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Measurement of Extracellular Ca2+ Influx and Intracellular H+ Efflux in Response to Glycerol and PEG6000 Treatments
测定响应甘油和PEG6000的胞外Ca2+流入量和胞内 H+流出量   

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Abstract

The characteristics of Ca2+ and H+ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H+ concentration and increase in cytoplasmic Ca2+ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca2+ and H+ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca2+ and H+ fluxes across transformed Pichia pastoris cells in response to glycerol and polyethylene glycol 6000 (PEG6000) treatments. The transformed yeast cells are loaded onto a coverslide pre-processed in the poly-L-lysine solution (0.1% w/v aqueous solution). After cell immobilization, microelectrodes are positioned above a monolayer of attached cell population. Micro-volts differences are measured at two excursion points manipulated by a computer. Micro-volts differences could be converted into ion fluxes using the ASET 2.0 and iFluxes 1.0 Software. The method is expected to promote the application of NMT in microbiology. We are very grateful to Younger USA (Xuyue Beijing) NMT Service Center for their critical reading of the manuscript.

Keywords: Ion flux(离子通量), Glycerol(甘油), Non-destructive measurement(非破坏检测), PEG(PEG), Signal(信号)

Materials and Reagents

  1. Transformed Pichia pastoris cells (Invitrogen, catalog number: V200-20 )
  2. Poly-L-lysine solution (0.1% w/v aqueous solution) (Sigma-Aldrich, catalog number: P4707 )
  3. Polyethylene glycol 6000 (PEG6000) (Merck KGaA, catalog number: 807491 )
  4. Glycerol (Sinopharm Chemical Reagent, catalog number: 10010692 )
  5. Yeast extract (Oxoid, catalog number: LP0021 )
  6. Peptone (Oxoid, catalog number: LP0037 )
  7. D-glucose (Sinopharm Chemical Reagent, catalog number: 10010592 )
  8. MES
  9. Standard medium buffer (pH 6.0) (see Recipes)
  10. Yeast extract peptone dextrose (YPD) medium (see Recipes)
  11. Calibration medium buffer (pH 7.0) (see Recipes)
  12. Calibration medium buffer (pH 5.0) (see Recipes)

Equipment

  1. Non-invasive Micro-test System (YoungerUSA, model: NMT100 series )
  2. Shaking incubator (Shanghai Anting Scientific Instrument Factory, model: HZQ-F160 )
  3. Centrifuge (Thermo Fisher Scientific, model: Fresco 21 )
  4. Microplate Reader Spectra (Molecular Devices, model: SpectraMax 190 )
  5. Glass coverslide (20 mm x 20 mm)
  6. Petri dish (35 mm in diameter)
  7. Micropipettor (Eppendorf, 100-1,000 μl and 10-100 μl)

Software

  1. JCal V3.2.1 (a free MS Excel spreadsheet, available at http://www.youngerusa.com or http://www.ifluxes.com)
  2. ASET 2.0 software (available at http://www.youngerusa.com)
  3. iFluxes 1.0 software (available at http://www.youngerusa.com)

Procedure

  1. The transformed cells are incubated in 10 ml YPD at 30 °C in a shaking incubator (200 rpm) for 12 h.
  2. Overnight cultures of different transformed cells are adjusted to an OD600nm of 0.2. OD600nm is monitored using microplate reader spectra.
  3. Fifty microliters of each are taken and added to 10 ml YPD containing a final concentration of 25% PEG6000 or 1 M glycerol or no exogenous osmolytes. The transformed yeast cells are grown to an OD600nm of 1.0 in YPD containing different exogenous osmolytes at 30 °C in a shaking incubator (200 rpm). OD600nm is monitored using microplate reader spectra.
  4. One milliliter of each is taken and pelleted at 2,000 rpm for 5 min at room temperature.
  5. Nine hundred microliters of supernatant are removed, and the cells are resuspended in the rest of YPD media.
  6. The coverslips are immersed in the poly-L-lysine solution (0.1% w/v aqueous solution) for 24 h.
  7. Prior to each flux measurement, the microelectrodes must be calibrated in calibration medium (pH 7.0 and pH 5.0), respectively, following to the same procedure and standards. Only Ca2+ electrodes with Nernstian slope > 26 mV/decade and H+ electrodes with Nernstian slope > 53 mV/decade are used in the protocol. Data are discarded if the post-test calibrations fail.
  8. Ten microliters of transformed cells are loaded on the coverslip for 5 min, washed off with standard medium to ensure a monolayer of attached cells and incubated in the standard medium for 5 min at room temperature.
  9. Microelectrodes are positioned 10 μm above the attached cell population consisting of 15 cells with equal size. Micro-volts differences are measured at two excursion points, one 10 μm above the cell population and the other 20 μm away, at a frequency of 0.05 Hz manipulated by a computer. The kinetics of net Ca2+ and H+ fluxes near each cell population are monitored for 10 min.
  10. For each sample, four clones are incubated in 10 ml YPD, and the resulting four cell populations are measured (see steps 1-9).
  11. Micro-volts differences are exported as raw data before they are converted into net Ca2+ and H+ fluxes by using the JCal V3.2.1. The ion flux assay around each type of transformed cells is replicated independently three times.


    Figure 1. Schematic diagram of ion flux detection (www.xuyue.net). The microelectrode tip is filled with liquid ion exchanger (LIX). A voltage gradient (dV) is measured by the electrometer between two positions over the travel range dx. A concentration gradient (dc) is calculated based on dV. Do, ion diffusion constant; J, net ion flux.

Recipes

  1. Standard medium (aqueous solution) buffer (pH 6.0)
    0.1 mM CaCl2
    0.1 mM KCl
    0.3 mM MES
    10 mM glucose
    pH is adjusted to 6.0 with HCl
    Stored at 4 °C
  2. Yeast extract peptone dextrose (YPD) medium (1 L)
    1% yeast extract
    2% peptone
    2% D-glucose (added to the medium after autoclave)
    10 g yeast extract
    20 g peptone are dissolved in 900 ml of water
    The medium is autoclaved for 20 minutes on liquid cycle, cooled to ~55 °C
    Mixed with 100 ml of 20% D-glucose
    The liquid medium is stored at room temperature
  3. Calibration medium (aqueous solution) buffer (pH 7.0)
    0.01 mM CaCl2
    0.1 mM KCl
    0.3 mM MES
    10 mM glucose
    pH is adjusted to 7.0 with HCl
    The medium is stored at 4 °C
  4. Calibration medium (aqueous solution) buffer (pH 5.0)
    0.1 mM CaCl2
    0.1 mM KCl
    0.3 mM MES
    10 mM glucose
    pH is adjusted to 5.0 with HCl
    The medium is stored at 4 °C

Acknowledgments

The protocol was adapted from our previously published paper Li et al. (2013). We wish to thank Younger USA (Xuyue Beijing) NMT Service Center for the technical support. This research was financially supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Project no. KZCX2-YW-BR-17) and National Natural Science Foundation of China (41371264, 41401281).

References

  1. Li, T., Hu, Y. J., Hao, Z. P., Li, H., Wang, Y. S. and Chen, B. D. (2013). First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices. New Phytol 197(2): 617-630.
  2. McLamore, E. S. and Porterfield, D. M. (2011). Non-invasive tools for measuring metabolism and biophysical analyte transport: self-referencing physiological sensing. Chem Soc Rev 40(11): 5308-5320.
  3. Shabala, L., Ross, T., McMeekin, T. and Shabala, S. (2006). Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment. FEMS Microbiol Rev 30(3): 472-486.
  4. Shabala, L., Ross, T., Newman, I., McMeekin, T. and Shabala, S. (2001). Measurements of net fluxes and extracellular changes of H+, Ca2+, K+, and NH4+ in Escherichia coli using ion-selective microelectrodes. J Microbiol Methods 46(2): 119-129. 
  5. Wang, Q., Zhao, Y., Luo, W., Li, R., He, Q., Fang, X., Michele, R. D., Ast, C., von Wiren, N. and Lin, J. (2013). Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization. Proc Natl Acad Sci U S A 110(32): 13204-13209.
  6. Xu, R. R., Qi, S. D., Lu, L. T., Chen, C. T., Wu, C. A. and Zheng, C. C. (2011). A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana. FEBS J 278(13): 2296-2306. 

简介

Ca 2+和H sup +通量的特征可反映水通道蛋白的活性,因为水通道蛋白活性的上调与细胞质H的减少直接相关> + 浓度和细胞质Ca 2+浓度的增加。较高的水通道蛋白活性可以通过改变进入和离开细胞的水流来保护细胞免受渗透压力。为了证实水通道蛋白对细胞对不同渗透胁迫的耐受性,使用非侵入性微测试技术(NMT)测量净Ca 2+和H + +通量, )。 NMT提供了跨膜的净离子迁移的实时原位检测。在这里,我们描述了原位检测穿过转化的巴斯德毕赤氏酵母的净Ca 2+和 + 通量的方案。 >细胞响应于甘油和聚乙二醇6000(PEG6000)处理。将转化的酵母细胞加载到在聚-L-赖氨酸溶液(0.1%w/v水溶液)中预处理的盖片上。细胞固定后,微电极位于单层细胞群上方。在由计算机操纵的两个偏移点处测量微伏电压差。使用ASET 2.0和iFluxes 1.0软件,微伏电压差可以转换为离子通量。该方法有望促进NMT在微生物学中的应用。我们非常感谢Younger USA(徐越北京)NMT服务中心对稿件的批判性阅读。

关键字:离子通量, 甘油, 非破坏检测, PEG, 信号

材料和试剂

  1. 转化的巴斯德毕赤酵母细胞(Invitrogen,目录号:V200-20)
  2. 聚-L-赖氨酸溶液(0.1%w/v水溶液)(Sigma-Aldrich,目录号:P4707)
  3. 聚乙二醇6000(PEG6000)(Merck KGaA,目录号:807491)
  4. 甘油(Sinopharm Chemical Reagent,目录号:10010692)
  5. 酵母提取物(Oxoid,目录号:LP0021)
  6. 蛋白胨(Oxoid,目录号:LP0037)
  7. D-葡萄糖(Sinopharm Chemical Reagent,目录号:10010592)
  8. MES
  9. 标准介质缓冲液(pH 6.0)(参见配方)
  10. 酵母提取物蛋白胨葡萄糖(YPD)培养基(参见配方)
  11. 校准介质缓冲液(pH 7.0)(参见配方)
  12. 校准介质缓冲液(pH 5.0)(参见配方)

设备

  1. 非侵入式微测试系统(YoungerUSA,型号:NMT100系列)
  2. 摇床培养箱(上海安亭科学仪器厂,型号:HZQ-F160)
  3. 离心机(Thermo Fisher Scientific,型号:Fresco 21)
  4. 微孔板读数器光谱(Molecular Devices,型号:SpectraMax 190)
  5. 玻璃盖片(20mm×20mm)
  6. 培养皿(直径35mm)
  7. 微量移液器(Eppendorf,100-1,000μl和10-100μl)

软件

  1. JCal V3.2.1(免费的MS Excel电子表格,可在 http://www.youngerusa.com 或< a target ="_ blank"href ="http://www.ifluxes.com/"> http://www.ifluxes.com )
  2. ASET 2.0软件(位于 http://www.youngerusa.com
  3. iFluxes 1.0软件(可从 http://www.youngerusa.com 获得)

程序

  1. 将转化的细胞在10ml YPD中在30℃下在振荡培养箱(200rpm)中孵育12小时。
  2. 将不同转化细胞的过夜培养物调整至0.2的OD 600nm。使用酶标仪光谱监测OD <600nm
  3. 各取50微升并加入到10ml终浓度为25%PEG6000或1M甘油或无外源性渗压剂的YPD中。转化的酵母细胞在30℃下在振荡培养箱(200rpm)中在含有不同外源性渗压剂的YPD中生长至OD 600nm为1.0。使用酶标仪光谱监测OD <600nm
  4. 取每份1毫升,在室温下以2,000rpm离心5分钟
  5. 除去9百微升的上清液,将细胞重悬浮于剩余的YPD培养基中。
  6. 将盖玻片浸没在聚-L-赖氨酸溶液(0.1%w/v水溶液)中24小时。
  7. 在每次通量测量之前,必须根据相同的程序和标准,分别在校准介质(pH 7.0和pH 5.0)中校准微电极。只有具有能斯脱斜率的Ca 2+电极。 26mV/decade和Hernon +电极,其Nernstian斜率>协议中使用53 mV/decade。如果后测试校准失败,则会丢弃数据。
  8. 将10微升转化细胞加载到盖玻片上5分钟,用标准培养基洗涤以确保附着细胞的单层,并在室温下在标准培养基中温育5分钟。
  9. 微电极位于由具有相等大小的15个细胞组成的附着细胞群上方10μm。在两个偏移点处测量微伏电压差,在细胞群体上方一个10μm 其它20μm,以0.05Hz的频率通过计算机操作。监测每个细胞群附近的净Ca 2+和 +通量的动力学10分钟。
  10. 对于每个样品,将四个克隆在10ml YPD中孵育,并测量所得的四个细胞群体(参见步骤1-9)。
  11. 微电压差在通过使用JCal V3.2.1转换成净Ca 2+和μH +通量之前被导出为原始数据。围绕每种类型的转化细胞的离子流测定独立地复制三次

    图1.离子通量检测示意图( www.xuyue.net )。微电极尖端填充有液体离子交换剂(LIX)。通过静电计在行程范围dx 上的两个位置之间测量电压梯度( dV 。基于dV计算浓度梯度( dc )。 ,离子扩散常数; J ,净离子流量。

食谱

  1. 标准介质(水溶液)缓冲液(pH 6.0)
    0.1mM CaCl 2/v/v 0.1 mM KCl
    0.3 mM MES
    10mM葡萄糖 用盐酸将pH调节至6.0 储存在4°C
  2. 酵母提取物蛋白胨葡萄糖(YPD)培养基(1L)
    1%酵母提取物
    2%蛋白胨
    2%D-葡萄糖(高压灭菌后加入培养基) 10g酵母提取物
    将20g蛋白胨溶解在900ml水中
    培养基在液体循环中高压灭菌20分钟,冷却至〜55℃ 与100ml 20%D-葡萄糖混合 液体介质在室温下贮存
  3. 校准介质(水溶液)缓冲液(pH 7.0)
    0.01mM CaCl 2·h/v 0.1 mM KCl
    0.3 mM MES
    10mM葡萄糖 用盐酸将pH调节至7.0 培养基在4℃下保存
  4. 校准介质(水溶液)缓冲液(pH 5.0)
    0.1mM CaCl 2/v/v 0.1 mM KCl
    0.3 mM MES
    10mM葡萄糖 用盐酸将pH调节至5.0 培养基在4℃下保存

致谢

该协议改编自我们先前发表的论文Li (2013)。我们要感谢美国(北京徐越)NMT服务中心的技术支持。这项研究得到了中国科学院知识创新计划(项目编号KZCX2-YW-BR-17)和中国国家自然科学基金(41371264,41401281)的资助。

参考文献

  1. Li,T.,Hu,Y.J.,Hao,Z.P.,Li,H.,Wang,Y.S.and Chen,B.D。(2013)。 首次克隆和表征来自丛枝菌根真菌的两种功能性水通道蛋白基因Glomus intraradices 。 New Phytol 197(2):617-630。
  2. McLamore,E. S.和P​​orterfield,D.M。(2011)。 用于测量代谢和生物物理分析物转运的非侵入性工具:自参考生理感测。 Chem Soc Rev 40(11):5308-5320。
  3. Shabala,L.,Ross,T.,McMeekin,T。和Shabala,S。(2006)。 用于研究微生物对环境的适应性反应的非侵入性微电极离子通量测量。 FEMS Microbiol Rev 30(3):472-486。
  4. Shabala,L.,Ross,T.,Newman,I.,McMeekin,T。和Shabala,S。(2001)。 净通量和H + ,Ca J Microbiol Methods 46(2):119-129。 
  5. Wang,Q.,Zhao,Y.,Luo,W.,Li,R.,He,Q.,Fang,X.,Michele,RD,Ast,C.,von Wiren,N.and Lin, 2013)。 单粒子分析揭示了拟南芥铵转运蛋白AMT1的截止控制; 3通过聚类和内化。 Proc Natl Acad Sci U S A 110(32):13204-13209。
  6. Xu,R.R.,Qi,S.D.,Lu,L.T.,Chen,C.T.,Wu,C.A。和Zheng,C.C。 DExD/H盒RNA解旋酶对于拟南芥中的K +剥夺反应和耐受性很重要 。 FEBS J 278(13):2296-2306。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, T. and Chen, B. (2013). Measurement of Extracellular Ca2+ Influx and Intracellular H+ Efflux in Response to Glycerol and PEG6000 Treatments. Bio-protocol 3(18): e911. DOI: 10.21769/BioProtoc.911.
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