Name: imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum
Uploaded: 2011-09-20T12:00:00
Description: Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

这项工作是由国立卫生研究院从NIAID的，壁间基金的支持。

1。 粘菌的趋化主管细胞的制备<ol><li>要生成四discoideum细胞趋化的趋化营，收获细胞增长，从一个惊天文化D3 - T的富媒体在22 ° C。 </li><li>非营养发育缓冲区清洗细胞两次（DB缓冲区含5毫米的Na 2 HPO 4，5毫米的KH 2 PO 4，MgCl 2的2毫米，0.1毫米氯化钙 2） 。 </li><li>重新暂停在DB缓冲区细胞密度在2 × 10 7细胞/ ml。 </li><li>摇在100转速在22 ° C 10毫升...

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深远的趋化细胞的主管阶段进程对于野生型D. discoideum细胞，大约需要5〜6小时脉冲在室温下的发展，诱导他们进入一个良好的趋化主管的阶段，在此期间，细胞中显示良好的极化的细胞形态和细胞快速迁移（图1）。几个因素，如cAMP的浓度为脉冲，温度和不同的遗传背景，可能会影响深远的趋化主管阶段的过程。一个营地梯度引导细胞走向cAMP的来源，细胞迁移的趋化指定...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> D3 - T的生长介质</td><td> KD医疗</td><td></td><td></td></tr><tr><td>咖啡因</td><td>西格玛</td><td></td><td></td></tr><tr><td> Latrunculin乙</td><td>分子探针</td><td></td><td></td></tr><tr><td> Alexa的594 </td><td>分子探针</td><td></td><td></td></tr><tr><td>腺苷</td><td>西格玛</td><td></td><td></td></tr><tr><td> ChronTrol XT的可编程定时器</td><td> ChronTrol公司</td><td></td><td></td></tr><tr><td> Miniplus 3蠕动泵</td><td> Gilbson </td><td></td><td></td></tr><tr><td>平台旋转摇床</td><td></td><td></td><td></td></tr><tr><td> FemtoJet microcapillary供给压力</td><td> Eppendorf公司</td><td></td><td></td></tr><tr><td>单和四以及实验室康二玻璃罩商会</td><td>国际Nalge NUNC </td><td></td><td></td></tr><tr><td> LSM 510 META或有同等作用的荧光显微镜</td><td>蔡司</td><td></td><td>一个40X 1.3 NA或60X 1.4无油DIC的计划NEOFLUAR物镜</td></tr><tr><td>奥林巴斯X81或同等学历</td><td>奥林巴斯</td><td></td><td>需要一个100X 1.47 NA TIRF物镜</td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

许多真核细胞中可以检测到在其环境中的化学信号的梯度迁移相应的 1 。这种引导细胞迁移被称为趋化作用，这是必不可少的各种细胞进行他们的功能，如免疫细胞和神经细胞2，3的图案贩运。一个大家族的G蛋白偶联受体（GPCRs的）检测变量，作为趋化因子的小分子肽， 直接在 体内 4细胞的迁移。趋化研究的最终目的是要了解一个GPCR的机械感官的趋化因子梯度和控制信号的事件，以趋化。为此，我们使用成像技术，实时监测，时空趋化因子，细胞运动的趋化因子梯度浓度，G蛋白偶联受体介导的异三聚体G蛋白的激活，细胞内的信号在 5-8真核细胞趋化涉及事件。简单的真核有机体，粘菌，显示chemotaxic行为类似白细胞和 D discoideum是一个重要的模型系统，为研究真核细胞趋化。由于自由生活的阿米巴，D discoideum细胞分裂丰富培养基。饥饿时，细胞进入一个发展的计划中，他们通过cAMP介导的趋化聚集，形成multicullular结构。趋化阵营中涉及的许多组件已经确定在 D discoideum。阵营的一个G蛋白偶联受体的结合（cAR1）诱导的异三聚体G蛋白解离成Gγ和Gβγ 亚基7，9，10 。 Gβγ亚基激活Ras，这反过来又激活的PI3K，转换成11-13细胞膜上的画中画画中画3 2。作为画中画3 pleckstrin同源性（PH值）域的蛋白质结合位点，从而招募这些蛋白膜14，15 。 cAR1受体的激活还控制PTEN基因， 这 3画中画画中画2月 16日，17去磷酸化膜 ​​协会。是进化上保守的G蛋白偶联受体介导的趋化因子，如中性粒细胞18的人体细胞趋化的分子机制。我们提出了下面的方法研究D 的趋化discoideum细胞 。 1。制备趋化组件细胞。 2。成像的细胞趋化的一个营地梯度。 3。监测的异三聚体G蛋白G蛋白偶联受体在单个活细胞中的诱导活化。 4。在单个活细胞实时成像趋化触发动态画中画3反应。我们开发的成像方法可以应用于研究人类白细胞趋化作用。

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

在这里，我们描述了详细的调查趋化活细胞成像的方法。我们目前的荧光显微方法来监测细胞迁移的信号事件的时空动态。测量信号的事件，使我们进一步了解如何GPCR的信令网实现了梯度感应的趋化因子和控制真核细胞的定向迁移。

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Research

JoVE Journal

Biology

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

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 ...

科研

生物学

Quantifying Agonist Activity at G Protein-coupled Receptors

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

调查Coronin A在早期饥饿反应中的作用<em&gt;粘菌</em&gt;通过聚集测定

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...