Name: in vivo quantification of protein turnover in aging c. elegans using photoconvertible dendra2
Uploaded: 2020-06-13T14:30:00
Description: Watch this Scientific Journal Video about In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2 at JoVE.com

我们感谢DFG（KI-1988/5-1到JK，神经库博士奖学金由神经治疗卓越集群到M MLP）的资金。我们还感谢柏林莱布尼茨分子药理学研究所（FMP）的成像核心设施提供成像设置。此外，我们要感谢迪奥戈·费莱西亚诺，他建立了Dendra2系统，并提供了指导。

1. 表达神经元亨廷顿-登德拉2融合蛋白的C.elegans的一代
<ol><li>通过传统的限制性酶消化10、吉布森组装11或任何选择方法，克隆线虫表达载体（即pPD95_75、Addgene #1494）中编码POI的基因。插入启动子，在所需的组织中或在所需的发育阶段驱动表达。将 Dendra2 荧光团（N-或C-终端）与POI一起插入框架中。</li>
	<li>生成表达聚变构造的转基因C.elegans（例如?...

<ol>
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要理解蛋白质的功能，了解其合成、位置和降解非常重要。随着新型、稳定、明亮的 FP 的发展，可视化和监控 POIs 变得更加简单和高效。基因表达聚变PAFP，如Dendra2具有独特的定位，可以研究POI的稳定性。当暴露在紫蓝色光下时，Dendra2在保存的氨基酸三合体内的精确位置断裂。荧光团经历了一个小的结构变化，导致光谱从绿色到红色23的完全转移。这种转变允许检测和监测与Dend...

生物体的蛋白质组在不断更新。蛋白质根据细胞的生理需求不断降解和合成。一些蛋白质很快被消除，而另一些则寿命更长。使用基因编码的荧光蛋白 （FPs） 时，监测蛋白质动力学是一项更简单、更准确、侵入性更小的任务。FPs以自动催化形式形成，可以融合到任何感兴趣的蛋白质（POI），但不需要酶折叠或需要辅助因子，但氧气1。新一代 FP 最近被设计为在照射时使用确定波长的光脉冲切换颜色。这些光激活的FP（PAFP）允许标记和跟踪POIS，或他们居住的细胞器或细胞，并检查其定量和/或定性参数2。FPs 能够跟踪任何 POI 的移动、方向性、运动速率、扩散系数、移动分数与移动分数、它驻留在一个蜂窝隔间中的时间以及其周转率。对于特定的细胞器，运动和运输，或裂变和融合事件可以确定。对于特定的单元格类型，可以建立单元格的位置、分割速率、体积和形状。至关重要的是，PAFP 的使用允许无需连续可视化，也无需任何新合成探头的干扰即可进行跟踪。细胞和整个生物体的研究已经成功地使用PAFP来解决体内的生物问题，如癌症和转移的发展，细胞骨架的组装或分解，以及RNA-DNA/蛋白质相互作用3。在这份手稿中，光显微镜和PAFP用于在神经退行性疾病的C.elegans模型中发现体内容易聚集的蛋白质亨廷顿蛋白（HTT）的周转率。
此处描述的协议量化了融合蛋白亨廷顿蛋白（HTT-D2）的稳定性和降解性。Dendra2 是第二代单体 PAFP4，它不可逆转地将其发射/激发光谱从绿色切换到红色，以响应紫外线或可见蓝光，其强度增加高达 4，000 倍5，,6。亨廷顿是导致亨廷顿舞蹈症（HD）的蛋白质，这是一种致命的遗传性神经退行性疾病。亨廷顿-1含有一段谷氨酰胺（CAG，Q）。当蛋白质以超过39Q表达时，它误入突变、有毒和致病性蛋白质。突变HTT容易聚集，导致神经元细胞死亡和退化，无论是短寡聚物种或较大的高度结构化淀粉样体7。
线虫是研究衰老和神经退化的模型系统，由于其操作方便性、等基因性、寿命短和光学透明度8。为了研究HTT体内的稳定性，在C.elegans的神经系统中表达了一种融合结构。HTT-D2 转基因含有 25Qs （HTTQ25-D2） 或 97Qs （HTTQ97-D2） 的病理拉伸，在整个线虫的生命周期9中，泛神经表达过度。通过将实时C. elegans接受简短且聚焦的光点，单个神经元被拍照切换，并随着时间的推移跟踪转换的 HTT-D2。为了确定HTT-D2的降解量，将刚转换的HTT-D2的红色信号与确定一段时间后HTT-D2的剩余红色信号进行比较。因此，与生理形态相比，发现亨廷顿蛋白的膨胀和毒性形式时，可以研究亨廷顿素是如何降解的;前神经元或后神经元对Q97与Q25的存在的反应如何不同，尤其是在较长的时间段内;以及蛋白质组菌网络 （PN） 在老化期间的崩溃如何导致降解率的差异。这些结果只描述了一组关于HTT-D2周转率的少量观测结果。然而，与蛋白质聚集和蛋白质结合领域相关的更多生物学问题可以通过这种体内应用加以解决。

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			<td>Agar-Agar Kobe I</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>5210.2</td>
			<td>NGM component</td>
		</tr>
		<tr>
			<td>Agarose, Universal Grade</td>
			<td>Bio &#38; Sell GmbH</td>
			<td>BS20.46.500</td>
			<td>Mounting slide component</td>
		</tr>
		<tr>
			<td>BD Bacto Peptone</td>
			<td>BD-Bionsciences</td>
			<td>211677</td>
			<td>NGM component</td>
		</tr>
		<tr>
			<td>Deckgl&#228;ser-18x18mm</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0657.2</td>
			<td>Cover slips</td>
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		<tr>
			<td>EC Plan-Neufluar 20x/0.50 Ph2 M27</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Objective</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ 1.52p</td>
			<td>NIH</td>
			<td></td>
			<td>Analysis Software</td>
		</tr>
		<tr>
			<td>Levamisole Hydrochloride</td>
			<td>AppliChem GmbH</td>
			<td>A4341</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>LSM710-ConfoCor3</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Laser Scanning Confocal Micoscope</td>
		</tr>
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			<td>Mounting stereomicroscope</td>
			<td>Leica Camera AG</td>
			<td></td>
			<td>Mounting microscope</td>
		</tr>
		<tr>
			<td>neuronal-HTTQ25-Dendra2</td>
			<td>this paper</td>
			<td></td>
			<td>C. elegans strain</td>
		</tr>
		<tr>
			<td>neuronal-HTTQ97-Dendra2</td>
			<td>this paper</td>
			<td></td>
			<td>C. elegans strain</td>
		</tr>
		<tr>
			<td>OP50 Escherichia coli</td>
			<td>CAENORHABDITIS GENETICS CENTER (CGC)</td>
			<td>OP50</td>
			<td>Nematode food source</td>
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			<td>Sodium Chloride</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>3957.2</td>
			<td>NGM component</td>
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			<td>Standard-Objekttr&#228;ger</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0656.1</td>
			<td>Glass slides</td>
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		<tr>
			<td>ZEN2010 B SP1</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Confocal acquisition software</td>
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in vivo quantification of protein turnover in aging c. elegans using photoconvertible dendra2

蛋白质在细胞内不断合成和降解，以保持平衡。能够监测感兴趣的蛋白质的降解，不仅是了解其生命周期的关键，也是发现蛋白质组网络中的不平衡的关键。此方法演示如何跟踪致病蛋白亨廷顿蛋白的降解。与Dendra2融合的两种亨廷顿蛋白在C.elegans神经系统中表达：一种是生理版本，另一种是含有扩大和致病性的谷氨酰胺。Dendra2是一种光可转换荧光蛋白;在短的紫外线（UV）辐照脉冲下，Dendra2将其激发/发射光谱从绿色切换为红色。与脉冲追逐实验类似，可以监控和量化转换红-Dendra2的周转率，而不管新合成的 green-Dendra2 的干扰。使用基于共体显微镜的，由于C.elegans的光学透明度，可以监测和量化亨廷顿-Dendra2在活体，老化的生物体的降解。神经元亨廷顿蛋白-Dendra2在转换后很快部分退化，并随着时间的推移进一步清除。控制降解的系统缺乏突变亨廷顿蛋白的存在，并且随着老化而进一步受损。同一神经系统内的神经元亚型表现出不同的转液能力，用于亨廷顿-登德拉2。总体而言，监测与Dendra2融合的任何感兴趣的蛋白质，不仅可以提供重要信息，包括其降解和所涉及的蛋白酶网络的玩家，而且可提供有关其位置、贩运和运输的重要信息。

通过微注射获得两种线虫菌株，在框架中表达亨廷顿-1蛋白片段，并结合光可转换蛋白Dendra2获得，质粒作为染色体外阵列保存。融合结构从整个老化过程中在整个C.elegans神经系统中表达。在这里，HTT-D2 包含生理 25 多聚聚聚三聚胺拉伸 （HTTQ25-D2，图 1A ） 或完全青素和致病性重复与 97 谷氨酰胺 （HTTQ97-D2，图 1</strong...

Watch this Scientific Journal Video about In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2 at JoVE.com

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

这里介绍的是一个协议，用于监测蛋白质亨廷顿蛋白的降解，该蛋白与光可转换的荧光素Dendra2有关。

使用光可转换的登德拉2，在老化的蛋白质周转量中，在体内C.Elegans

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is ...

该协议允许跟踪和定量对活和老化的C.elegans感兴趣的蛋白质的降解。这种体内和非侵入性技术只需要很少的显微镜设置，以可靠地将Dendra2转化为明亮和稳定的荧光。该方法适用于哺乳动物系统以及斑马鱼。研究，把营业额在原型统计网络以及运输和贩运。使用蓝光激光扫描时，注意不要引发不需要的 Dendra2 转换，并测试和优化每个系统和融合蛋白的采集设置和转换设置。从在20摄氏度下将年龄匹配的线虫长到实验所需的年龄开始。在成像实验当天，在双蒸馏水中以3%浓度熔化一般品位的阿加罗斯。当气糖冷却时，切开一毫升移液器尖端，以方便吸入大约 400 微升的熔化气液。小心地在干净的玻璃滑梯上添加几滴阿加糖，并立即将另一张幻灯片放在水滴的顶部，在两个玻璃片之间形成一个薄薄的阿加糖垫。当加糖干燥时，小心地拆下顶部滑轨。准备好足够的幻灯片后，打开共和显微镜软件，并设置激发和发射光路。对于绿色 Dendra2 添加 486 到 553 纳米，对于红色 Dendra2 添加 580 到 740 纳米。根据荧光团的强度调整两个通道的功率和增益，将针孔完全打开。选择顺序通道模式并设置轨道以切换每个帧。将扫描模式设置为帧，帧大小设置为 1024 by 1024，线步数为 1。将平均值设置为 2，通过单向线的均值方法和模式将平均值设置为平均值。将位深度设置为 8 位。定义用于 Dendra2 转换的多维采集设置。对于转换和漂白，请使用 405 纳米二极管激光器设置为 60% 的能量功率。选择两个周期的时间序列，周期之间间隔为 0.0 毫秒，并且正常启动和停止。将漂白设置为扫描后开始两个之一，重复 30 次迭代，并在强度降至 50% 时停止，然后，定义采集像素停留速度以快速进行转换，并定义介质以捕获快照图像。要将线虫安装到幻灯片上，请使用永久标记绘制每个幻灯片上的 agarose 垫对面的窗口，并编号四个方块。将 15 微升的 levamisole 添加到 agarose 垫的中间，并使用立体显微镜和线挑将年龄匹配的线虫转移到液滴中。使用睫毛选择轻轻地将每个线虫移动到正方形的一个窗口中。当所有线虫都重新定位后，等待蠕虫停止移动，然后再在液体上放置盖滑，以在成像过程中固定线虫。然后，将幻灯片放在共合显微镜舞台上。对于绿色 Dendra2 转换，请使用绿色荧光下 20 X 目标的目镜定位第一线虫并聚焦在头部或尾部。切换到共和模式，增加或减少增益和激光功率，以获得饱和但不会过度曝光的图像。在所选神经元周围绘制一个感兴趣区域，并在尾部周围绘制第二个较大的感兴趣区域，其中包括第一个感兴趣区域。设置要获取、漂白和分析的第一个区域。设置要获取和分析的第二个感兴趣区域，但不得漂白。将扫描速度设置为最大值，并开始扫描以转换选定的 Dendra2 神经元。转换后，立即开始使用绿色 561 纳米激光进行实时扫描，以在红色通道中可视化 Dendra2，并找到转换神经元的焦点和最大投影。快速将扫描速率设置为较低的像素驻用速度，以更高的分辨率获取两个通道的快照图像。此图像在转换后的时间为零。然后，使用可识别的名称和或数字保存扫描，然后保存时间零标签。要跟踪 Dendra2 随时间而退化，请打开相应线虫神经元的时间零图像。使用相同的图像设置，开始实时扫描红色通道，并将转换后的红色神经元聚焦。然后，在不更改任何采集参数的情况下，以与第一个图像相同的速度获取快照。请务必仔细定义转换设置，以便高效、充分地转换 Dendra2，并在整个不同时间点中保持相同的采集参数。要分析转换后的 Dendra2 图像，请打开斐济的时间零点和第二个时间点图像，然后打开"分析和设置测量"。选择"面积"和"集成密度"功能，然后选择使用红色通道获得的图像。使用多边形选择工具，在零转换神经元的时间周围绘制一个感兴趣区域。要正确识别神经元的轮廓，请选择"图像、调整"和"阈值"以突出显示强度阈值。定义选择后，单击"分析和测量"。将显示一个弹出结果窗口，包括所选感兴趣区域的区域、集成密度和原始集成密度值。从第二个时间点对图像执行相同的选择和测量过程。然后，将值复制到电子表格中。在转换前的此代表性分析中，当样本在红色通道中兴奋时，看不到红色信号。辐照后，绿色信号随着HTT-D2的转换而减弱，随后出现红色信号。HTT-D2表达会随着时间而下降，导致红色HTT-D2信号水平降低，绿色HTT-D2信号可能随着新合成更多的融合蛋白而增加。值得注意的是，与头部神经元相比，尾部区域的神经元明显更活跃。此外，在转换后24小时，头部和尾部神经元的控制HTTQ25-D2明显退化。然而，在致病性HTTQ97-D2中未观察到这种差异，这表明蛋白位虫网络无法去除含有较长谷氨酸的HTT-D2在整个神经系统中延伸。HTTQ97-D2在非常古老的线虫中并没有像HTTQ25-D2那样有效降解，这突出地突出了蛋白虫网络无法去除较老动物中可能有毒的亨廷顿蛋白的聚合和可能有毒的物种。重要的是，尾部神经元能够应对年轻线虫中的有毒HTTQ97-D2，这表明各种伴随蛋白细胞网络机制可能正在工作，以消除HTT-D2。对于每个样本，在转换后立即获取和未过度曝光和非饱和图像，并在整个时间重复使用该样本的相同设置。有了这个协议，可以测试蛋白质统计网络的变化，还可以在超分辨率显微镜的帮助下，跟踪疾病相关蛋白质的传播。

Research

JoVE Journal

Biology

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

Antibody Staining in C. Elegans Using "Freeze-Cracking"

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Measurements of Physiological Stress Responses in C. Elegans

Measurements of Physiological Stress Responses in C. Elegans

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Measuring RAN Peptide Toxicity in C. elegans

Measuring RAN Peptide Toxicity in C. elegans

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...