Name: using caenorhabditis elegans to screen for tissue-specific chaperone interactions
Uploaded: 2020-06-07T14:00:00
Description: Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

我们感谢由NIH国家研究中心（NCRR）资助的卡诺哈布迪炎遗传学中心，感谢其一些线虫菌株。爱泼斯坦H.F.开发的单克隆抗体来自由NICHD主持、由爱荷华大学生物系维护的发展研究杂交瘤库。这项研究得到了以色列科学基金会的赠款（第278/18号赠款）和以色列科技部以及意大利共和国国家促进总局外交和国际合作部的赠款（第3-14337号赠款）的支持。我们感谢本-兹维实验室的成员帮助编写这份手稿。

1. 为 RNAi 准备线虫生长介质板
<ol><li>在 1 L 瓶中，加入 3 克 NaCl、2.5 克巴克托-佩普顿、17 克醋和蒸馏水至 1 L 和高压灭菌液。</li>
	<li>冷瓶至 55 °C。</li>
	<li>加入 25 mL 的 1 M KH2PO4、 pH 6.0、 1 mL 的 1 M CaCl2、 1 mL 的 1 M MgSO4和 1 mL 的胆固醇溶液 （表 1）来制作线虫生长介质 （NGM）。</li>
	<li>加入1 mL的安培素（100毫克/毫升）和0.5兆升的1M IPTG（表1），...

一个反映它是如何组织起来的，并在不同的元子细胞和组织中发挥作用的蛋白石网络的综合图片仍然缺乏。为了解决这一缺陷，需要提供关于该网络各组成部分（如分子伴郎）在发育和老化过程中特定组织中相互作用的具体信息。在这里，我们展示了组织特异性扰动的使用如何使我们能够检查给定组织中的伴郎网络。为了探索组织特异性伴郎遗传相互作用，考虑了三种不同的方法。在第一种方法?...

细胞通过使用质量控制机器来修复、封存或去除任何受损的蛋白质1，2来应对蛋白质的损害。蛋白质复合物的折叠和组装由分子伴奏支持，这是一组高度保存的蛋白质，可以修复或封存受损的蛋白质3，4，5，6，7。去除受损蛋白质由无处不在的蛋白系统（UPS）8或自噬机械9与伴奏10，11，12合作进行调解。因此，蛋白质平衡（蛋白石）是由折叠和降解机3，13组成的质量控制网络维持的。然而，了解体内蛋白石网络各组成部分之间的相互作用是一项重大挑战。虽然蛋白质-蛋白质相互作用屏幕有助于提供关于物理相互作用和伴郎复合物的重要信息14，15，了解组织特定的伴郎网络在体内的组织和补偿机制是缺乏的。
基因相互作用经常被用作一种强大的工具，用来研究参与共同或补偿性生物通路16、17、18的基因对之间的关系。这种关系可以通过结合一对突变和量化一个基因突变对第二个基因16突变引起的表型严重性的影响来测量。虽然大多数此类组合在表型方面没有表现出任何效果，但某些遗传相互作用可能加剧或减轻所测量表型的严重程度。当双删除突变体的表型比组合单个删除突变体时看到的预期表型更严重时，观察到加重突变，这意味着两个基因在平行通路中工作，共同影响给定函数。相比之下，当双删除突变体的表型不如单个删除突变体中出现的表型严重时，则观察到缓解突变，这意味着两个基因作为一个复合体协同作用或参与同一通路16、18。因此，可以量化的多种表型，包括广泛的表型，如致命性、生长率和育雏大小，以及特定表型（如转录记者）已用于识别遗传相互作用。例如，Jonikas等人依靠ER应激记者使用配对基因删除分析19来检查糖核酸细胞ER展开蛋白反应蛋白体网络的相互作用。
基因相互作用屏幕涉及系统地交叉配对删除突变，以产生一套全面的双突变体20。然而，在动物模型中，特别是在C.elegans中，这种大规模的方法是不可行的。相反，突变菌株可以通过使用RNA干扰（RNAi）21对基因表达进行向下调节来测试其遗传相互作用模式。C. elegans是一个强大的系统，基于 RNAi22，23屏幕。在C.elegans中，双链RNA（dsRNA）的传递是通过细菌喂养实现的，导致dsRNA分子扩散到许多组织。通过这种方式，引入的dsRNA分子通过快速和简单的程序21影响动物。因此，使用RNAi的基因相互作用屏幕可以揭示使用RNAi库24对一组基因或大多数C.elegans编码基因进行下调节的影响。在这样的屏幕上，影响感兴趣的突变体的行为，但不是野生类型菌株的命中是被监测的表型的潜在修饰剂25。在这里，我们应用突变和RNAi筛选的组合，系统地绘制C.elegans组织特异性伴郎相互作用的地图。

<table>
	<tbody>
		<tr>
			<td>12-well-plates</td>
			<td>SPL</td>
			<td>BA3D16B</td>
			<td></td>
		</tr>
		<tr>
			<td>40 mm plates</td>
			<td>Greiner Bio-one</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm plates</td>
			<td>Greiner Bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well 2 mL 128.0/85mm</td>
			<td>Greiner Bio-one</td>
			<td>780278</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGA03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Formedium</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Sigma</td>
			<td>BO126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck</td>
			<td>1.02382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Qimaging</td>
			<td>q30548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Amresco</td>
			<td>0433-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Leica</td>
			<td>DM5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m)</td>
			<td>Sigma</td>
			<td>SCGPUO2RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Leica</td>
			<td>MZ165FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Frutarom</td>
			<td>2355519000024</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Formedium</td>
			<td>367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>1.04873.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Bio-Lab</td>
			<td>001649029100</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>22189-08-8</td>
			<td>Gift from the Morimoto laboratory</td>
		</tr>
		<tr>
			<td>Myosin MHC A (MYO-3) antibody</td>
			<td>Hybridoma Bank</td>
			<td>5-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Sigma</td>
			<td>s-0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio-Lab</td>
			<td>001903029100</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td>61930705001730</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate pouring pump</td>
			<td>Integra</td>
			<td>does it p920</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAi Chaperone library</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>VWR Life Science</td>
			<td>0837-500</td>
			<td></td>
		</tr>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Bio world</td>
			<td>41300000-1</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Duchefa Biochemie</td>
			<td>64-75-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Bio-Lab</td>
			<td>002009239100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

using caenorhabditis elegans to screen for tissue-specific chaperone interactions

蛋白质和蛋白质复合物的正确折叠和组装对细胞功能至关重要。细胞采用质量控制途径，纠正、封存或消除受损蛋白质，以维持健康的蛋白质组，从而确保细胞蛋白石和防止进一步的蛋白质损伤。由于蛋白酶网络中的多余功能，使用多细胞生物 体Caenorhabdd炎的 伴随编码基因的敲击或突变来筛选可检测表型，在大多数情况下，可检测到小或无表型。我们制定了有针对性的筛选策略，以确定特定功能所需的陪护人员，从而弥合表型和功能之间的差距。具体来说，我们使用RNAi合成交互屏幕监测新的伴郎互动，敲击伴郎表情，一次一个伴郎，在携带伴郎编码基因突变或过度表达兴趣伴郎的动物中。通过破坏两个单独呈现无严重表型的陪护，我们可以识别在两者都扰动时加重或暴露特定表型的陪护。我们证明，这种方法可以识别出特定的伴郎组合，这些伴郎可以协同工作，调节与给定表型相关的蛋白质或蛋白质复合物的折叠。

分别使用 UNC-45 中的温度敏感突变来筛选在允许或限制条件下加重或减轻相互作用的情况 肌肉组装和维护提供了一个有效的系统来研究组织特定的伴郎相互作用。收缩肌肉的功能单元，肉瘤，呈现一个水晶般的结构和调节蛋白排列。运动蛋白肌素的稳定性及其融入收缩肌瘤的厚丝取决于伴郎和UPS组件30的合作。一个例子，这样的陪护是保存和专门肌素伴奏UNC-45，主...

Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

为了研究伴奏和伴奏基底相互作用，我们使用RNA干扰与轻度突变或伴郎过度表达相结合，在有机体水平上监测组织特异性蛋白质功能障碍，在 卡诺哈布德炎中 执行合成相互作用屏幕。

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

The overall goal of the following experiment in genetically trackable design Caenorhabditis elegans is to identify tissue-specific chaperone interactions. The main advantage of this technique is that it combines easy to use assays such as fitting Golan AI and behavioral readouts to expose novel tissue specific interactions. For a non-stressful embryo synchronization, use a worm pick to move about 100 eggs from an unsynchronized worm plate to a newly seated NGM plate and cultivate the animals according to the appropriate experimental schedule.When the animals reach the first day of egg-laying, slowly add one milliliter of M9 buffer to the plate, distant to the bacterial lawn, and rotate the plate so that the buffer completely covers the plate. Tilt the plate to one side to remove the liquid and wash the animals from the plate. When all of the worms have been washed from the plate, use a standard plastic pipette tip to cut a square of Agar around where the eggs are concentrated and place the piece of Agar onto a newly seated NGM plate.Cultivate the eggs under the same culture conditions. At the end of the incubation, the plate should be covered with synchronized eggs. After washing all of the animals from the plate as just demonstrated, add one milliliter of fresh M9 buffer and use a cell scraper to release the eggs from the plate.When all of the eggs have been detached, transfer the entire volume of buffer from the plate into a conical tube for centrifugation. Centrifuge the tube and discard the supernatant. After removing the supernatant, add up to one milliliter of fresh M9 buffer to the plate and pipette to resuspend the eggs.Wash the eggs five more times as just demonstrated. After the last wash, the egg pellet should appear white and all but the last 200 microliters of supernatant can be removed. To cultivate synchronized nematodes for an experiment, place a drop of about 30 eggs close to the bacterial lawn and each interference RNA seated plate and about 30 eggs onto a plate seated with empty vector containing bacteria.Then cultivate the animals under the appropriate culture conditions according to the experimental protocol. To assess the embryonic lethality of the synchronized animals, when the animals begin to lay eggs, transfer about 100 eggs to an empty plate and spread the eggs into rows to simplify the counting. After 24 to 48 hours, score the percent of unhatched eggs.For reference, compare the experimental animal eggs to the eggs of animals treated with empty vector control bacteria. To set up a paralysis assay, cultivate age-synchronized animals on empty vector control bacteria, until the animals reach adulthood, but before egg-laying starts. Use a fine marker to draw a line on the back of a regular NGM Agar plate, and place five to ten animals on the marked line.Set a timer for 10 minutes and score the percentage of animals remaining on the line as paralyzed worms. For reference, compare this data to the percentage of mutant animals grown on empty vector control bacteria. For Protein Knockdown Validation, place 250 to 300 synchronized eggs onto a 60 millimeter NGM interference RNA plate seated with the relevant double stranded RNA expressing or empty vector containing bacteria, and cultivate the eggs for the appropriate experimental period.At the end of the incubation, transfer a total of 200 young adult animals into the cap of a 1.5 milliliter tube containing 200 microliters of PBST maintained at the animals cultivation temperature. Close the cap carefully, and centrifuge the animals at 1000 times G for 1 minute. At the end of the centrifugation, add 800 microliters of fresh PBST to the tube and centrifuge the worms again.Carefully remove the top 900 microliters of the supernatant, and wash the nematodes in fresh PBST 3 more times as just demonstrated. After the last wash, remove all but the last 100 microliters of the supernatant and add 25 microliters of 5X sample buffer to the animals. Heat the samples for 10 minutes at 92 degrees Celsius and 1000 revolutions per minute, before loading 20 microliters of each sample onto an SDS page gel.Then perform Western blot analysis using the appropriate antibodies to determine the relative stability of the protein of interest, and use densitometric software to determine the intensity of the bans. HSP6 knocked down during development results in a strong developmental arrest in wild type animals. At the same time, HSP6 knocked down in a muscle specific interference RNA strain expressing wild-type RDE1 in muscle tissue does not cause developmental arrest.Whereas HSP6 knocked down an intestine specific RNAI strain expressing wild type RDE1 in intestinal cells, results in a strong developmental arrest and phenotype. A mutation in HSP6 MG585 that causes a mild growth delay, can, therefore, be used to screen for aggravating or alleviating chaperone interactions by crossing a strain carrying that mutated gene into an intestinal specific interference RNA strain, and screening the chaperone interference RNA library. Myosin organization and UNC45 mutant animals treated with STI1 AHSA1 or DAF41 interference RNA, is similar to that of wild type worms at the fourth larval stage.Although the mutants exhibit disrupted sarcomeres after reaching adulthood. In contrast, both UNC45 mutant animals treated with an empty vector control and wild type animals remain unaffected. Genetic interactions are not indicative of physical interaction.Thus, follow-up experiments using genetic interaction screen must be performed to validate and directly examine the nature of the identified interactions.

Research

JoVE Journal

Biology

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

科研

生物学

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

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 ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Automating Aggregate Quantification in Caenorhabditis elegans

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用