Name: the microscopy-based assay to study and analyze the recycling endosomes using snare trafficking
Uploaded: 2022-02-12T15:00:00
Description: Watch this Scientific Journal Video about The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking at JoVE.com

这项工作得到了生物技术部的支持（BT / PR32489 / BRB / 10 / 1786 / 2019到SRGS）;科学与工程研究委员会（CRG/2019/000281给SRGS）;DBT-NBACD（BT/HRD-NBA-NWB/38/2019-20 至 SRGS）和 IISc-DBT 合作计划（至 SRGS）。该部门的基础设施得到了DST-FIST，DBT和UGC的支持。AMB 由 DBT-JRF （DBT/2015/IISc/NJ-02） 提供支持。

该协议涉及黑素细胞的接种，然后转染质粒。进一步的步骤包括固定，免疫染色，成像和分析细胞，以测量RE的长度和数量。该协议的详细说明如下。
1. 在预处理的盖玻片上接种小鼠黑素细胞
<ol><li>用基底膜基质培养基（在完全RPMI培养基中为1：20：20：1：20）涂覆在培养皿（即35毫米培养皿中4- 5个培养皿）中的玻璃盖玻片，并在组织培养罩中干燥15分钟。使用前...

<ol>
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回收内体是一组内吞细胞器，它们介导所有细胞类型中货物到细胞表面的回收21，22，23，24，25。在黑色素细胞等特殊细胞类型中，这些细胞器部分地将其运输路线转移到黑素体以进行生物发生3，16，26。?...

黑色素色素的生物合成发生在黑素体中，黑素体是一种与常规溶酶体共存的黑素细胞特异性溶酶体相关细胞器（LRO）。内吞系统在黑色素体的生物发生中起着关键作用，黑素体是肤色和防止电离辐射的光保护所必需的1，2，3。在此过程中，黑色素合成酶在早期/分选内体上被分选，然后通过称为循环内体（REs）的管状或囊泡内体运输到过早的黑素体4，5，6，7，8，9，10。这些细胞器的靶向和融合调节全功能色素沉着黑素体的成熟7，11，12，13，14。在Hermansky-Pudlak综合征中观察到，这些细胞器形成或对这些细胞器的货物分拣中的缺陷导致眼皮白化病和其他临床表型15，16。
在这里，我们描述了一种基于显微镜的简单技术来研究和分析RE。在这种方法中，我们利用了一种跨膜蛋白Qa-SNARE Syntaxin（STX）13，它存在于循环内体17上，并且在黑素细胞中分选内体和黑素体之间的循环12，18。此外，删除N端非结构化调节域（即SynN或STX13Δ129）允许SNARE卡在黑素体中，黑素体测量向黑素体的前向运输途径12。我们在研究中使用了已知的回收内体标志物Rab GTPase（Rab）11111111。对野生型黑素细胞中的蛋白质GFP-STX13WT，GFP-STX13Δ129，mCherry-Rab11和TYRP1进行荧光成像，然后定量其相对定位，除了它们靶向黑素体外，还将提供REs的性质和动力学。因此，这是一种简单的技术，可用于可视化和测量黑素细胞中RE的动力学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-TYRP1 antibody (TA99)</td>
			<td>ATCC</td>
			<td>HB-8704</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>ThermoFisher Scientific</td>
			<td>11668-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>022600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol-12-myristate-13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>ThermoFisher Scientific</td>
			<td>31800-022</td>
			<td></td>
		</tr>
	</tbody>
</table>

the microscopy-based assay to study and analyze the recycling endosomes using snare trafficking

回收内体（REs）是由所有细胞类型中的早期/分选内体产生的管状囊泡细胞器。这些细胞器在黑素体的生物发生中起着关键作用，黑素体是由黑素细胞产生的溶酶体相关细胞器。RE在形成期间将黑素细胞特异性货物输送到过早的黑素体。在Hermansky-Pudlak综合征的几个突变体中观察到的REs的产生阻塞，导致皮肤，头发和眼睛色素减退。因此，研究REs的动力学（参见数量和长度）对于了解这些细胞器在正常和疾病条件下的功能是有用的。在这项研究中，我们的目标是使用常驻SNARE STX13测量RE动力学。

STX13Δ129突变体定位到黑素体的定量分析 STX13在小鼠野生型黑素细胞中的免疫荧光显微镜显示，GFP-STX13WT定位为囊泡和管状结构，GFP-STX13Δ129定位为细胞表面外的环状结构（图1A）。此外，细胞内环状GFP-STX13Δ129显示出与黑素体蛋白TYRP1（图1A）和明场成像黑素体（数据未显示）的共定位<sup c...

Watch this Scientific Journal Video about The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking at JoVE.com

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

回收内体是内体管网络的一部分。在这里，我们提出了一种使用GFP-STX13作为细胞器标记来量化回收内体动力学的方法。

基于显微镜的测定，用于使用SNARE运输研究和分析回收内体

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

基于显微镜的测定，使用SNARE运输来研究和分析回收内体。这些是皮肤黑素细胞。你在细胞内看到的黑色细胞器被称为黑素体。黑素体被称为一类称为溶酶体相关细胞器或LRO的细胞器。回收内体是来自所有细胞类型的早期或分类内体的管状囊泡细胞器。这些细胞器在黑色素体的生物发生中起关键作用，黑素体是由黑素细胞产生的溶酶体相关细胞器。回收内体在形成过程中将黑素细胞特异性货物输送到过早的黑素体。在几种综合征中观察到的循环内体的产生是皮肤，头发和眼睛色素沉着过度的细胞。因此，研究循环内体的动力学有助于了解这些细胞器在正常和疾病条件下的功能。本研究旨在使用阻止SNARE语法in-13来测量循环内体的动力学。第一步是在预处理的盖玻片上接种小鼠黑素细胞。将玻璃盖玻片涂在基底膜基质介质的培养皿中。并在组织培养罩中干燥15分钟。使用前用1X PBS清洗盖玻片一次。在基底膜介质上接种细胞，以50%至60%的能力接种盖玻片。始终在完整的RPMI培养基中向电镀的细胞悬浮液中加入200纳摩尔的PMA。接种细胞后，将含有细胞的培养皿在CO2培养箱中孵育12至24小时。第二步是用语法蛋白-13质粒转染细胞。孵育含有DNA和转染试剂的吸头，混合五分钟，无需重复移液。在室温下，每10分钟用手敲击管子30分钟。在孵育期间，用1X PBS洗涤细胞两次，一次用OPTI-MEM洗涤细胞，然后向细胞中加入1密耳的OPTI-MEM。孵育30分钟后，通过覆盖培养皿以滴滴方式将转染试剂DNA混合物加入细胞中。将细胞在37摄氏度下孵育六个小时。用转染试剂吸出OPTI-MEM培养基，并加入补充200纳摩尔PMA的完整RPMI培养基。将细胞在37摄氏度下孵育48小时。第三步是细胞的固定。请注意，以下程序在组织培养罩外进行。转染48小时后，用1X PBS洗涤细胞两次，然后用3%甲醛固定细胞30分钟。固定后，用1X PBS洗涤细胞两次，并将盖玻片储存在1X PBS中直至进一步使用。或者，电池可以安装在玻璃板上或储存在四摄氏度。第四步是细胞的免疫染色。准备一个潮湿的腔室。将石膏膜切割片放在培养皿中的鼠标滤纸上。盖上铝箔。制备25微升一抗溶液。以1至200的稀释液加入抗体。将此溶液作为滴剂添加到潮湿室中的parafilm上。用镊子小心地抬起盖玻片。将其倒置在一抗染色液的滴上，然后盖上潮湿室的盖子。在室温下孵育30分钟。同样，以1至500的稀释度制备二抗溶液，并将其放在潮湿室盖玻片旁边的副膜上。为了染色细胞核，以120，000至130，000的稀释度向溶液中加入DAPI。使用镊子，小心地从一抗溶液中取出盖玻片，并将其浸入1X PBS中两次。轻触薄纸上的盖玻片，以清除盖玻片上多余的PBS。将其放在潮湿室中的二抗染色溶液上，并且由于溶液中存在荧光染色抗体，因此不要将其暴露在光线下。孵育后，小心地从二抗溶液中取出盖玻片，然后将其浸入1X PBS中两次。此外，点击薄纸上的盖玻片以除去盖玻片上多余的PBS。将12微升成像试剂放在载玻片上，并小心地将染色的盖玻片放在安装试剂上。倒置薄纸上的载玻片，然后轻轻按压。接下来，使用荧光显微镜对盖玻片进行成像，并使用ImageJ分析图像。第六步是定量回收内体局部蛋白质和黑素体之间的重叠。用黑素体定量语法蛋白-13 delta-129共定位。小鼠野生型黑素细胞中 syntaxin-13 的免疫荧光显微镜检查显示 GFP-syntaxin-13 野生型定位为囊泡和管状结构。GFP-syntaxin-13 delta-129除了细胞表面外，还定位环状结构。此外，细胞内环状GFP-syntaxin-13 delta-129在明场成像的黑素体中显示出与黑素体蛋白TYRP1的共定位。如前所述，在黑素体中观察到一组过度表达的GFP-syntaxin-13野生型。为了测量GFP-syntaxin-13野生型和GFP-syntaxin-13 delta-129对黑素体的相对定位，使用斐济软件并使用JACOP插件进行了分析。与具有TYRP1的GFP-syntaxin-13野生型相比，测量的Mander重叠系数（即MOC）与具有TYRP1的GFP-syntaxin-13 wild类型相比高出约1.5倍。有趣的是，与GFP-syntaxin-13 wild类型相比，TYRP1的GFP-syntaxin-13 delta-129的MOC值高出2.9倍。这些数据表明，在稳态下，GFP-syntaxin-13 delta-129黑素体的定位相对高于GFP-syntaxin-13野生型。定量synan-13野生型定位的回收内体。GFP-syntaxin-13野生型的免疫荧光显微镜检查显示与已知的循环内体蛋白Rab11共定位。与具有GFP-syntaxin-13野生型的mCherry-Rab11相比，具有mCherry-Rab11的GFP-syntaxin-13野生型之间的MOC大约高出1.4倍。为了测量GFP-syntaxin-13野生型阳性内体小管的数量和长度，使用了斐济软件，如方案部分所述。mCherry-Rab11在实验中使用阳性对照。与表达mCherry-Rab11的细胞相比，转染GFP-syntaxin-13野生型的黑素细胞显示出每个细胞的肾小管数量更多。然而，当细胞中GFP-syntaxin-13野生型和mCherry-Rab11共表达时，肾小管减少。第七步是定量回收内体的管状数量和长度。当细胞中GFP-syntaxin-13野生型和mCherry-Rab11共表达时，肾小管减少。有趣的是，GFP-syntaxin-13野生型和mCherry-Rab11的平均肾小管长度在单独或一起表达的细胞中彼此相当。总之，这些数据表明GFP-syntaxin-13野生型定位为与Rab11相似的回收内体。这项研究表明，syntaxin-13突变体的N端递送，即GFP-syntaxin-13 delta-129，定位于黑素体GFP-syntaxin-13 delta-129可以用作从循环内体到细胞表面和LRO的水泡运输的报告。相比之下，GFP-syntaxin-13野生型定位为与Rab11相似的循环内体。本研究表明，GFP-syntaxin-13野生型也可用于标记黑素细胞中的循环内体。GFP-syntaxin-13野生型可能比Rab11更好地回收内体标记，因为Rab11改变了内体动力学。总而言之，GFP-syntaxin-13野生型作为潜在的回收内体标记，以研究稳态条件下的动力学。

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

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 ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...