Name: application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria
Uploaded: 2015-01-02T14:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1.合成10nm的纳米金（金纳米粒）15 <ol><li>准备解决方案答:加2毫升1％水溶液氯化金成160毫升的Milli-Q，或双蒸水。 </li><li>制备溶液B:加入8毫升1％柠檬酸三钠×2的H 2 O和160微升1％的单宁酸成32毫升的Milli-Q，或双蒸水。 </li><li>热身方案A和B到60°C和混合他们同时进行搅拌。立即观察到深蓝色。约15分钟后，观察到红色。然后升温至95℃，保持5分钟，冷却溶液至室温。 </li><li>添加一滴的NP悬浮到碳包覆的网格并允许在空气中干燥。检查纳米粒的通过透射电子显微镜的大小和形态。 </li></ol> 2.涂料黄金纳米粒子与BSA和标签罗丹明N-羟基酯（NHS）16 <ol><li>添加900微升金纳米粒到1.5ml的Eppendorf管中，离心机以15000×g离心30分钟。欧普倚重，准备多个管可以一次。 </li><li>弃去上清液，重新悬浮沉淀在900微升灭菌的Milli-Q水，并添加100微升2毫克/毫升BSA / PBS中，混合在Vortex以800rpm进行30分钟。 </li><li>以除去过量的BSA，离心制备以15,000×g离心60分钟。 </li><li>弃去上清液，并重新暂停金纳米粒BSA在125微升PBS，加入12.5微升1M的碳酸氢盐。 </li><li>在即将使用前，制备10毫克/毫升的溶液罗丹明NHS / DMSO中，加入30微升罗丹明NHS / DMSO溶液至1ml金 - 牛血清白蛋白纳米粒混悬液。孵育2小时（或O / N）在室温时800转混合反应，避免暴露在光线下。 </li><li>通过净化透析对PBS的黄金BSA罗丹明纳米粒在4ºC超过36期5缓冲变化 - 48小时。 </li><li>为了稳定纳米颗粒，加10微升的200毫克/毫升BSA / PBS到每个1毫升金 - 牛血清白蛋白 - 若丹明纳米粒。以除去游离的或释放BSA的若丹明，离心机15，000 XG 60分钟。重悬沉淀在2毫克/毫升BSA / PBS，测量OD 520，并储存在4℃下，以避免暴露于光。 </li><li>稀释的纳米颗粒10次用Milli-Q或双蒸水，加一滴到碳包覆的网格，并允许在空气中干燥。检查纳米粒的大小和形态由透射电子显微镜（TEM）。 注意:用于NP制剂的所有材料进行灭菌和操作被在细胞培养罩或在火焰旁长凳进行。 </li></ol> 3.文化HeLa细胞<ol><li> HeLa细胞永久表达LAMP1-GFP在DMEM中培养，用10％胎牛血清（FCS），在37℃下在含有5％的CO 2的气氛中生长。 </li><li>种子细胞在50000每孔的密度在8孔室滑动（Ibidi）和孵化O / N。 </li></ol>细菌4.文化<ol><li>使用沙门伤寒NCTC 12023 WILd型（WT）菌株。为了便于比较，使用突变株ΔSSAV缺陷在SPI2-T3SS和Δ 西法缺乏关键效应西法为SIF。使用的菌株窝藏质粒pFPV25.1增强GFP的组成型表达。 注:细菌菌株是常规培养在LB肉汤（LB）并加入50微克/毫升羧苄青霉素（WT）和LB并加入50微克/毫升羧苄青霉素和/或50微克/毫升卡那霉素（ΔSSAV，ΔSIFA ）的要求保持质粒。 </li><li>接种细菌的单个菌落3毫升的LB用适当的抗生素和摇动的条件下进行曝气，在37ºC生长的O / N，则稀释1点31分在新鲜LB，并继续生长3.5小时。在这个时间点，将培养达到晚期对数期和细菌是高度侵入。 A&#39;辊筒&#39;方便孵化试管培养与通风。 </li></ol> 5.通过感染HeLa细胞沙门氏菌和金BSA-罗丹明纳米粒脉冲大通标签（见图1计划） <ol><li>测量子培养细菌的OD 600，并稀释至OD 600 = 0.2的1ml的PBS（〜3×10 8 CFU / ml）的，在8孔室玻片中添加细菌HeLa细胞的适当的量来实现的多个感染的100（MOI）。 </li><li>孵育在细胞培养箱30分钟，洗3次，用PBS以除去未内在化的细菌（该时间点被设定为0小时后的感染，或0小时圆周率）。加入300微升新鲜培养基含有100μg/ ml庆大霉素和维持1小时。然后用含10微克/毫升庆大霉素的温育时间，其余的新鲜培养基更换培养基。 </li><li>孵育与含有100微克/毫升庆大霉素1小时培养基后，将培养基替换为成像介质（鹰MEM不含FCS，L-谷氨酰胺，酚红和碳酸氢钠，用30毫米的HEPES，pH值7.4）共ntaining 10微克/毫升庆大霉素。金-牛血清白蛋白-若丹明纳米粒加入到HeLa细胞中，以获得外径520 = 0.1的终浓度。 注:金 - 牛血清白蛋白 - 若丹明纳米粒也可加入到细胞中感染之前，或在不同的时间点PI </li><li>经过1小时温育后，除去介质，洗3次，用PBS中，并加入300微升含有10％FCS和10μg/ ml的庆大霉素的温育时间的其余部分新鲜成像介质。 注:温育持续时间可以根据所用NP和细胞系的浓度而变化。对于RAW264.7巨噬细胞，我们观察到，30分钟孵化与纳米粒子在OD 520 = 0.05的允许浓度足够的内在。 </li></ol> 6.成像<ol><li>使用共焦成像系统，例如共焦激光扫描（CLSM）或旋转式圆盘（SD）的显微镜用的潮湿环境室，用于高分辨率成像在不同的时间点。 </li><li>开关上的温度下ONTROL系统和等待，直到它是稳定的。优化成像设置，例如放大，扫描速度，分辨率，Z-栈等使用适当的激发/发射设置GFP和金BSA-罗丹明纳米粒。对于这个协议，使用400 Hz的扫描速度，分辨率为512×512像素和0.25微米的Z-步长。激发GFP和使用Ar激光（488纳米）和氦氖激光器（543纳米），分别金 - 牛血清白蛋白 - 若丹明。包括一个亮场（BF）信道用于观察细胞的形状。对于其他显微镜系统和感染的条件下，设置有作相应的调整。 注:使用相同的Ar激光作为高炉信道和信号的光源与光电倍增（PMT）检测器进行检测。 </li><li>在指定的时间点PI，包含安装在显微镜舞台和影像记录受感染的细胞的8孔玻片。 </li></ol>图像7.分析<ol><li>采用显微图像分析软件（见<stron克&gt;材料和设备的数据表）通过细胞内沙门氏菌分析内溶酶体系统的重塑。通过点击"打开"，然后选择文件打开数据。 注:另外，开源软件如ImageJ的或FIJI可用于定量图像分析。 </li><li>在图标上超越视图中点击工具栏上的物体<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" />以添加新的表面项目。点击<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （下一个）。 </li><li>为了分析的感兴趣区域（ROI）的区域，选择一个细分的投资回报率。在一个可视面积，覆盖在图像矩形边框部分代表的投资回报率。在相应的X轴输入值，y-和z-域，或者直接点击箭头在预览矩形来修改大小和ROI的位置。然后点击<img alt="图标" fo:content-width=".1in" src<html> ="/文件/ ftp_upload / 52058 / 52058icon2.jpg"/&gt;（下一个）。 </li><li>作为源通道，选择频道3（信号金BSA-罗丹明NPS）。检查平滑选项来设置所得到的区域的平滑度。手动定义的值或接受自动生成的值。对于阈值，选择绝对强度的选项。 </li><li>对于阈值的调整，选择手动选项，并设置一个值。在可视面积，表面门槛预览显示为灰色。 </li><li>上的标签分类曲面所得表面可通过各种标准来过滤。默认过滤器是，和其他过滤器"体素&gt; 10号"也可以包括通过点击"添加"。如果一个过滤是没有必要的，然后通过点击删除按钮删除所有过滤器。点击<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （下一个）。 </li><li>要完成表面创建，点击/files/ftp_upload/52058/52058icon3.jpg"/&gt;（完成）。在对象列表，目前未检查的项目量和新创建面框显示在可视面积。 </li><li>出口统计数据。现在，在超越视图根据其区域中物体的颜色变化从紫色到红色。和"情节数字区"表显示的统计信息（ 如身份证号码和对象的面积）。出口由点击Excel文件表面1所有统计<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> （保存）。 </li></ol>

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No conflicts of interest declared.

哺乳动物细胞内溶酶体系统控制重要的生理过程，包括营养吸收，激素介导的信号转导，免疫监视和抗原呈递17。到现在为止，各种标记已经用于内吞途径和跟踪研究的标记。例如，LysoTracker探针是由Molecular Probes公司（Life Technologies公司，美国）为溶酶体标记开发荧光acidotropic探针，其可以选择性地积聚在细胞区室与低内pH和有效地在纳摩尔浓度18标记的活细胞。然而，LysoTracker探针...

荧光纳米粒子（纳米粒子），包括金属纳米粒子，量子点，聚合物纳米颗粒，二氧化硅纳米颗粒，碳圆点， 等等 ，都在过去几十年1,2-引起了相当的重视。相比传统的有机染料，荧光纳米颗粒显示出理想的化学，光学和机械性质，如强的信号强度，耐漂白和高生物相容性3,4。这些优势使他们的首选细胞内检测和活细胞成像的方法。此外，各种电子致密纳米粒子是通过电子显微镜（EM）可见，促进它们的使用为相关显微分析，这使得活细胞在光学显微镜（LM）和更高的分辨率跟踪在超微结构水平使用EM 5的组合。例如金纳米粒已经有效地用作生物传感器在活细胞中对敏感的诊断，以及在免疫标记6的字段长的时间。近期s案例研究表明，金纳米粒具有不同的尺寸和形状可容易地通过大量的各种细胞系摄取并通过内体途径常规输送，因此有很大的潜力被施加于细胞内囊泡运输跟踪和内溶酶体系统贴标7,8- 。 微生物病原体，如沙门氏菌 ， 志贺氏菌和李斯特菌 ，开发不同的机制侵入非吞噬宿主细胞9。被内化后，病原体，无论是定位于细胞质或螯合在膜结合隔间，广泛的互动与他们的主机环境和调节这些有利于自己的生存10。例如， 沙门氏菌驻留并复制感染11后，在细胞内的吞噬体舱称为含沙门氏菌的空泡（SCV）。成熟SCV贩卖朝向高尔基体，进行与内吞途径连续的相互作用，并诱导形成广泛的管状结构，例如沙门氏菌诱导长丝（SIF），分拣连接蛋白肾小管， 沙门氏菌诱导分泌载体膜蛋白3（SCAMP3）小管等12-14。研究这些细菌病原体如何操纵宿主细胞途径是必不可少的理解传染病。 这里，金-牛血清白蛋白-若丹明纳米粒被用作流体示踪剂标记的宿主细胞内溶酶体系统，以及人体胃肠病原体沙门鼠伤寒（ 沙门氏菌 ）用作模型细菌来研究所述病原体与相互作用主机内吞途径。在非感染细胞和感染的细胞与WT 沙门氏菌或突变株细胞内的金-牛血清白蛋白-若丹明纳米粒通过共焦激光扫描显微镜（CLSM）成像。然后了Imaris软件用于定量纳米粒的分布，表明沙门氏菌感染引起的内涵体/溶酶体极端重排。下面该方法的描述中，类似的实验，可以设计成跟踪内在化纳米颗粒的长期命运，并调查各外源物质或内源性因子对真核细胞的内吞途径的影响。

<table><tbody><tr><td>Gold chloride</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Tannic acid</td><td>Sigma-Aldrich</td><td>403040</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Sigma</td><td>C8532</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>NHS-Rhodamine</td><td>Pierce</td><td>46406</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Gentamicin</td><td>Applichem</td><td>A1492</td><td></td></tr><tr><td>Kanamcyin</td><td>Roth</td><td>T832</td><td></td></tr><tr><td>Carbenicillin</td><td>Roth</td><td>6344</td><td></td></tr><tr><td>8-well chamber slides</td><td>Ibidi</td><td>80826</td><td>tissue culture treated, sterile</td></tr><tr><td>Imaris Software</td><td>Bitplane</td><td>version 7.6</td><td>various configurations available</td></tr></tbody></table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

荧光纳米粒子（纳米粒子）具有理想的化学，光学和机械性能是有希望的工具来标记胞内细胞器。在这里，我们介绍利用金-牛血清白蛋白-若丹明纳米粒标记的真核细胞的内溶酶体系统以及由细胞内病原体沙门氏菌监测的宿主细胞途径的操纵的方法。该纳米粒子易于被HeLa细胞内化和本地化下旬内涵体/溶酶体。 沙门氏菌感染引起的沙门氏菌中引起膜结构的囊泡和纳米颗粒的积累重排。我们部署的共聚焦显微镜图像定量分析的了Imaris软件包。对象的数目和在非感染细胞的大小分布是从那些在沙门氏菌 -感染细胞明显，指示由WT 沙门氏菌极其重塑内溶酶体系统。

通过完善的通过由柠檬酸和鞣酸减少氯金酸的方法生成的黄金纳米粒子。 如图2A所示 ，合成的金纳米粒是准球形的形状具有大约10nm的尺寸。 BSA的涂层和罗丹明-标记不影响它们的形态或尺寸（ 图2B）。 已经报道了金纳米粒可容易地采取了由各种哺乳动物细胞和内吞系统7结束了。按照以前的作品，明亮的红色荧光信号，观察在HeLa细胞后1小时?...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

本文介绍了合成和纳米粒子的荧光标记（NPS）的方法。所述纳米颗粒是在脉冲追踪实验施加标记的真核细胞的内溶酶体系统。内溶酶体系统的细胞内病原体沙门氏菌的活动操纵其次是活细胞成像和量化。

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  Universität Osnabrück. 本文介绍了合成和纳米粒子的荧光标记（NPS）的方法。所述纳米颗粒是在脉冲追踪实验施加标记的真核细胞的内溶酶体系统。内溶酶体系统的细胞内病原体沙门氏菌的活动操纵其次是活细胞成像和量化。 

视频: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

培养细胞功能化纳米化对跨 golgi 网络的内分泌吸收和逆行迁移分析

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

科研

生物化学

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

免疫与感染

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

神经科学

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

生物学

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

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

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

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 ...

An Introduction to Endocytosis and Exocytosis

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become defective. This video will first briefly review a few pivotal discoveries in the history of endo- and exocytosis research. Next, some key questions will be examined, followed by a discussion of the prominent methods used to investigate these problems, including cell labeling, fusion assays, and fluorescence imaging. Finally, it will explore current research being conducted by scientists in the field today.

Endocytosis and Exocytosis; Clathrin Coated Pits and SNAREs

内吞作用和胞吐过程简介

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes ...

教育

JoVE Science Education

高级生物学

细胞生物学

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app.jove.com/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

使用荧光成像可视化神经元中的囊泡运动

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...