback. This work was funded through P20GM104936 and P20GM103418 (to P.A.).

Competing interests

We have no conflict of interest or competing interests to declare.

References

  1. Avasthi, P., Onishi, M., Karpiak, J., Yamamoto, R., Mackinder, L., Jonikas, M. C., Sale, W. S., Shoichet, B., Pringle, J. R. and Marshall, W. F. (2014). Actin is required for IFT regulation in Chlamydomonas reinhardtii. Curr Biol 24(17): 2025-2032.
  2. Cerutti, H., Johnson, A. M., Gillham, N. W. and Boynton, J. E. (1997). Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9(6): 925-945.
  3. Courtemanche, N., Pollard, T. D. and Chen, Q. (2016). Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat Cell Biol 18(6): 676-683.
  4. Detmers, P. A., Carboni, J. M. and Condeelis, J. (1985). Localization of actin in Chlamydomonas using antiactin and NBD-phallacidin. Cell Motil 5(5): 415-430.
  5. Farhat, Y. (2013). Reagent Preparation: HEPES Stock Solution (0.1 M, pH 7.4). Protocol Place Dec 2013.
  6. Flores, L. R., Keeling, M. C., Zhang, X., Sliogeryte, K. and Gavara, N. (2019). Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour. Sci Rep 9(1): 3241.
  7. Harper, J. D., McCurdy, D. W., Sanders, M. A., Salisbury, J. L. and John, P. C. (1992). Actin dynamics during the cell cycle in Chlamydomonas reinhardtii. Cell Motil Cytoskeleton 22(2): 117-126.
  8. Onishi, M., Pringle, J. R. and Cross, F. R. (2016). Evidence that an unconventional actin can provide essential F-Actin function and that a surveillance system monitors F-Actin integrity in Chlamydomonas. Genetics 202:977-996.
  9. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.

简介

该方案旨在可视化莱茵衣藻(Chlamydomonas reinhardtii)中的丝状肌动蛋白网络。 我们使用F-肌动蛋白探针鬼笔环肽改善了固定细胞标记条件。 我们通过将鬼笔环肽孵育时间减半,选择最佳固定条件并选择健康细胞群来创建 Chlamydomonas 优化方案。 该鬼笔环肽方案快速,有效,并且是迄今为止唯一允许在固定的营养性衣原体细胞中进行可靠的肌动蛋白丝检测的标记方法。 该方法揭示了 Chlamydomonas 中先前未鉴定的肌动蛋白结构和对细胞骨架动力学的新见解。
【背景】莱茵衣藻(Chlamydomonas reinhardtii)是一种单细胞绿藻,广泛用于研究光合作用,纤毛,叶绿体生物学和生物燃料的合成。它是这些领域的领先模型系统,因为有机体的两个顶端鞭毛,廉价和简单的培养条件,以及完全测序的单倍体基因组允许进行优秀的遗传研究。了解 Chlamydomonas 细胞骨架将在推进这些研究领域中发挥重要作用。然而,莱茵衣藻(Chlamydomonas reinhardtii)的丝状肌动蛋白网络仍然未被表征,部分原因是难以看到该真核藻类中的细丝。

最近,在检测 Chlamydomonas 中的丝状肌动蛋白方面取得了一些成功。通过表达荧光标记的肌动蛋白结合肽,LifeAct,活细胞成像揭示了基于肌动蛋白的核周结构(Avasthi et al。,2014; Onishi et al。,2016)。然而,越来越多的证据表明LifeAct改变细胞形态,肌动蛋白组织,肌动蛋白依赖性功能,并优先标记肌动蛋白结构的一个子集(Courtemanche et al。,2016,Flores et al。 ,2019)。此外,由于外源基因的表观遗传沉默, Chlamydomonas 难以实现稳定表达(Cerutti et al。,1997)以及需要将肌动蛋白探针与固定细胞器标记共定位对于理解 Chlamydomonas 肌动蛋白生物学的复兴领域至关重要。因此,肌动蛋白可视化的有效且特异的固定细胞方法将导致该领域的显着进步并提供对基本肌动蛋白生物学的新颖见解。

可用的肌动蛋白抗体不区分单体和丝状肌动蛋白。先前已经表明,广泛使用的荧光肌动蛋白探针鬼笔环肽在营养性衣藻属细胞中标记丝状肌动蛋白方面无效(Harper et al。,1992)。然而,鬼笔环肽确实标记了称为受精管的游离细胞中的肌动蛋白致密结构(Detmers et al。,1985)。通过优化鬼笔环肽染色条件,我们现在能够在营养细胞中可视化 Chlamydomonas 肌动蛋白网络(图1)。具体而言,将染色孵育时间从30分钟减少到16分钟对于获得最佳信噪比至关重要,这允许在 Chlamydomonas 的自发荧光叶绿体上方进行检测。使用更亮和更光稳定的荧光团Atto 488对于获得高信噪比和均匀标记也是至关重要的。该协议使我们能够了解细胞体肌动蛋白丝如何首次在配子和营养细胞中分布。以前未鉴定的丝状肌动蛋白结构对于探索肌动蛋白依赖性行为以及如何在 Chlamydomonas 中共表达肌动蛋白基因以调节细胞骨架动力学至关重要。这些策略的开发可能对于肌动蛋白可视化也具有挑战性的其他原生生物中的肌动蛋白丝的研究是有用的。


图1.野生型植物莱茵衣藻(Chlamydomonas reinhardtii)中的鬼笔环肽染色。 A.使用我们的优化方案对特定丝状肌动蛋白染色的代表性原始单一宽视野图像。 B.在A.C中可视化的场的Z-堆叠(0.3μm步长)的去卷积最大强度投影。明场和荧光通道的叠加,其中鬼笔环肽信号以绿色表示。 D.解卷积单个细胞的Z-堆叠(0.3μm步长)的最大强度投影,证明有效的鬼笔环肽染色。比例尺=5μm。

关键字:莱茵衣藻, 肌动蛋白, 鬼笔环肽, 细胞骨架, 纤维

材料和试剂

  1. 的Kimwipes
  2. 封口膜
  3. 疏水标记(Elite PAP Pen,Diagnostic Biosystems参考:K039)
  4. 1.5毫升Microcentrifuge管
  5. Columbia Jars(DWK Life Sciences Wheaton TM Columbia Jars for Coverslips,目录号:02-912-637)
  6. 10μl接种环
  7. 培养管(硼硅酸盐玻璃管13 x 100 mm项目)(Globe Scientific,目录号:1510)
  8. 微管吸头
  9. 显微镜载玻片
  10. 盖玻片(22 x 22 mm正方形,0.13 mm至0.16 mm厚)(Corning,目录号:2845-22)
  11. 一次性培养皿60 x 15 mm(VWR ®,目录号:25384-164)
  12. Whatman ®定性滤纸,1级(Whatman,目录号:1001 125)
  13. Chlamydomonas 菌株
    注意:野生型菌株CC-125交配型+来自 Chlamydomonas 资源中心(明尼苏达大学)。
  14. Atto 488鬼笔环肽(Sigma,目录号:49409),储存于-20°C,避光
  15. 聚L-赖氨酸溶液(Sigma,目录号:P8920)
  16. PBS片剂(Research Products International PBS 100 ml Tablets SKU P32080-100T)
  17. PFA水溶液16%(电子显微镜科学,目录号:15710)
  18. HEPES(康宁,目录编号:61-034-RM)
  19. NaOH颗粒(J.T. Baker,目录号:3722)
  20. HCl(Fischer Chemical,目录号:A481-212)
  21. 丙酮(组织学)(Fisher Chemical,目录号:A16P-4)
  22. Fluoromount-G TM (Invitrogen,编号:00-4958-02)
  23. 去水
  24. BD Biosciences Difco TM 琼脂,颗粒状(参考号:214530)
  25. 猎人的微量元素(可以从 Chlamydomonas 资源中心订购)
  26. 乙酸,冰川(认证ACS)(Fisher化学,目录号:A35S-500)
  27. 甲醇
  28. Tris碱(C 4 H 11 NO 3 ),超纯
  29. 氯化铵(NH 4 Cl)
  30. 硫酸镁七水合物(MgSO 4 ·7H 2 O)
  31. 二水氯化钙(CaCl 2 ·2H 2 O)
  32. 磷酸氢二钾无水(K 2 HPO 4 )
  33. 磷酸二氢钾(KH 2 PO 4 )
  34. 1x PBS(见食谱)
  35. 7.5 mM HEPES缓冲液中4%PFA(参见食谱)
  36. 鬼笔环肽原料制剂(见食谱)
  37. TAP液体培养基(Tris Acetate Phosphate Media)(见食谱)
    1. 100x Tris缓冲液(见食谱)
    2. 100x TAP盐(见食谱)
    3. 1000x磷酸盐溶液(见食谱)
  38. 1.5%TAP琼脂平板(见食谱)

设备

  1. 化学铲
  2. 刻度量筒
  3. 烧杯
  4. 搅拌盘
  5. 搅拌棒
  6. pH计
  7. 规模
  8. 镊子
  9. 移液器
  10. Gusto ®高速迷你离心机(Heathrow Scientific,目录号:HEA10050)
  11. 空手套箱,盖遮挡样品避光
  12. 用于图像采集的显微镜(我们使用配备QiIMAGING QICAM的Nikon Eclipse Ti-S)
  13. 滚筒(Cel Gro Tissue Culture Rotator)(Thermo Scientific,目录号:1640Q)
  14. -20°C冰柜

软件

  1. 惠更斯必备(科学体积成像)
  2. ImageJ(Schneider et al。,2012)

程序

  1. 细胞培养和实验装置
    1. 从Tris醋酸磷酸盐(TAP)琼脂平板上取2个Tris醋酸磷酸盐(TAP)液体培养基,在滚筒(40转/分钟)上培养两个接种CC-125交配型+细胞的接种环(~16小时) )在室温下生长照明。
    2. 第二天,建立一个封口膜工作区(图2)。使用疏水标记在盖玻片上绘制一个圆圈,最后将移液细胞以及实验中使用的所有未来培养基。确保每次试验都适当地标记盖玻片,我们发现在每个盖玻片的角落写上“F”有助于从后面表示正面。在室温下将200μl聚-L-赖氨酸加入疏水环中,10分钟后快速浸入蒸馏水中洗涤。一旦将聚L-赖氨酸浸入水中,它将立即从盖玻片上脱落,快速将盖玻片放回封口膜上,并使用Kimwipe去除盖玻片边缘周围的任何多余水分。盖住工作区,以防止可能粘附在Poly-L-Lysine涂层盖玻片上的碎屑。未使用的聚L-赖氨酸盖玻片可以在室温下储存以备将来使用。


      图2. Parafilm工作区

    3. 通过以1,800rpm(203 x g )离心1ml细胞培养物1.5分钟来选择健康细胞是必要的。弃去上清液并将细胞重悬于600μl新鲜TAP培养基中。慢慢地上下移动媒体几次然后让文化坐下来。健康的游泳细胞应在约10分钟后迁移至培养物的顶部。然后移取(从培养物顶部)200μl重悬浮细胞至聚-L-赖氨酸包被的盖玻片5分钟并遮挡样品避光。细胞被光线遮挡,因为它们会光照并向上游向光线,从而减少了粘附在盖玻片上的细胞数量。

  2. 细胞固定和透化
    1. 5分钟后,将盖玻片上的液体倾斜,并用7.5mM HEPES pH 7.4中的200μl4%新鲜多聚甲醛(PFA)置换至疏水圈的中间。让细胞在室温下在固定剂中孵育15分钟。
      注意:如果存在非特异性叶绿体或明亮的pyrenoid信号(图3A和3B),则稀释PFA溶液。我们不用粉末制成PFA,但是通过简单地将未固化的16%PFA溶液的固定剂稀释至4%来改善标记。我们发现使用PFA 2-3周后,标记效率降低并使pyrenoid信号非常突出。从新鲜的安瓿中稀释固定剂缓解了这个问题。


      图3.使用非最佳条件进行鬼笔环肽染色。 A.如果细胞在染色液中孵育时间过长或PFA过期,细胞将显示强烈的pyrenoid信号(黄色箭头)和非特异性细胞质染色。这种类型的信号可以很容易地与Atto 488信号区分开,因为没有丝状核周或顶端染色,而是在整个细胞体中发现昏暗的模糊信号,如上面A和B所示。比例尺=5μm。 C. Chlamydomonas 细胞的一般图,突出显示粉红色的丝状肌动蛋白,蓝色的pyrenoid和绿色的叶绿体。

    2. 将PFA溶液倾斜到Kimwipe中并将盖玻片放入含有1x PBS的Columbia瓶中洗涤3分钟。使用足够的PBS,使盖玻片完全浸没在液体中。 
    3. 对于细胞透化,将盖玻片浸没在含有80%预冷丙酮(在水中稀释并在-20℃下储存)的哥伦比亚罐中,然后在-20℃下孵育5分钟。
    4. 快速将盖玻片转移到含有100%预冷丙酮的第二个哥伦比亚罐中,并在-20℃下再孵育5分钟。
    5. 将盖玻片放回封口膜上,如果需要,让它们风干至少2分钟或更长时间。

  3. 鬼笔环肽染色
    1. 通过将盖玻片转移到含有1x PBS的Columbia瓶中并孵育5分钟来再水化细胞。
    2. 将盖玻片放回封口膜上。用Atto 488鬼笔环肽(Sigma)在黑暗中染色盖玻片16分钟。这比推荐的染色时间短,可显着降低背景,提高信噪比。
      注意:与Alexa-488相比,Atto 488鬼笔环肽试剂大大提高了光稳定性和亮度,可以实现更均匀和可重复的细丝标记。由于光稳定性,Atto 488标记的幻灯片可以重新成像。
    3. 通过将盖玻片转移到含有1x PBS的Columbia罐中,快速倾斜染色溶液并洗涤细胞。洗一次5分钟。
    4. 用Kimwipe清除盖玻片上多余的液体,但要小心不要打扰疏水圈内部(细胞粘附的地方)。尽可能快地安装具有自密封Fluoromount-G TM (Invitrogen)的盖玻片。该密封剂设计用于染色实验,其中最终步骤是含水的。

  4. 图像采集
    使用配备QiIMAGING QICAM的Nikon Eclipse Ti-S,在明场和宽场荧光通道(GFP滤光片组)中捕获0.3μm步进Z-堆叠。使用Huygens Essential deconvolution软件对ImageJ中的荧光图像进行去卷积。

数据分析

Hyugens Essential deconvolution软件(Scientific Volume Imaging)用于后图像分析。使用以下参数对图像进行去卷积:

  1. 算法:经典MLE
  2. PSF
    模式:理论
    最大迭代次数:10
  3. 迭代模式:优化 
  4. 质量变化Thresh。 (%):0.001
  5. 信噪比:20
  6. 背景模式:自动
  7. 背景估计半径:0.7
  8. 相对背景:0.0
  9. 背景校正:如果可能
  10. 砖块模式:自动
  11. 每砖模式的PSF:自动
  12. 每砖的PSF,手动模式:1

食谱

  1. 1x PBS
    1. 将1片PBS溶于80ml纯净水中,加入100ml,1x溶液。搅拌至溶解
    2. 1片含有137 mM氯化钠,2.7 mM氯化钾和11.9 mM磷酸盐缓冲液
  2. pH7.4的7.5mM HEPES缓冲液中的4%PFA
    1. PFA水溶液16%(电子显微镜科学)
    2. HEPES Stock Solution 10x(0.1 M,pH 7.4)(Farhat,2013)
      1. 对于100毫升0.1 M HEPES,pH 7.4,将2.38克HEPES加入适当的烧杯中(在这种情况下为100-200毫升)
      2. 向烧杯中加入80ml去离子水
      3. 在烧杯中加入搅拌棒,将其放在搅拌盘上直至完全溶解(约1分钟)
      4. 加入一个NaOH颗粒使pH值升至7.4。添加一个颗粒将使pH值达到7左右
      5. 一旦第一个沉淀完全溶解,如果需要,加入第二个NaOH颗粒,将pH升至7.4。仔细监测,如果在颗粒完全溶解之前pH值接近7.3 / 7.4,用干净的刮刀小心地取出NaOH颗粒
        注意:根据我们的经验,大约1.5颗颗粒适合将pH值提高到7.4,因此需要回收第二颗颗粒才能达到合适的pH值。
      6. 如果pH值过高,请小心加入少量HCl将其降至pH值7.4,同时监测pH值 注意:戴上手套,保护眼睛,并使用这种酸性溶液时要格外小心。
      7. 一旦溶液的pH值为7.4,加入足量的去离子水使体积升至100毫升
      8. 如果可能的话,过滤(如果在这里使用Whatman滤纸),并在冰箱中储存长达4个月或等分并在-20°C下冷冻以备将来使用
      9. 对于50ml,将37.5ml的1x HEPES溶液加入到12.5ml新鲜的16%PFA中,最终浓度为7.5%HEPES缓冲液中的4%PFA。轻轻涡旋并在4°C下储存
  3. 鬼笔环肽原料制备
    1. 按照制造商的说明,将冻干的鬼笔环肽试剂重悬于0.5 ml预冷甲醇中,并在-20°C下保存。避光
    2. 为了制备工作原料,每个盖玻片加入5μl鬼笔环肽原液至200μl1xPBS。例如,如果您需要染色4个盖玻片,请将20μl的原液添加到800μl的1x PBS中。保持鬼笔环肽在任何时候都不受光照影响
  4. TAP液体介质(1升)
    1. 将约800毫升去离子水放入烧杯,烧瓶或瓶中
    2. 添加以下解决方案:
      1. 10毫升100倍Tris缓冲液
        在最终体积100 ml H 2 O中溶解24.2 g tris(游离碱,不含tris HCl)
      2. 10毫升100倍TAP盐
        溶解18.75克NH 4 Cl,5克MgSO 4 ·7H 2 O(七水合物)和2.5克CaCl 2 ·2H 2 O(二水合物),终体积为500ml dH 2 O.
      3. 1毫升1,000倍磷酸盐溶液
        溶解21.6g K 2 HPO 4 和10.8g KH 2 PO 4 ,最终体积为200ml dH <子> 2 0
      4. 1毫升猎人微量元素
      5. 1毫升冰醋酸
    3. 加入去离子水至总体积为1升
    4. 测试溶液的pH值。它应该在7到7.3之间。如果需要,使用乙酸调节pH。如果最初pH值低于7,则从头开始重新开始
    5. 高压灭菌,使用前冷却
  5. 1.5%TAP琼脂平板
    1. 按照配方4a-d制备TAP液体培养基
    2. 对于1.5%TAP平板,加入15 g Difco TM 琼脂,造粒至1 L TAP溶液
    3. 加入搅拌棒和高压灭菌器
    4. 从高压灭菌器中取出琼脂溶液,让溶液冷却。搅拌琼脂,使其均匀分布在整个容器中
    5. 将30-40ml琼脂倒入每个培养皿中。 1升琼脂应制成~2个1.5%TAP琼脂平板的套管,可储存在4°C冰箱或室温下短期使用

致谢

感谢Avasthi实验室的成员进行故障排除和手稿反馈。这项工作是通过P20GM104936和P20GM103418(至P.A.)资助的。

利益争夺

我们没有利益冲突或竞争利益申报。

参考

  1. Avasthi,P.,Onishi,M.,Karpiak,J.,Yamamoto,R.,Mackinder,L.,Jonikas,M.C.,Sale,W。S.,Shoichet,B.,Pringle,J。R. and Marshall,W.F。(2014)。 肌动蛋白是 Chlamydomonas reinhardtii 的IFT监管所必需的。 Curr Biol 24(17):2025-2032。
  2. Cerutti,H.,Johnson,A.M.,Gillham,N.W。和Boynton,J.E。(1997)。 Chlamydomonas 的核转化体中外源基因的表观遗传沉默。 植物细胞 9(6):925-945。
  3. Courtemanche,N.,Pollard,T。D.和Chen,Q。(2016)。 在LifeAct与荧光蛋白融合的活细胞中计数聚合肌动蛋白时避免人工制品。 Nat Cell Biol 18(6):676-683。
  4. Detmers,P.A.,Carboni,J.M。和Condeelis,J。(1985)。 使用抗肌动蛋白和NBD-phallacidin在 Chlamydomonas 中定位肌动蛋白。 Cell Motil 5(5):415-430。
  5. Farhat,Y。(2013)。 试剂准备:HEPES储备溶液(0.1 M,pH 7.4)。 Protocol Place Dec 2013。
  6. Flores,L.R.,Keeling,M.C.,Zhang,X.,Sliogeryte,K。和Gavara,N。(2019)。 Lifeact-GFP改变F-肌动蛋白组织,细胞形态和生物物理行为。 Sci Rep 9(1):3241。
  7. Harper,J.D.,McCurdy,D.W.,Sanders,M.A.,Salisbury,J.L。和John,P.C。(1992)。 莱茵衣藻(Chlamydomonas reinhardtii)细胞周期中的肌动蛋白动力学。 Cell Motil Cytoskeleton 22(2):117-126。
  8. Onishi,M.,Pringle,J。R. and Cross,F.R。(2016)。 非常规肌动蛋白可提供必需的F-肌动蛋白功能且监测系统监测F-肌动蛋白的证据 Chlamydomonas 中的完整性。 Genetics 202:977-996。
  9. Schneider,C.A.,Rasband,W。S.和Eliceiri,K.W。(2012)。 NIH Image to ImageJ:25年的图像分析。 Nat Methods 9(7):671-675。
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引用:Craig, E. W. and Avasthi, P. (2019). Visualizing Filamentous Actin in Chlamydomonas reinhardtii. Bio-protocol 9(12): e3274. DOI: 10.21769/BioProtoc.3274.
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