作者支持由瑞典研究委员会，瑞典战略研究，瑞典癌症协会，瑞典的儿童癌症协会，阿克Wibergs基础和Jeanssons基础的基础。

1。探头引入到细胞<ol><li>一个基于FRET的探头和质粒抗共转染的细胞。选择转染的方法，该方法是有效的，在你感兴趣的细胞类型。对于U2OS细胞，标准的磷酸钙转染方法给予足够的结果15。 </li><li>选择适当的抗生素至少7天的细胞。这丰富的数额细胞表达探头和限制有毒的表达水平，表达水平，严重影响细胞周期的细胞量。 </li><li> （可选）选择稳定的克隆。 </li><li>使用固定或活细胞，验证这两个CFP和YFP在大约相同比例的所有细胞中。 </li><li>细胞的情况下，一些只包含CFP或YFP的，有可能发生重组。重组可以发生在细菌生长或转染后的探针，并可能在后一种情况下，依靠质粒质量。重复质粒p赔偿和线性质粒转染前，如果问题仍然存在。 </li></ol> 2。监测比例的FRET <ol><li>玻璃底菜，或安装在一腔盖玻片上的种子细胞。确保菜/盖玻片没有1.5（170微米厚）。 </li><li>山无酚红机动epifluorescence显微镜设置为37 ° C温度控制细胞生长培养基确保媒体CO 2或使用CO 2独立的媒体，如利博维茨- 15，pH调节。 </li><li>使用透射光细胞聚焦。别看在使用荧光灯细胞。 </li><li>开始通过收购12位或16位图像YFP的激励（YFPex）和YFP的排放（YFPem）过滤器和一个合适的分色镜。使用最大分级，仍允许亚细胞所需的决议。使用高分级，以减少曝光时间或强度是至关重要的，以避免光毒性。 </li><li>我通过测量或估计粗糙的平均，最小和最大像素值，在一个地区缺乏细胞asure或估计的背景强度和近似噪音。更改曝光时间和中性密度滤光片，在细胞内的平均的信号大约是背景强度加五到十倍的最大和最小的背景强度之间的差异。 </li><li>检查图像不接近饱和。在图像的最大强度应小于允许强度的差异，当细胞进入有丝分裂的动态范围（例如最大2000）在12位图像的一半。 </li><li>重复步骤2.4至2.6使用CFP激发和YFP排放过滤器和一个合适的分色镜。 </li><li>选择与转染细胞的多个地区，不包括点2.4至2.7的地区，并确保最大强度是不到一半在所有细胞中的动态范围。 </li><li>启动时间推移实验，获得两个图像，每45分钟使用YFPex - YFPem和​​CFPex - YFPem过滤器设置。 </li></ol> 3。验证和优化条件<ol><li>打开采集到的图像和监控探针转染的细胞表达有丝分裂有丝分裂的细胞周期时间。 </li><li>测量的细胞周期时间比较公布的数据用的细胞类型。另外，电影未转染细胞获得一个图像只透射光（如DIC或相衬）每隔一小时，参考细胞周期时间。 </li><li>在情况下，从有丝分裂有丝分裂的时间相对应的参考细胞周期的时间，继续步骤4。另外，重新获取图像更难曝光，更多的时间点或多个Z -水平并重复步骤3。 </li><li>如果从有丝分裂有丝分裂的时间不符合参考细胞周期的时间，电影转染细胞在年底取得一个图像只透射光（如DIC或相衬），其次是一个图像的每一个小时实验，以确定转染细胞。 </li><li>如果仍然显示转染细胞细胞周期较长时间，重复第1步，但转染少探头或选择包含探头的数量非常低的稳定的克隆。 </li><li>如果转染的细胞显示正常的细胞周期时间，在荧光灯下，重复第2步，但降低风险，通过​​修改分级，中性密度滤镜，曝光时间，以及图像之间的时间。 </li></ol> 4。分析的FRET <ol><li>专门到显微镜的大多数软件，可以进行分析。在这里，我们描述了如何执行分析使用免费ImageJ（ <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> ）利用插件比加（ <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus 。HTML</a> ）。 </li><li>打开ImageJ的图像。如果您的图片格式的多色STACK（例如Deltavision），首先将它们转换为一个hyperstack单独的个别通道: 图像hyperstack栈hyperstack，随后分裂单通道堆栈使用: 图像hyperstack减少维 </li><li>乘以YFPex - 3使用YFPem栈: 过程数学乘法这一步是可选的，因为它是唯一旨在确保该比率在0和1之间的范围，增加图像的视觉清晰度。 </li><li>在YFPex - YFPem栈，绘制细胞缺乏的区域（ROI）兴趣的地区，但接近到要测量的细胞。不同的细胞在相同的图像可能需要不同的地区，作为背景和信号强度通常是最强的，在图像的中心。 </li><li>经理的投资回报率的投资回报率分析工具的投资回报率经理 。 </li><li>测量都YFPex的投资回报率的平均强度- YFPem和CFPex - YFPem堆栈:分析测量</em&gt;。 </li><li>在多个时间点重复和验证，背景强度接近细胞的利益是整个电影类似。 </li><li>设置包括最小强度: 分析设置测量最小和最大灰度值的测量 。 </li><li>画一个涵盖了细胞的大部分利益的地区和措施，在双方的YFPex最小强度 - YFPem和​​CFPex - YFPem堆栈。从4.6测得的最小强度和背景强度之间的差异和除以二。这提供了一个剪切值开始估计。 </li><li>打开比加。选择YFPex - YFPem和​​CFPex - YFPem栈。 FRET的比例，选择CFPex - YFPem栈1，倒FRET的比例可视化探针FRET后磷酸化效率降低，选择YFPex - YFPem栈1。 </li><li>将测得的背景强度和剪切值。 </li><li>设置缩放比例栈和应用合适的外观üP表（LUT）的可视化比例的变化。 </li></ol> 5。代表性的成果 PLK1活动是第一次在G2期和有丝分裂期间的峰细胞核中可见。图3显示了一个实验，用最小的光毒性设置在第2所述。请注意，这是一个有代表性的初步结果，并曝光条件或图像之间的时间可以进行修改，以提高信号的信噪比或时间分辨率。图3D显示，多数表达探头细胞增殖与细胞周期时间的20至25小时，表明探头成像条件和表达水平不影响细胞周期的时间。虽然有相当大的噪音，PLK1活动增加的趋势在G2和有丝分裂14调峰清晰可见（图3A）。加工的原始数据，这里所讲均值滤波作为一个kymograph（图3B），或量化的平均倒RA<html>氧化钛（图3C）可以提高清晰度。 <img alt="图1" src="/files/ftp_upload/3410/3410fig1.jpg" /> 图1:一个基于FRET的探头原则，以监察激酶和磷酸酶的活动。两个荧光基团，通常CFP（蓝色）和YFP（绿色），是由一个磷酸结合结构域（橙色）和phosphorylatable序列（黄色）连接。磷酸化（红色）介导的磷酸化结合结构域结合，从而诱导在探头的构象变化。的两个荧光基团，从而影响了FRET CFP和YFP之间的效率之间的距离或方向不同的构象变化的结果。 FRET技术可以通过令人兴奋的CFP和监测YFP的发射（虚线）的可视化。 <img alt="图2" src="/files/ftp_upload/3410/3410fig2.jpg" /> 图2实验过程的示意图大纲。 p_upload/3410/3410fig3.jpg"/&gt; 图3。PLK1活动首先在G2期和有丝分裂期间的峰细胞核中可见。 U2OS细胞表达一个基于FRET的探头监控PLK1活动9,14摄制60小时使用一个Deltavision百吉成像系统，配备了20倍不适用0.7空中目标和汞灯。成像条件造成最小的光毒性，在第2条所述，使用4X binning和中性密度滤光片块99％的入射光。倒的FRET比虚假的颜色表示，通过4个师的一个细胞。 B，在A所示的应用均值滤波后的细胞kymograph。 C，量化的倒FRET在A D所示的细胞的比例，累计有丝分裂条目50的FRET探针表达的细胞，包括子细胞的分裂。

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我们什么都没有透露。

监测苦恼整个细胞周期需要评估短期应对外部刺激时，是同样重要的的考虑。首先，细胞周期的进程很容易扰动压力信号，要求保持在最低限度，光毒性。其次，所有的记者可能影响滴定激酶，磷酸或相互作用域的细胞过程。可能是最简单的方法来评估，如果有足够的实验条件来衡量细胞周期的长度从有丝分裂有丝分裂和独立实验的荧光成像探针表达比较。 在监测的关键因?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 目录编号 </td><td> 公司 </td></tr><tr><td>莱博维茨的L - 15，无酚红</td><td> 21083-027 </td><td> GIBCO公司，生命科技</td></tr><tr><td>的DMEM + Glutamax，我</td><td> 31966 </td><td> GIBCO公司，生命科技</td></tr><tr><td>胎牛血清（FBS） </td><td> SV30160.03 </td><td> Hyclone公司</td></tr><tr><td> 0.05％胰蛋白酶EDTA </td><td> SH30236.01 </td><td> Hyclone公司</td></tr><tr><td>青霉素链霉素</td><td> SV30010 </td><td> Hyclone公司</td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO公司，生命科技</td></tr><tr><td>嘌呤霉素</td><td> P8833 </td><td> Sigma - Aldrich公司</td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

福斯特共振能量转移（FRET）为基础的记者1允许在活细胞中的内源性蛋白激酶和磷酸酶活性的评估。这种探头通常由CFP和YFP的变种，由phosphorylatable序列和磷酸结合结构域进行干预。磷酸化后，探头改变构象，CFP和YFP之间的距离或方向的变化，结果，导致在变化FRET效率（图1）。几个探头在过去十年中，已公布的监测多个激酶和磷酸酶的活性的平衡，其中包括，PKD的5 4 3 PKB，PKC，ERK的6，7 JNK 的 CDK 18， 极光B 9 2，PKA的记者和 PLK1 9 。鉴于模块化设计，额外的探头有可能出现在不久的将来10。 通过细胞周期的进展是受压力signali吴途径11。值得注意的是，不同的细胞周期调节在不受干扰的增长，而当细胞恢复应力 12。定时通过细胞周期的细胞成像，因此需要特别小心。这将成为一个问题，尤其是当用人比例成像，因为两个图像具有很高的信噪比，需要正确地解释结果。成比例的FRET成像细胞周期依赖激酶和磷酸酶活性的变化为主被限制在细胞周期 8,9,13,14分节。 在这里，我们讨论的方法来监视基于FRET的探头使用整个人类的细胞周期的比例成像。方法依赖于设备，是在生命科学领域的许多研究人员，并不需要显微镜或图像处理专业知识。

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

基于FRET的记者越来越多地用于监测活细胞中的激酶和磷酸酶的活动。在这里，我们描述了如何使用基于FRET的记者，以评估目标磷酸化细胞周期依赖性变化的方法。

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K 次观看.  Karolinska Institutet. 基于FRET的记者越来越多地用于监测活细胞中的激酶和磷酸酶的活动。在这里，我们描述了如何使用基于FRET的记者，以评估目标磷酸化细胞周期依赖性变化的方法。

视频: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

从老鼠血液通过大隐静脉和心脏穿刺“

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

科研

生物学

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

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 ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

转基因的产生<em&gt;水润</em&gt;通过胚胎显微注射

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

在活细胞悬浮液荧光测定G蛋白选择性GPCR构象测量使用FRET传感器

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

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