Name: spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells
Uploaded: 2012-03-09T13:00:00
Description: Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

这项研究是由美国国立卫生研究院的研究经费（DK090868和GM092930要德州仪器）的支持。有一个在笼中的雷帕霉素模拟待批专利。

1。转染质粒DNA <ol><li>加入质粒DNA，0.5微克膜栓联邦储备银行（以下简称为的LDR）和0.5微克FKBP-Tiam 1 mRNA（37.5微升DH 2 O荧光蛋白标记的T-细胞淋巴瘤侵袭转移诱导蛋白1：激活RAC）加入1μLFuGENE HD。 </li><li>涡，在室温下孵育20分钟。同时，进行步骤1.3-1.8。 </li><li>每8井室以及50μL聚-D-赖氨酸洗净。 </li><li>每口井的中心洗DH 2 O </li><li> Trypsinize 80-85％汇合NIH-3T3细胞，并稀释至10毫升培养?...

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我们描述了一个技术，它采用一种新型的笼状化合物与FKBP联邦储备银行的异二聚体系统一起为了操纵Rho GTP酶的活性，在几秒钟的时间尺度上精确的亚细胞定位。 本办法有三个限制。首先，因为该方法的基础上，对防止或允许跨质膜dimerizer扩散的目标细胞的位置需要质膜或附近。可能使该化合物进入细胞，而直到光线照射诱导二聚体的的CRB化学结构的进一步优化。随着这种dim...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>雷帕霉素</td><td> tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE高清</td><td>罗氏公司</td><td> 04709691001 </td></tr><tr><td>抗生物素蛋白</td><td>西格玛</td><td> A9275-10MG </td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D2650-5X5ML </td></tr><tr><td>体积排阻柱</td><td> GE医疗集团</td><td>自旋捕集的G-25 </td></tr><tr><td>玻璃底8室</td><td> Thermo Scientific的</td><td> 12-565-47 </td></tr><tr><td>倒置荧光显微镜</td><td>蔡司</td><td> Axiovert200M </td></tr><tr><td>紫外LED光源</td><td>拉普光电电子有限公司</td><td> UVILED </td></tr><tr><td>共焦旋转盘</td><td>横河电机公司</td><td> CSU10 </td></tr><tr><td> CCD相机</td><td>滨松</td><td> ORCA的-ER </td></tr><tr><td> 100X目的镜头</td><td>蔡司</td><td>计划复消色差透镜</td></tr></tbody></table> CRB综合计划（版权ACS2011）6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="计划1" /></a> 合成条件：（一）BnBr，钾2 CO 3，二甲基甲酰胺（二）（五），三氟乙酸（C）（D）丙炔-BR K 2 CO 3，二甲基甲酰胺，3 f.HNO甘油，猫。 PTSA，甲苯（F）硼氢化钠3 CN的四氯化钛 ，乙腈（G）TF 2 O的吡啶（H），甲醇盐酸，（我）LiAlH 4，四氢呋喃（J）敦沛证券，吡啶，CH 2 CL 2（K）为NaN 3，二甲基甲酰胺（L）8，2,6 -二叔丁基吡啶，CH 2 CL 2（米）13， 硫酸铜 ，抗坏血酸，2 -丙醇，H 2 O CH 2 CL 2。

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Rho家族的小的鸟嘌呤triphosphatases与伟大的时空精度（GTP酶）的动态监管是必不可少的各种细胞功能和事件1，2。已经揭示了他们的活动可视化和实时3本地化他们spatiotemporally动态性质。为了获得更深入地了解自​​己的角色在不同的细胞功能，在分子水平上，下一步应该是摄蛋白质的活动在一个精确的亚细胞定位和定时。 为了实现这一目标，我们已经开发出一种光致，时空，时间控制的小GTP酶激活两种技术结合的方法：（1）雷帕霉素诱导FKBP联邦储备银行的异二聚体和（2）的雷帕照片囚禁的方法。与使用雷帕霉素介导的FKBP-FRB异二聚体，我们已经开发出一种快速诱导includi小GTP酶的激活或灭活方法吴RAC 4，Cdc42的4 4 RhoA蛋白和Ras 5，其中雷帕霉素诱导易位的的FKBP融合GTP酶，或他们的活化，在联邦储备银行锚定在细胞膜。与此异二聚体系统的耦合，我们还制定了雷帕霉素类似物的照片囚禁系统。图片笼状化合物是一种小分子，被称为作为一个囚禁组与photocleavable保护组抑制其活动。为了抑制异二聚体活性完全，我们设计了1笼中雷帕霉素，被拴1高分子这样认为，由此产生的大量复杂的可以不穿过细胞膜，导致1细胞6内的化学dimerizer几乎没有背景活动。我们计划图1所示系统。与这两个系统的结合，我们在本地招募了RAC激活质膜上几秒钟的时间表，并取得了在亚利光致RAC激活EL 6。

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

被描述为轻小GTPase活性的时空控制的方法。这种方法是基于FKBP-FRB雷帕霉素诱导的异二聚体和照片囚禁系统。优化的光照射，使时空，时间控制在亚细胞水平的小GTP酶激活。

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Research

JoVE Journal

Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...