Name: f&#246;rster resonance energy transfer measurements in living plant cells
Uploaded: 2021-06-28T15:00:00
Description: Watch this Scientific Journal Video about FÃƒÂ¶rster Resonance Energy Transfer Measurements in Living Plant Cells at JoVE.com

实验在比勒费尔德大学生物学院的光学显微镜技术平台（LiMiTec）进行。这项工作由比勒费尔德大学资助。

注意：对于以下方案，如前所述，进行了原生质体的瞬时转染12。下面给出了简要说明。
1. 原生质体的瞬时转染
<ol><li>将拟 南芥 生态型哥伦比亚的约4克健康叶子切成1毫米切片，并将它们转移到20毫升酶溶液中（1.5%纤维素酶;0.4%马克酶;0.1%牛血清白蛋白V级分;0.4米甘露醇;20mM KCl;20mM 2-（N-吗啉）乙磺酸（MES），pH 5.7;10mM CaCl2）。</li>
	<li>?...

<ol>
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N., Bisseling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+FRET-microscopy+in+living+plant+cells.">GFP-based FRET-microscopy in living plant cells.</a> Trends in Plant Science. 4, 287-291 (1999).</li><li>Van Rheenen, J., Langeslag, M., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+confocal+acquisition+to+optimize+imaging+of+fluorescence+resonance+energy+transfer+by+sensitized+emission.">Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission.</a> Biophysical Journal. 86, 2517-2529 (2004).</li><li>Seidel, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+C-termini+of+V-ATPase+subunits+by+in+vivo-FRET+measurements.">Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements.</a> FEBS Letters. 579, 4374-4382 (2005).</li><li>Seidel, T., Schnitzer, D., Golldack, D., Sauer, M., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+iso-enzymes+of+plant+V-ATPase+as+revealed+by+in+vivo-FRET.">Organelle-specific iso-enzymes of plant V-ATPase as revealed by in vivo-FRET.</a> BMC Cell Biology. 9, 28 (2008).</li><li>Schnitzer, D., Seidel, T., Sander, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+energization+state+affects+peripheral+stalk+stability+of+plant+vacuolar+H+-ATPase+and+impairs+vacuolar+acidification.">The cellular energization state affects peripheral stalk stability of plant vacuolar H+-ATPase and impairs vacuolar acidification.</a> Plant Cell Physiology. 52, 946-956 (2011).</li><li>Roshchina, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+autofluorescence:+application+to+the+study+of+plant+living+cells.">Vital autofluorescence: application to the study of plant living cells.</a> International Journal of Spectroscopy. 2012, 124672 (2012).</li><li>Holtorf, S., Apel, K., Bohlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+constitutive+and+inducible+promoters+for+the+overexpression+of+transgenes+in+Arabidopsis+thaliana.">Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.</a> Plant Molecular Biology. 29, 637-646 (1995).</li><li>Seidel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+and+FRET-analysis+of+subunits+c+and+a+of+the+vacuolar+H+-ATPase+in+living+plant+cells.">Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells.</a> Journal of Biotechnology. 112 (1-2), 165-175 (2004).</li><li>Beemiller, P., Hoppe, A. 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供体淬灭和敏化受体发射的特点是线性关系，允许基于供体或受体计算FRET。线性的相应因子称为 G 因子（供体对受体）或 xi（受体对供体），它们是倒数值4。由于荧光蛋白的广泛吸收和发射光谱，通过荧光显微镜测量荧光蛋白之间的FRET通常需要校正DSBT和ASBT。但是，许多校正依赖于可测量的 ASBT，这是协议第 7 节中方程分母的一个因素，α = 0 会导致未定义的方程5<...

Förster共振能量转移（（F）RET）通常通过荧光光谱观察，尽管该过程本身并不局限于荧光团之间发生。潜在的偶极子-偶极子耦合只需要一个发光的供体分子和一个光吸收受体。这是从归一化供体发射和受体吸光度谱1所需的光谱重叠积分J导出的。然而，由于RET与荧光竞争，能量转移可以通过荧光发射的改变来测量：RET诱导供体猝灭和敏化受体发射。
基于荧光团的RET被称为荧光共振能量转移（FRET），以将其与生物发光共振能量转移（BRET）分开。RET在很大程度上取决于供体和受体之间的距离，其范围广泛为0.5-10 nm2 ，因此与蛋白质及其复合物的尺寸范围相同。其次，RET取决于偶极子-偶极子取向kappa的平方。结合蛋白质结合的荧光团由于分子量和缓慢的旋转弛豫而可以忽略的事实，RET允许分析构象改变3。
所谓的Förster半径基于光谱重叠积分和重叠的波长范围，因此吸收红光的发色团比蓝光吸收染料产生更长的Förster半径。由于FRET测量的动态范围受到0.5×R0 和1.5×R0的限制，FRET对ECFP-EYFP的动态范围为2.5-7.3 nm，因为它的R0 为4.9 nm4。
荧光团的亮度由其摩尔消光系数和量子产率的乘积给出。对于FRET测量，选择亮度几乎相似的荧光团是有利的。这增强了对供体淬火和敏化受体发射的检测。它还有利于显微镜系统的校准。观察常用的FRET对青色和荧光蛋白，青色荧光蛋白的较低亮度变得明显（图1A）。
但是，受体的寿命必须低于供体的寿命，从而确保受体可用于能量转移。如果受体的寿命超过供体的寿命，则当供体再次激发时，受体可能仍处于激发状态。先进的青色荧光蛋白（如mTurquoise）显示出更长的使用寿命，因此有助于增加FRET的可能性（图1B）。FRET的概率还取决于受体的摩尔消光系数。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well slides</td>
			<td>Ibidi</td>
			<td>80821</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil Immersol&#160; W2010</td>
			<td>Zeiss</td>
			<td>444969-0000-000</td>
			<td>refraction index of water</td>
		</tr>
		<tr>
			<td>LSM 1: AxioObserver with LSM 780 scan head, confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 2: AxioObserver with LSM 5 scan head, confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

f&#246;rster resonance energy transfer measurements in living plant cells

基于敏化发射的Förster共振能量转移（FRET）实验很容易完成，但取决于微观设置。共聚焦激光扫描显微镜已成为生物学家的主力军。商用系统在激光功率调整和探测器灵敏度方面具有高度的灵活性，并且通常组合不同的探测器以获得完美的图像。然而，由于这种灵活性，通常不可能比较来自不同实验和设置的基于强度的数据。生物学家友好的程序具有优势，可以简单可靠地调整激光和探测器设置。 
此外，由于活细胞中的FRET实验受到蛋白质表达和供体 - 受体比的变异性的影响，因此必须考虑蛋白质表达水平以进行数据评估。这里描述的是用于可靠和可重复的FRET测量的简单方案，包括用于估计蛋白质表达和调整激光强度和检测器设置的例程。数据评估将通过校准已知FRET效率的荧光团融合来执行。为了提高简单性，已经比较了在细胞中通过测量重组荧光蛋白获得的校正因子。

共聚焦激光扫描显微镜的调整 激光调整显示，随着激光强度的增加，发射线性增加（图2 和 表1）。正如氩离子激光器所预期的那样，514 nm线的发射远高于458 nm线的发射，正如更陡峭的斜率所证明的那样。在随后的实验中，分别为514 nm线和458 nm线选择了4.5%和6.5%的激光功率。这导致1123（514 nm）和1141（458 nm）的发射强度几乎相等。
<p class="jove_cont...

Watch this Scientific Journal Video about FÃƒÂ¶rster Resonance Energy Transfer Measurements in Living Plant Cells at JoVE.com

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

提供了一个方案，用于设置标准的共聚焦激光扫描显微镜，用于 体内 Förster共振能量转移测量，然后进行数据评估。

活植物细胞中的Förster共振能量转移测量

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

该协议提供了一个简单的程序，使通过敏化排放的FRET实验更具可重复性和可比性。FRET的主要优点是实时测量，因此可以解决动态过程。对于使用透射光电倍增管进行激光调整，将含有转染原生质体的八孔载玻片放在显微镜下。选择空孔后，选择线扫描模式和直方图视图，将激光强度降至最低，并将检测器增益调整为可检测的背景噪声。接下来，在记录相应信号的同时，以0.5%的步长增加激光强度。要使用反射模式进行激光调整，请选择一个空孔，然后应用反射滤光片。打开反射模式，确保探测器波长范围覆盖激光器的波长。选择线扫描模式和直方图视图，将激光强度降至最低，并将探测器增益调整为可检测的背景噪声。接下来，将物镜移动到最低位置，然后将其向上移动，直到盖玻片的反射可见，然后在记录相应信号的同时以0.5%的步长增加激光强度。对于数据评估，请按信号强度对数据进行制表和排序，然后将信号强度与相对激光功率绘制，并选择产生相似信号强度的激光强度。为了调整光电倍增管，选择一个空孔并应用反射滤光片，切换到反射模式，并确保探测器波长范围覆盖激光器的波长。选择线阵扫描模式和直方图视图，将探测器增益降低到最大值的一半，并将激光强度调整到可检测的背景噪声。接下来，将物镜移动到最低位置，然后将其向上移动，直到盖玻片的反射可见，然后以50至100伏步长增加检测器增益并记录相应的信号。对于数据评估，将强度与每个探测器的探测器增益绘制，然后选择单个探测器增益以获得相似的灵敏度。对于图像采集，选择合适的滤光片或二向色镜，并对所有通道使用相同的二向色镜，以实现逐行扫描，然后选择用于活细胞成像的水浸物镜，并选择12位或16位扫描，扫描速度适中。接下来，定义检测范围，优选470至510纳米用于供体检测，530至600纳米用于受体检测，在FRET对增强青色荧光蛋白或ECFP和增强型黄色荧光蛋白或EYFP的情况下。如前所述应用探测器和激光器设置后，根据需要根据获得的激光功率表修改激光强度。确保信噪比覆盖探测器的整个动态范围。在对针孔直径进行微调，同时保持激光强度和检测器增益恒定后，执行FRET测量，拍摄至少20个细胞的图像。为了确定串扰校正，仅对表达供体荧光团的细胞和仅表达受体荧光团的细胞进行FRET测量。为了校准测量，使用表达供体受体融合的细胞进行FRET测量。对于数据评估，获取像元的线轮廓，确保每个图谱包含不超过一个像元。将配置文件另存为文本文件。然后使用数据部分中的文本文件导入选项，将文本文件导入电子表格。接下来，通过应用 max 函数读出最大值，然后在表中列出获得的值，该表中各有一列供体排放 ID、FRET 发射 IF、受体排放 IA，以及至少四个数据集（仅限供体、仅接受者、接受者融合和测量）。激光调整显示，随着激光强度的增加，发射线性增加。如陡峭的斜率所示，514纳米线的发射远高于458纳米线的发射。在恒定激光功率下改变探测器增益揭示了两个被分析探测器的指数行为。用重组纯化蛋白分析的供体和受体的差异和光谱渗漏，以及用表达这些蛋白的细胞分析的差异和光谱渗漏表明，不可能省略可能由细胞色素引起的活细胞中光谱渗漏的测定。标记的空泡ATPA亚基，VHA AECFP和VHA AEYFP之间的FRET效率随着信号强度的增加而降低。相反，VHA E1 ECFP和VHA CEYFP之间的相互作用与信号强度无关。选择合适的光学元件对于检测供体和受体至关重要。该协议也可以应用于活细胞中的比例传感器，以提高这些实验中的可重复性。

Research

JoVE Journal

Biochemistry

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

科研

生物化学

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...