这项工作得到了国家卫生研究院[R01GM121847]的支持;纪念斯隆·凯特林癌症中心（MSKCC）支持赠款/核心赠款（P30 CA008748）;和 MSKCC 功能基因组学倡议。

作者没有什么可透露的。

与典型的体外TIRF 单分子实验相比，此处描述的检测的单分子成像由于使用细胞提取物和高浓度荧光标记抗体而异常复杂。与一轮表面PEGylation的更常见做法相比，第二轮PEGylation（第2步）大大减少了非特异性抗体对检测表面15的结合。扩散荧光抗体的高浓度会导致极高的荧光背景，掩盖单分子检测抗体结合。为了减少这种荧光背景，激光事件角度远高于临界角（第 3.4 步）?...

在真核系统的翻译主要通过7甲基瓜诺辛（m7G）帽依赖通路1发生。研究表明，真核翻译的启动步骤是限速的，也是第2、3、4条规则的共同目标。利用遗传5、生化6、7、8、结构9和基因组10大块方法，对上限依赖转化机制进行了广泛的研究。虽然这些方法确定了调节依赖上限启动的多种机制，但其分辨率仅限于异质和异步启动事件信号的合奏平均值。最近，在体内翻译事件中，通过测量荧光抗体与新生多肽11、12、13、14上的表皮结合的方法，对个体进行了可视化。然而，这些新方法在解决单个启动事件的能力方面也受到限制，因为多个荧光抗体必须结合一种新生肽，以便从高细胞内荧光背景中解决单个转化事件。在许多生物相互作用中，解决的个体动能事件为理解在分子水平上无法同步的复杂多步骤和重复的生物过程提供了重要的见解。需要采用新的方法来跟踪各个翻译事件的动态，以便更好地了解依赖上限的启动和监管。
我们最近开发了一种体外测定，用单分子分辨率15测量依赖上限的启动动力学。考虑到这个启动途径3、16中涉及大量已知和未知的蛋白质因子，单分子测定被开发为与现有的体外无细胞翻译系统兼容，从而受益于细胞因子的保存和强大的翻译活性17、18、19、20、21、22、23、24、25。此外，使用无细胞翻译系统可以更兼容地比较单分子观测结果和以前的批量结果。这种方法将新的单分子动力学见解直接整合到现有的依赖上限启动的机械框架中。为了建立单分子测定，传统的无细胞翻译系统被修改为三种方式:在记者mRNA的开放读取帧（ORF）的开头插入表位编码序列:记者mRNA的3+末端是生物素化，以促进mRNA末端系绳到单分子检测表面:和荧光标签抗体补充翻译提取物。这些修改只需要基本的分子生物学技术和常用的试剂。此外，这些修饰和单分子成像条件保留了无体细胞翻译反应的翻译动力学15。
在此测定（图1），5+端封顶和3+端生物素化记者mRNA被固定在流室的链球菌涂层检测表面。然后，流室中将填充一种无细胞的转化混合物，并辅以荧光标记的抗体。在mRNA翻译发生在表位序列26、27下游的大约30-40个子元之后，表位从核糖体出口隧道中浮现出来，并变得能够与荧光标记的抗体相互作用。这种相互作用是快速的，它通过单分子荧光成像技术进行检测，能够在无活性细胞转化过程中跟踪具有单分子分辨率的翻译动力学。这种检测在体外研究依赖上限的翻译动力学及其调节方面应大有裨益，特别是对于体外分析中具有工作量的系统。
建立这种单分子检测的先决条件是工作散装无细胞翻译分析，这可以通过翻译提取物实现，无论是商业上可用的或按照先前描述的方法28准备。真核转化提取物可以从不同的细胞中获取，包括真菌、哺乳动物和植物28。对于成像，此测定需要配备可调谐激光强度和事件角度的 TIRF 显微镜、电动样品阶段、机动流体系统和样品温度控制装置。这种要求对于现代 体外 单分子TIRF实验来说是通用的，而且可能以不同的方式实现。这里介绍的实验使用了一个客观类型的TIRF系统，该系统由商用显微镜、软件和配件组成，全部列在 材料表中。

<table><tbody><tr><td>100X oil objective, N.A. 1.49</td><td>Olympus</td><td>UAPON 100XOTIRF</td><td></td></tr><tr><td>Acryamide/bis (40%, 19:1)</td><td>Bio-Rad</td><td>161-0144</td><td></td></tr><tr><td>Alkaline liquid detergent</td><td>Decon</td><td>5332</td><td></td></tr><tr><td>Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)</td><td>UCT Specialties, LLC</td><td>A0700</td><td></td></tr><tr><td>Andor ixon Ultra DU 897V EMCCD</td><td>Andor</td><td>DU-897U-CSO-#BV</td><td></td></tr><tr><td>Andor Solis software</td><td>Andor</td><td></td><td>For controlling the Andor EMCCD</td></tr><tr><td>Band-pass filter</td><td>Chroma</td><td>532/640/25</td><td></td></tr><tr><td>Band-pass filter</td><td>Chroma</td><td>NF03-405/488/532/635E-25</td><td></td></tr><tr><td>Biotin-PEG-SVA</td><td>Laysan Bio Inc</td><td>Biotin-PEG-SVA</td><td></td></tr><tr><td>Coenzyme A free acid</td><td>Prolume</td><td>309-250</td><td></td></tr><tr><td>Coolterm software</td><td></td><td></td><td>For controlling the syringe pump</td></tr><tr><td>Desktop computer</td><td>Dell</td><td></td><td>For controlling the microscope, camera, stage, and pump.</td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>R405/488/532/635</td><td></td></tr><tr><td>Direct-zol RNA microprep 50RNX</td><td>Fisher Scientific</td><td>NC1139450</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Epoxy</td><td>Devcon</td><td>14250</td><td></td></tr><tr><td>Firefly luciferin D-Luciferin free acid</td><td>Prolume</td><td>306-250</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>BP1185500</td><td></td></tr><tr><td>Hydrogen perioxide</td><td>Sigma-Aldrich</td><td>216763-500ML</td><td></td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Z-81226A</td><td>Low auto-fluorescence</td></tr><tr><td>Luciferase Assay System</td><td>Promega</td><td>E1500</td><td></td></tr><tr><td>MEGASCRIPT T7 Transcription Kit</td><td>Thermo fisher</td><td>AM1334</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>MMX04751</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>Microscope slide</td><td>Thermo Scientific</td><td>3048</td><td></td></tr><tr><td>Monoclonal anti-FLAG M2-Cy3</td><td>Sigma-Aldrich</td><td>A9594</td><td></td></tr><tr><td>mPEG-SVA</td><td>Laysan Bio Inc</td><td>mPEG-SVA-5000</td><td></td></tr><tr><td>MS(PEG)4</td><td>Thermo Scientific</td><td>22341</td><td></td></tr><tr><td>NaCl (5M)</td><td>Thermo Scientific</td><td>AM9760G</td><td></td></tr><tr><td>No 1.5 microscope Cover glass</td><td>Fisherband</td><td>12-544-C</td><td></td></tr><tr><td>Olympus Laser, 532nm 100mM</td><td>Olympus digital Laser system</td><td>CMR-LAS 532nm 100mW</td><td></td></tr><tr><td>Olympus TirfCtrl software</td><td>Olympus</td><td></td><td>For controlling the laser intensity and incident angle</td></tr><tr><td>Optical table</td><td>TMC vibration control</td><td>63-563</td><td>With vibration isolation</td></tr><tr><td>Phenol chloroform isoamyl alcohol mix</td><td>Sigma-Aldrich</td><td>77617-100ml</td><td></td></tr><tr><td>Pierce RNA 3&#039; End Biotinylation Kit</td><td>Thermo Scientific</td><td>20160</td><td></td></tr><tr><td>Potassium hydroxide pellets</td><td>Sigma-Aldrich</td><td>P1767-500G</td><td></td></tr><tr><td>Prior motorized XY translation stage</td><td>Prior</td><td>PS3J100</td><td></td></tr><tr><td>Prior PriorTest software</td><td>Prior</td><td></td><td>For controlling the Prior motorized stage</td></tr><tr><td>Recombinant RNasin RNase Inhibitor</td><td>Promega</td><td>N2515</td><td></td></tr><tr><td>Stage top Incubator</td><td>In vivo scientific (world precision Instruments)</td><td>98710-1</td><td>With a custom built acrylic cage</td></tr><tr><td>Staining jar</td><td>Fisher Scientific</td><td>08-817</td><td></td></tr><tr><td>Streptavidin</td><td>Thermo Scientific</td><td>43-4301</td><td></td></tr><tr><td>Sulfuric acid</td><td>Fisher Scientific</td><td>A300212</td><td></td></tr><tr><td>SYBR green II</td><td>Fisher Scientific</td><td>S7564</td><td></td></tr><tr><td>Syringe</td><td>Hamilton</td><td>1725RN</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard apparatus</td><td>55-3333</td><td></td></tr><tr><td>Tris (1M), pH = 7.0</td><td>Thermo Scientific</td><td>AM9850G</td><td></td></tr><tr><td>Ultrasonic Bath</td><td>Branson</td><td>CPX1800H</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378-500G</td><td></td></tr><tr><td>Vaccinia Capping system</td><td>New England Biolabs</td><td>M2080S</td><td></td></tr><tr><td>Zymo-Spin IC Columns</td><td>Zymo Research</td><td>C1004</td><td></td></tr></tbody></table>

an in vitro single-molecule imaging assay for the analysis of cap-dependent translation kinetics

依赖上限的蛋白质合成是真核细胞的主要转化途径。虽然各种生化和遗传方法允许对依赖上限的翻译及其调节进行广泛的研究，但这种翻译途径的高分辨率动能特征仍然缺乏。最近，我们开发了一种 体外 测定法，以单分子分辨率测量依赖上限的转化动力学。该检测基于荧光标记的抗体结合到新生的表皮标记的多肽。通过成像抗体与新生肽（核糖体）mRNA复合物的结合和分离，可以跟踪单个 mRNA 的翻译进展。在这里，我们提出了建立此测定的协议，包括 mRNA 和 PEGylated 幻灯片制备、翻译的实时成像以及单分子轨迹分析。此分析能够跟踪单个依赖上限的翻译事件，并解决关键的翻译动力学，如启动和拉长率。该测定可广泛应用于不同的翻译系统，在依赖上限的翻译动力学和转化控制机制的 体外研究中 应大有裨益。

按照所描述的协议，在3'末端系记者mRNA（图1）的活性无细胞翻译过程中，能够与新生的N终端标记多肽进行单个抗体相互作用的成像，具有单分子分辨率。报告使用三种合成 mRNA 进行最小演示实验:LUC（编码未标记的路西法酶）、LUCFLAG（编码 3xFLAG 标记的路西法酶）和hp-LUCFLAG（LUC FLAG，在 5' 领先区...

Watch this Scientific Journal Video about An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics at JoVE.com

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

跟踪单个翻译事件允许对依赖上限的翻译机制进行高分辨率动能研究。在这里，我们演示了基于荧光标记抗体和表皮标记的新生肽之间的成像相互作用的 体外 单分子检测。该方法使启动和肽伸长动力学的单分子特征 在主动体外 帽依赖翻译。

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

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Research

JoVE Journal

Biochemistry

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

3.0K 次观看.  Memorial Sloan Kettering Cancer Center. 跟踪单个翻译事件允许对依赖上限的翻译机制进行高分辨率动能研究。在这里，我们演示了基于荧光标记抗体和表皮标记的新生肽之间的成像相互作用的 体外 单分子检测。该方法使启动和肽伸长动力学的单分子特征 在主动体外 帽依赖翻译。

视频: An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

科研

生物化学

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s&#8722;1 and 0.35 s&#8722;1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

荧光RNA适配体 外和细胞 开启动力学的测定

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

免疫与感染

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

生物学

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

用于分析依赖帽的翻译动力学的体外单分子成像测定

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定量荧光测量全球本地化翻译

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

荧光RNA适配体外和细胞开启动力学的测定

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

单分子成像的基因调控

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

摘要

探索更多视频

此视频中的章节

定量荧光测量全球本地化翻译

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

荧光RNA适配体 外和细胞 开启动力学的测定

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

单分子成像的基因调控

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

用于分析依赖帽的翻译动力学的体外单分子成像测定

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

荧光RNA适配体外和细胞开启动力学的测定

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术