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Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling
免疫荧光标记定位蓝藻鱼腥藻属PCC7120中的蛋白质   

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Abstract

Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the background fluorescence for non-specific attachments. In this protocol, rabbit antibodies were raised against the synthetic peptide of CyDiv protein (Mandakovic et al., 2016). The secondary antibody conjugated to the fluorophore Alexa488 was used due to its different emission range in comparison to the autofluorescence of the cyanobacterium.

Keywords: Cell division(细胞分裂), Cyanobacteria(蓝藻), CyDiv(CyDiv), Anabaena(鱼腥藻属), Protein immunolocalization (蛋白质免疫定位)

Background

The immunofluorescence of cyanobacteria has been used extensively in cell identification and counting studies (Jin et al., 2016). However, immunolocalization of proteins has not been achieved efficiently in cyanobacteria. The most recurrent method to localize proteins is by fusing the protein of interest to a fluorescent protein such as GFP (Green Fluorescent Protein) that has a different emission wavelength (compared with cyanobacterial autofluorescence), and subsequent visualization using epifluorescence or confocal microscopy (Flores et al., 2016; Santamaria-Gomez et al., 2016).

The structural properties of cyanobacterial cells are the main challenges for applying immunofluorescence techniques. They consist of an inner membrane (IM), a peptidoglycan layer (PG) and an outer membrane (OM) (Rippka, 1988; Baulina, 2012; Jin et al., 2016), with an additional exopolysaccharide layer (sheath). The sheath is found in both unicellular and filamentous cyanobacteria (Kehr and Dittmann, 2015), and their thickness, composition and appearance depend on growth conditions, metabolic status, cell differentiation and other external and internal parameters (Jin et al., 2016). The sheath tends to trap antibodies by unspecific interactions. To avoid this problem, the washing and membrane permeabilization steps are the key to a successful immunofluorescence technique in cyanobacteria.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml tubes (Eppendorf)
  3. 50 ml tubes (Falcon tubes)
  4. Poly-L-lysine coated glass slides (Sigma-Aldrich, catalog number: P0425-72EA )
  5. Cover slips
  6. Petri dish
  7. Filter with a pore size of 0.2 µm
  8. Filamentous cyanobacterium, Anabaena sp. PCC7120
  9. BG-11 liquid supplied with 10 mM NaHCO3 (Rippka, 1988)
  10. Sodium hydrogen carbonate (NaHCO3) (EMD Millipore, catalog number: 106329 )
  11. Ethanol (EMD Millipore, catalog number: 1.00983.2500 )
  12. Triton X-100 (Winkler Limitada, catalog number: BM-2020 )
  13. Bovine serum albumin (BSA) (Divbio Science, catalog number: 41-903-100 )
  14. Tween-20 (Winkler Limitada, catalog number: TW-1652 )
  15. Secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, Invitrogen, catalog number: A11008 )
  16. ProLong Gold Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36930 )
  17. Nail varnish
  18. Primary polyclonal antibody against All2320 peptide (Mandakovic et al., 2016)
  19. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
  20. Potassium chloride (KCl) (EMD Millipore, catalog number: 104938 )
  21. Sodium dihydrogen phosphate (Na2HPO4) (EMD Millipore, catalog number: 106559 )
  22. Potassium phosphate monobasic (KH2PO4) (EMD Millipore, catalog number: 529568 )
  23. PBS buffer (pH 7.4) (see Recipes)

Equipment

  1. Pipettes
  2. Hydrophobic PAP pen (Thermo Fisher Scientific, catalog number: 008877 )
  3. Freezer at -20 °C
  4. Incubator at 4 °C
  5. Incubator at 55 °C
  6. Incubator at 24 °C with white light
  7. Olympus Fluoview FV1000 Confocal Microscope (Olympus, model: FluoviewTM FV1000 ) and objectives of 60x/1.35 NA oil immersion and 100x/1.40 NA oil immersion. Laserline Argon 488 (Excitation 495 nm, Emission 509 nm) and Laserline DPSS (Excitation 565 nm, Emission 590 nm)
  8. Moisture chamber (A dark plastic box with a moistened paper inside, PolarSafeTM Polypropylene Freezer Storage Box) (Argos Technologies, catalog number: R3130 )

Software

  1. ImageJ software (https://imagej.net)

Procedure

  1. Organism and growth conditions
    1. Anabaena sp. PCC7120 is grown axenically in BG-11 liquid medium at 24 °C under white light (25 µmol m-2 sec-1) and shaking at 90 rpm.

  2. Fixation and permeabilization
    1. 50 µl of cyanobacterial culture (OD750 = 0.3) is added to a poly-lysine microscopy slide and dried for 20 min at 55 °C. Do not fix the cells with organic solvents or aldehydes.
    2. Fix the cell spots in 70% ethanol and incubate for 30 min at -20 °C. The slide is immersed in cold 70% ethanol contained in a Petri dish.
    3. The slides are air-dried for 20 min at room temperature.
    4. Use a hydrophobic PAP pen to draw a circle around the slide-mounted cell spot and let it dry for 15 min at room temperature.

  3. Labeling procedure
    1. Permeabilize the cells by adding a drop of 0.05% Triton X-100 in PBS for 2 min at room temperature, and repeat it three times by removing the drop each time with a pipette.
    2. Incubate with a drop of 3% BSA, 0.2% Triton X-100 in PBS for 1 h at 4 °C in a moisture chamber and remove this blocking solution.
    3. The cells are incubated with the primary antibody diluted 1:100 in a solution with 1% BSA, 0.05% Tween-20 in PBS. Pre-immune serum diluted 1:100 in a solution with 1% BSA, 0.05% Tween-20 in PBS was used as a control to ensure that the primary antibody is working. The cells with the solutions are incubated for 2 h at 4 °C, in a moisture chamber.
    4. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature, and repeat three times.
    5. Incubate with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (diluted in PBS with 1% BSA and 0.05% Tween-20, final concentration 10 µg/ml) for 45 min at 4 °C, in a moisture chamber.
    6. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature for three times.
    7. Add a drop of Prolong Antifade reagent to the sample slide, and then cover this with a cover slip while taking care not to create air bubbles. Seal with nail varnish.
    8. The slides are visualized with a Fluoview FV1000 Confocal Microscope and images are acquired in 16 bits. Alexa Fluor 488 is excited at a wavelength of 495 nm and emission is measured at 509 nm. To visualize autofluorescence due to phycobilisomes, samples are excited using 565 nm and fluorescence emission is monitored at 590 nm (Figure 1).


      Figure 1. Immunolocalization of CyDiv in Anabaena sp. PCC7120. Deconvoluted image of a Z-stack. A. Autofluorescence; B. Image signal derived from primary antibody anti-CyDiv and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG; C. Merged image of the autofluorescence and CyDiv-Alexa Fluor 488 fluorescence. White scale bar = 5 µm.

Data analysis

Images of Z-stacks were processed using ImageJ software (Schneider et al., 2012). For each channel of images, the point-spread function (PSF) was calculated using the Born and Wolf model within the PSF Generator plugin (Kirshner et al., 2013). Image deconvolution was performed with the Deconvolution Lab plugin with Richardson-Lucy algorithm using 10 iterations (Vonesch and Unser, 2008).

Recipes

  1. PBS buffer (pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    1.4 mM Na2HPO4
    1.4 mM KH2PO4
    Note: The PBS is filtered through a filter with a pore size of 0.2 µm and stored at room temperature.

Acknowledgments

The protocol described has been modified from (Plominsky et al., 2013; Miyagishima et al., 2014). This work was supported by Fondecyt grants #1131037, 1161232 and Fellowships for Graduate Student of Chilean Government # 21100780 and 21150983.

References

  1. Baulina, O. I. (2012). Ultrastructural plasticity of cyanobacteria. Springer Science & Business Media.
  2. Flores, E., Herrero, A., Forchhammer, K. and Maldener, I. (2016). Septal junctions in filamentous heterocyst-forming cyanobacteria. Trends Microbiol 24(2): 79-82.
  3. Jin, C., Mesqutia, M., Emelko, M., and Wong, A. (2016). Automated enumeration and size distribution analysis of Microcystis aeruginosa via fluorescence imaging. J Comput Vis Imaging Syst 2(1).
  4. Kehr, J. C. and Dittmann, E. (2015). Biosynthesis and function of extracellular glycans in cyanobacteria. Life (Basel) 5(1): 164-180.
  5. Kirshner, H., Aguet, F., Sage, D. and Unser, M. (2013). 3-D PSF fitting for fluorescence microscopy: implementation and localization application. J Microsc 249(1): 13-25.
  6. Mandakovic, D., Trigo, C., Andrade, D., Riquelme, B., Gomez-Lillo, G., Soto-Liebe, K., Diez, B. and Vasquez, M. (2016). CyDiv, a conserved and novel filamentous cyanobacterial cell division protein involved in septum localization. Front Microbiol 7: 94.
  7. Miyagishima, S. Y., Kabeya, Y., Sugita, C., Sugita, M. and Fujiwara, T. (2014). DipM is required for peptidoglycan hydrolysis during chloroplast division. BMC plant biology 14(1): 57.
  8. Plominsky, Á. M., Larsson, J., Bergman, B., Delherbe, N., Osses, I. and Vásquez, M. (2013). Dinitrogen fixation is restricted to the terminal heterocysts in the invasive cyanobacterium Cylindrospermopsis raciborskii CS-505. PloS one 8(2): e51682.
  9. Rippka, R. (1988). Recognition and identification of cyanobacteria. Methods enzymol 167: 28-67.
  10. Santamaria-Gomez, J., Ochoa de Alda, J. A., Olmedo-Verd, E., Bru-Martinez, R. and Luque, I. (2016). Sub-cellular localization and complex formation by aminoacyl-tRNA synthetases in cyanobacteria: Evidence for interaction of membrane-anchored ValRS with ATP synthase. Front Microbiol 7: 857.
  11. 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.
  12. Vonesch, C. and Unser, M. (2008). A fast thresholded landweber algorithm for wavelet-regularized multidimensional deconvolution. IEEE Trans Image Process 17(4): 539-549.

简介

诸如免疫荧光的技术被广泛用于确定蛋白质的亚细胞分布。在这里我们报告一种免疫定位蛋白质的方法。 PCC7120通过荧光显微镜检测荧光团结合的抗体。该方法改善了蓝细菌细胞的透化性,并使非特异性附着物的背景荧光最小化。在该方案中,针对CyDiv蛋白质的合成肽(Mandakovic等人,2016)产生兔抗体。使用与荧光团Alexa488缀合的二抗,因为与蓝细菌的自发荧光相比,其发射范围不同。

背景 蓝细菌的免疫荧光已广泛用于细胞鉴定和计数研究(Jin等人,2016)。然而,在蓝细菌中蛋白质的免疫定位尚未有效地实现。定位蛋白质最复发的方法是通过将感兴趣的蛋白质融合到具有不同发射波长的荧光蛋白(绿色荧光蛋白)(与蓝细菌自发荧光相比))以及随后使用落射荧光或共聚焦显微镜(Flores < em,et al。,2016; Santamaria-Gomez等人,2016)。
&NBSP;蓝细菌细胞的结构性质是应用免疫荧光技术的主要挑战。它们由内膜(IM),肽聚糖层(PG)和外膜(OM)组成(Rippka,1988; Baulina,2012; Jin等人,2016),附加外多糖层(鞘)。鞘细胞均存在于单细胞和丝状蓝细菌中(Kehr and Dittmann,2015),其厚度,组成和外观取决于生长条件,代谢状态,细胞分化及其他外部和内部参数(Jin et al。 em>。,2016)。护套倾向于通过非特异性相互作用捕获抗体。为了避免这个问题,洗涤和膜透化步骤是蓝细菌成功免疫荧光技术的关键。

关键字:细胞分裂, 蓝藻, CyDiv, 鱼腥藻属, 蛋白质免疫定位

材料和试剂

  1. 移液器提示
  2. 1.5 ml管(Eppendorf)
  3. 50ml管(Falcon管)
  4. 聚-L-赖氨酸包被的载玻片(Sigma-Aldrich,目录号:P0425-72EA)
  5. 封面
  6. 培养皿
  7. 过滤器孔径为0.2μm
  8. 丝状蓝细菌,鱼尾草 sp。 PCC7120
  9. 用10mM NaHCO 3(Rippka,1988)
    提供的BG-11液体
  10. 碳酸氢钠(NaHCO 3)(EMD Millipore,目录号:106329)
  11. 乙醇(EMD Millipore,目录号:1.00983.2500)
  12. Triton X-100(Winkler Limitada,目录号:BM-2020)
  13. 牛血清白蛋白(BSA)(Divbio Science,目录号:41-903-100)
  14. Tween-20(Winkler Limitada,目录号:TW-1652)
  15. 第二抗体Alexa Fluor 488山羊抗兔IgG(Thermo Fisher Scientific,Invitrogen,目录号:A11008)
  16. ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM ,目录号:P36930)
  17. 指甲油
  18. 针对All2320肽的原代多克隆抗体(Mandakovic等人,2016)
  19. 氯化钠(NaCl)(EMD Millipore,目录号:106404)
  20. 氯化钾(KCl)(EMD Millipore,目录号:104938)
  21. 磷酸二氢钠(Na 2 HPO 4)(EMD Millipore,目录号:106559)
  22. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:529568)
  23. PBS缓冲液(pH 7.4)(见配方)

设备

  1. 移液器
  2. 疏水PAP笔(Thermo Fisher Scientific,目录号:008877)
  3. -20°C冷藏柜
  4. 4℃培养箱
  5. 孵化器在55°C
  6. 孵化器在24°C与白光
  7. Olympus Fluoview FV1000共聚焦显微镜(Olympus,型号:Fluoview TM FV1000)和60x / 1.35 NA油浸和100x / 1.40 NA油浸的目标。激光氩弧488(激发495nm,发射509nm)和激光线DPSS(激发565nm,发射590nm)
  8. 水分箱(内置湿纸的深色塑料盒,PolarSafe TM聚丙烯冷冻储存盒)(Argos Technologies,目录号:R3130)

软件

  1. ImageJ软件( https://imagej.net

程序

  1. 生物和生长条件
    1. 鱼尾鱼 sp。 PCC7120在BG-11液体介质中24℃,白光(25μmol/平方米/秒)下生长并以90rpm摇动。 >
  2. 固定和透化
    1. 将50μl蓝细菌培养物(OD 750×0.3)加入到聚赖氨酸显微镜载玻片中,并在55℃下干燥20分钟。不要用有机溶剂或醛固定细胞。
    2. 将细胞斑点固定在70%乙醇中,并在-20℃下孵育30分钟。将载玻片浸入含有培养皿的70%乙醇中
    3. 将载玻片在室温下风干20分钟。
    4. 使用疏水PAP笔在载玻片安装的细胞斑点周围绘制一圈,并在室温下干燥15分钟。

  3. 标签程序
    1. 通过在PBS中加入0.05%Triton X-100滴定2分钟使细胞渗透稳定,每次用移液管移除滴液,重复三次。
    2. 在4℃下,在潮湿室中,用PBS中的3%BSA,0.2%Triton X-100滴加1小时,并除去该封闭溶液。
    3. 将细胞与1%BSA稀释的1%BSA,0.05%Tween-20在PBS中的溶液一起稀释。在含有1%BSA,0.05%Tween-20的PBS溶液中以1:100稀释的预免疫血清用作对照,以确保一抗能正常工作。将溶液的细胞在4℃下,在水分室中孵育2小时
    4. 用PBS中的0.05%Triton X-100在室温下洗涤2分钟,重复三次
    5. 与第二抗体Alexa Fluor 488山羊抗兔IgG(在含有1%BSA和0.05%Tween-20的PBS中稀释,最终浓度为10μg/ ml)在4℃下在水分室中孵育45分钟。 >
    6. 用PBS中的0.05%Triton X-100在室温下洗涤3次
    7. 在样品载玻片上加入一滴Prolong Antifade试剂,然后盖上盖子,同时注意不要产生气泡。用指甲油密封。
    8. 使用Fluoview FV1000共焦显微镜可视化幻灯片,并以16位获取图像。 Alexa Fluor 488在495nm的波长下激发,并且在509nm处测量发射。为了使由phycobillisomes引起的自发荧光可视化,使用565nm激发样品,并在590nm监测荧光发射(图1)。


      图1. Cydiv在鱼腥藻中的免疫定位 PCC7120。 Z-stack的解卷积图像。 A.自发荧光B.来源于一抗抗体CyDiv和二抗Alexa Fluor 488山羊抗兔IgG的图像信号; C.自发荧光和CyDiv-Alexa Fluor 488荧光的合并图像。白色比例尺=5μm。

数据分析

使用ImageJ软件(Schneider等人,2012)处理Z堆叠的图像。对于每个图像通道,使用PSF Generator插件(Kirshner等人,2013年)中的Born和Wolf模型计算点扩散函数(PSF)。使用具有Richardson-Lucy算法的Deconvolution Lab插件,使用10次迭代进行图像去卷积(Vonesch和Unser,2008)。

食谱

  1. PBS缓冲液(pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    1.4mM Na 2 HPO 4
    1.4mM KH 2 PO 4
    注意:PBS通过孔径为0.2μm的过滤器过滤,并在室温下储存。

致谢

所描述的方案已经从(Plominsky等人,2013; Miyagishima等人,2014)修改。这项工作得到Fondecyt拨款#1131037,1161232和智利政府研究生奖学金#21100780和21150983的支持。

参考

  1. Baulina,OI(2012)。&nbsp; 蓝藻的超微结构可塑性。 Springer Science&amp;商业媒体
  2. Flores,E.,Herrero,A.,Forchhammer,K.and Maldener,I.(2016)。&nbsp; 丝状异型细菌形成蓝细菌的间隔点。趋势微生物 24(2):79-82。
  3. Jin,C.,Mesqutia,M.,Emelko,M.,and Wong,A.(2016)。&nbsp; 通过荧光成像自动枚举和大小分析绿脓杆菌 计算Vis Vis成像系统 em> 2(1)。
  4. Kehr,JC和Dittmann,E.(2015)。蓝藻中细胞外聚糖的生物合成和功能。生活(巴塞尔) 5(1):164-180。
  5. Kirshner,H.,Aguet,F.,Sage,D。和Unser,M。(2013)。用于荧光显微镜的3-D PSF拟合:实施和定位应用 J Microsc 249(1) :13-25。
  6. Mandakovic,D.,Trigo,C.,Andrade,D.,Riquelme,B.,Gomez-Lillo,G.,Soto-Liebe,K.,Diez,B.and Vasquez,M。(2016)。&nbsp;一个class =“ke-insertfile”href =“http://journal.frontiersin.org/article/10.3389/fmicb.2016.00094/full”target =“_ blank”> CyDiv,一种保守和新颖的丝状蓝细菌细胞分裂蛋白,涉及隔膜定位。前端微生物 7:94.
  7. Miyagishima,SY,Kabeya,Y.,Sugita,C.,Sugita,M.and Fujiwara,T。(2014)。&lt; a class =“ke-insertfile”href =“http://bmcplantbiol.biomedcentral.com /片段10.1186/1471-2229-14-57“target =”_ blank“> DipM是叶绿体分裂期间肽聚糖水解所必需的。 BMC植物生物学 14(1):57。
  8. Plominsky,Á。 M.,Larsson,J.,Bergman,B.,Delherbe,N.,Osses,I. andVásquez,M。(2013)。&nbsp; 氮固定限于侵入性蓝细菌Cylindrospermopsis raciborskii CS-505中的末端异种囊。 PloS one 8(2):e51682。
  9. Rippka,R。(1988)。&nbsp; 识别和识别蓝细菌。方法酶蛋白167:28-67。
  10. Santamaria-Gomez,J.,Ochoa de Alda,JA,Olmedo-Verd,E.,Bru-Martinez,R.and Luque,I.(2016)。&nbsp; 蓝细菌中氨基-tRNA合成酶的亚细胞定位和复合物形成:膜锚定ValRS与ATP合成酶相互作用的证据。前端微生物 7:857。
  11. Schneider,CA,Rasband,WS and Eliceiri,KW(2012)。&nbsp; NIH Image to ImageJ:25年的图像分析。 Nat方法 9(7):671-675。
  12. Vonesch,C。和Unser,M.(2008)。&nbsp; 用于小波正则化多维去卷积的快速阈值Landweber算法。 IEEE Trans Image Process 17(4):539-549。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Trigo, C., Andrade, D. and Vásquez, M. (2017). Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling. Bio-protocol 7(11): e2318. DOI: 10.21769/BioProtoc.2318.
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