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α2β1-integrin Clustering and Internalization Protocol
α2β1-整合素的群集和内化实验方案   

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Abstract

α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the extracellular epitope) to the α2β1-integrins and then to cluster the α2β1-integrin-bound primary antibodies together by the means of the secondary antibody. Binding is done on ice so that the α2β1-integrins will not internalize before both sets of antibodies are bound. Clustering is known to trigger α2β1-integrin internalization efficiently from the cell surface to the cytoplasm. In this protocol we used antibody-induced clustering of α2β1-integrin in order to quantitate the amount of internalized α2β1-integrins in comparison to cell surface-associated α2β1-integrin.

Keywords: integrin(整合素), collagen binding(胶原结合), enterovirus(肠道病毒), internalization(内化), clustering(聚类)

Material and Reagents

  1. Adherent Cells (e.g. A549, Hela, SAOS) (on small rounded coverslips, grown to subconfluency)
  2. Ice
  3. Fraction V (Sigma-Aldrich, catalog number: 85040C )
  4. Primary antibody (binds to the α2-integrin ectodomain) (e.g. AbD Serotec, catalog number: MCA2025 ) (diluted in medium containing 1 % serum; use: 4-5 µg/ml)
  5. Two different secondary antibodies that recognize the primary antibody (example: goat anti mouse Alexa- 488 and 555; Life Technologies, catalog numbers: A-11001 and A-21424 ) (diluted in medium containing 1 % serum; use: 1.3 µg/ml)
  6. Serum (Life Technologies, catalog number: 10270-106 )
  7. DAPI prolong gold mounting media (Life Technologies, catalog number: P36935 ) or any other mounting media
  8. Phosphate buffered saline (PBS)
  9. Culture medium with 0-10% serum (see Recipes)
  10. 4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P-6148 ) (see Recipes)

Equipment

  1. Coverslip (Thermo Fisher Scientific, Menzel-Gläzer)
  2. CO2 incubator
  3. Confocal fluorescence microscope

Software

  1. Data analysis by BioImage XD (open-source, http://www.bioimagexd.net/)
  2. ImageJ

Procedure

  1. Subculture suitable adherent cells (e.g. A549 or Hela cells) on round coverslips one or two days before the experiment: place sterile coverslips on a suitable cell culture dish and plate the cells on top of them. Before the experiment you may transfer coverslips with adherent cells to a suitable support, e.g. 4-well, 6- well or 12-well plates.
  2. Cool the cells on ice for a few minutes (in order to inhibit endocytosis/α2β1-integrin uptake) under a cover.
  3. Discard the medium and add primary antibody: 30 µl/coverslip, 60 min on ice.
  4. Wash the unbound antibody gently, by adding BSA-PBS carefully to the cells, not to cause detachment (3x ice-cold PBS containing 0.5 % albumin, 5 min each). For albumin, Fraction V is applicable.
  5. Secondary antibody (Alexa 555) incubation: 30 µl/coverslip, 30 min on ice (keep the coverslips under cover in the darkness).
  6. Wash the unbound antibody (3x BSA-PBS, on ice).
  7. α2β1-integrin internalization: add medium (with 10% serum) on cells and incubate the cells at 37 °C in CO2 incubator for the preferred time (example 2 h).
  8. Labeling the uninternalised/cell surface-associated α2β1-integrin: cool the cells on ice and do the secondary antibody incubation as in step 4 but now with different fluorescent tag (e.g. Alexa 488).
  9. Wash the unbound antibody (3x BSA-PBS).
  10. Fix the cells with 4% PFA for 20 min at room temperature.
  11. Mount the cells with DAPI prolong gold (or any other mounting media).
    1. As you image the cells, you will have your internalized α2β1-integrin in “red” (labelled with Alexa 555 conjugated secondary) and the cell surface-associated α2β1-integrin in “green/yellow” (labelled with both Alexas 555 and 488).
    2. The ratio of intracellular α2β1-integrin can be analyzed by comparing the intensities of the total α2β1-integrin pool (Alexas 555 and 488 colocalizing) and the intracellular α2β1-integrin (Alexa 555).
    3. The intensity analysis can be done for example with ImageJ or BioImage XD.
    4. In addition, BioImageXD contains a simple algorithm for calculating the ratio of surface/internalized antigen. The formula is Ch1/(Ch2 - Coloc), where Ch1 is number of voxels that are cell-surface associated and Ch2 is the total pool of antigens (cell surface and intracellular) and Coloc is number of colocalized voxels.

Recipes

  1. Culture medium with 0-10% serum
    1. Any normal cell culture medium can be used. 1% serum should be included in the binding steps.
    2. Complete cell culture medium (with 10% serum) may be used for the internalization step.
  2. Recipe for 4% PFA
    Dissolve 4 g paraformaldehyde powder to 40 ml of heated water, then add 1 M NaOH dropwise until the solution clears out, cool the solution, add 50 ml 0.2 M phosphate buffer and adjust pH to 7.4.

Acknowledgments

The method is a modification of the protocol described in Karjalainen et al. (2008). This work was supported by funding from the Finnish Academy.

References

Reference for exact protocol

  1. Karjalainen, M., Kakkonen, E., Upla, P., Paloranta, H., Kankaanpää, P., Liberali, P., Renkema, G. H., Hyypiä, T., Heino, J. and Marjomäki, V. (2008). A Raft-derived, Pak1-regulated entry participates in alpha2beta1 integrin-dependent sorting to caveosomes. Mol Biol Cell 19(7): 2857-2869.

References with modifications from the protocol

  1. Liberali, P., Kakkonen, E., Turacchio, G., Valente, C., Spaar, A., Perinetti, G., Böckmann, R. A., Corda, D., Colanzi, A., Marjomaki, V. and Luini, A. (2008). The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS. EMBO J 27(7): 970-981.
  2. Rintanen, N., Karjalainen, M., Alanko, J., Paavolainen, L., Mäki, A., Nissinen, L., Lehkonen, M., Kallio, K., Cheng, R. H., Upla, P., Ivaska, J. and Marjomäki, V. (2012). Calpains promote alpha2beta1 integrin turnover in nonrecycling integrin pathway. Mol Biol Cell 23(3): 448-463.
  3. Siljamäki, E., Rintanen, N., Kirsi, M., Upla, P., Wang, W., Karjalainen, M., Ikonen, E. and Marjomäki, V. (2013). Cholesterol dependence of collagen and echovirus 1 trafficking along the novel alpha2beta1 integrin internalization pathway. PLoS One 8(2): e55465.
  4. Turkki, P., Makkonen, K. E., Huttunen, M., Laakkonen, J. P., Ylä-Herttuala, S., Airenne, K. J. and Marjomäki, V. (2013). Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization. J Virol 87(17): 9822-9835. 

简介

α2β1整合素聚类实验可用于触发α2β1整联蛋白的内化。 当通过顺序施用原发性和荧光二抗进行聚类时,可以在细胞中进入整联蛋白的进入动力学。 该想法是首先允许一级抗体(识别胞外表位)与α2β1整联蛋白的结合,然后通过二级抗体将α2β1整联蛋白结合的一级抗体聚集在一起。 在冰上进行结合,使得α2β1-整联蛋白在两组抗体结合之前不内化。 聚类已知能够有效地从细胞表面向细胞质触发α2β1整合素内化。 在这个协议中,我们使用抗体诱导聚类的α2β1整合素,以定量内化的α2β1整合素的数量与细胞表面相关的α2β1整合素相比。

关键字:整合素, 胶原结合, 肠道病毒, 内化, 聚类

材料和试剂

  1. 贴壁细胞(例如A549,Hela,SAOS)(在小圆形盖玻片上,生长至亚汇合)
  2. 冰块
  3. 馏分V(Sigma-Aldrich,目录号:850-40℃)
  4. (在含有1%血清的培养基中稀释;使用:4-5μg/ml)的第一抗体(结合α2-整合素胞外域)(例如AbD Serotec,目录号:MCA2025)
  5. 识别一级抗体(例如:山羊抗小鼠Alexa-488和555; Life Technologies,目录号:A-11001和A-21424)(在含有1%血清的培养基中稀释;使用:1.3μg/ml )
  6. 血清(Life Technologies,目录号:10270-106)
  7. DAPI延长金色固定介质(Life Technologies,目录号:P36935)或任何其他固定介质
  8. 磷酸盐缓冲盐水(PBS)
  9. 含0-10%血清的培养基(参见配方)
  10. 4%多聚甲醛(PFA)(Sigma-Aldrich,目录号:P-6148)(参见配方)

设备

  1. Coverslip(Thermo Fisher Scientific,Menzel-Gläzer)
  2. CO <2>孵化器
  3. 共聚焦荧光显微镜

软件

  1. BioImage XD的数据分析(开源, http://www.bioimagexd.net/
  2. ImageJ

程序

  1. 在实验前一或两天将合适的贴壁细胞(例如A549或Hela细胞)在圆盖玻片上培养:将无菌盖玻片置于合适的细胞培养皿上,并将细胞铺在其上。 在实验之前,您可以将贴壁细胞的盖玻片转移到合适的支架上,例如4孔,6孔或12孔平板。
  2. 在冰上冷却细胞几分钟(以抑制内吞作用/α2β1整联蛋白吸收)。
  3. 弃去培养基并加入一抗:30μl/盖玻片,在冰上60分钟
  4. 轻轻地洗涤未结合的抗体,小心地将BSA-PBS加入细胞,不引起脱离(每次5x白蛋白的3x冰冷PBS)。 对于白蛋白,第V部分适用
  5. 二抗(Alexa 555)孵育:30μl/盖玻片,在冰上30分钟(在黑暗中保持盖玻片覆盖)。
  6. 洗涤未结合的抗体(3x BSA-PBS,在冰上)
  7. α2β1-整联蛋白内化:在细胞上加入培养基(含10%血清),并在37℃在CO 2培养箱中孵育细胞优选的时间(实施例2h)。
  8. 标记未内化的/细胞表面相关的α2β1整联蛋白:冷却冰上的细胞,并按照步骤4进行第二抗体温育,但现在使用不同的荧光标签(例如Alexa 488)。
  9. 洗涤未结合的抗体(3x BSA-PBS)
  10. 在室温下用4%PFA固定细胞20分钟
  11. 用DAPI延长金(或任何其他安装介质)安装细胞。
    1. 当你成像的细胞,你会有你的内化的α2β1整合素在"红色"(标记与Alexa 555共轭次级)和细胞表面相关的α2β1整合素在"绿色/黄色"(标记与Alexas 555和488) 。
    2. 可以通过比较总α2β1整联蛋白池(Alexa 555和488共定位)和细胞内α2β1整联蛋白(Alexa 555)的强度来分析细胞内α2β1整联蛋白的比率。
    3. 强度分析可以例如使用ImageJ或BioImage XD进行。
    4. 此外,BioImageXD包含一个简单的算法,用于计算表面/内化抗原的比率。公式是Ch1 /(Ch2-Coloc),其中Ch1是与细胞表面相关的体素的数目,Ch2是抗原的总池(细胞表面和细胞内),Coloc是共定位体素的数目。

食谱

  1. 含0-10%血清的培养基
    1. 可以使用任何正常细胞培养基。 1%血清应包括在结合步骤中。
    2. 完全细胞培养基(含10%血清)可用于内化步骤
  2. 4%PFA的配方
    将4g多聚甲醛粉末溶解于40ml热水中,然后滴加1M NaOH直至溶液澄清,冷却溶液,加入50ml 0.2M磷酸盐缓冲液并将pH调节至7.4。

致谢

该方法是Karjalainen等人(2008)中描述的方案的修改。 这项工作得到了芬兰学院的资助。

参考文献

精确协议的参考

  1. Karjalainen,M.,Kakkonen,E.,Upla,P.,Paloranta,H.,Kankaanpää,P.,Liberali,P.,Renkema,GH,Hyypiä,T.,Heino,J.andMarjomäki, )。 A 筏衍生的Pak1调节的条目参与α2β1整联蛋白依赖性排序到caveosomes。 Mol Biol Cell 19(7):2857-2869。

从协议修改的引用

  1. Liberali,P.,Kakkonen,E.,Turacchio,G.,Valente,C.,Spaar,A.,Perinetti,G.,Böckmann,RA,Corda,D.,Colanzi,A.,Marjomaki, ,A。(2008)。 Pak1依赖性大染色体的闭合需要CtBP1/BARS的磷酸化。 EMBO J 27(7):970-981。
  2. Rintanen,N.,Karjalainen,M.,Alanko,J.,Paavolainen,L.,Mäki,A.,Nissinen,L.,Lehkonen,M.,Kallio,K.,Cheng,RH,Upla,P.,Ivaska ,J。和Marjomäki,V。(2012)。 钙蛋白酶促进非循环整合素途径中的α2β1整合蛋白转换。 Mol Biol Cell 23(3):448-463。
  3. Siljamäki,E.,Rintanen,N.,Kirsi,M.,Upla,P.,Wang,W.,Karjalainen,M.,Ikonen,E。和Marjomäki, 胆固醇依赖的胶原和艾柯病毒1贩运沿着小说alpha2beta1整合素内化途径。 em> PLoS One 8(2):e55465。
  4. Turkki,P.,Makkonen,K.E.,Huttunen,M.,Laakkonen,J.P.,Ylä-Herttuala,S.,Airenne,K.J.andMarjomäki, 细胞对杆状病毒转导和艾柯病毒感染的易感性被蛋白激酶C磷酸化和波形蛋白组织修饰。 J Virol 87(17):9822-9835。 
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Marjomäki, V., Karjalainen, M., Upla, P., Siljamäki, E., Rintanen, N. and Turkki, P. (2014). α2β1-integrin Clustering and Internalization Protocol. Bio-protocol 4(7): e1088. DOI: 10.21769/BioProtoc.1088.
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