我们想感谢资助的伍斯特理工学院化学与生物化学系。

1。转染细胞系转染法1 （龙沙Nucleofection为增加原代细胞的转染效率） <ol><li>细胞应该是最近通过的，并在转染前数期增长。洗净phophate细胞缓冲液（PBS）和收获细胞由胰蛋白酶。 </li><li>在1500 XG 5分钟，离心收集细胞。 </li><li>添加消毒盖玻片的6孔​​板。盖玻片，可增强细胞粘附涂层。广场1.5毫升培养基（在这种情况下贝科的改良Eagle培养基（DMEM），10％胎牛血清（FBS），）每孔和平衡在孵化器的媒体。 </li><li>收获细胞胰蛋白酶和悬浮在最小的体积的磷酸盐缓冲液（PBS）。 </li><li>计数细胞，离心再悬浮的正确金额，提供 5％〜5X10样品细胞。 </li><li>仔细的细胞重悬于100μL室温每个样品Nucleofector解决方案。 </li><li>联合收割机100μL的细胞悬液与5μg质粒DNA和样品转移到Nucleofector试管，小心不要包括气泡。 </li><li>选择适当Nucleofector的程序（这可能需要以前的测试，以确定最佳的转染效率与死亡率最低）。 </li><li> Nucleofector试管架含有悬浮细胞/ DNA插入试管，并开始选择的程序。 </li><li>完成后，取出试管，并添加〜500μL预平衡文化传媒（6孔板）。 </li><li>立即转移到6孔板终体积为1.5毫升，每孔样品。细胞应该是镀在任一混合种群或单独控制。 </li></ol>转染法2 （Qiagen公司Effectene转染细胞系） <ol><li>准备使用Qiagen公司Effectene试剂的转染复合物，由先加入每个质粒DNA样本的0.8微克至200μL欧共体缓冲区。 </li><li>每个样品中加入6.4μL，增强试剂，混合短暂弹管，为2分钟，在室温下孵育。 </li><li>每个样品中，混合加入20μLEffectene试剂弹管，15分钟在室温下孵育。 </li><li>在此孵化，准备转染细胞系的利益。最近传代细胞（&lt;80％汇合）洗一次在磷酸盐缓冲液（PBS）。在这种情况下加回1.5毫升新鲜的温暖和平衡增长中期（贝科改良Eagle培养基（DMEM），10％胎牛血清（FBS））。 </li><li>一旦转染复合物孵育15分钟，1.2毫升的媒体转移到每个样品。混合吹打，并立即传送〜700μL的配合，在6孔板每两个水井。加入滴加，旋流板，轻轻混匀。允许细胞转染至少3个小时（可能会随细胞类型）来完成。 </li><li>消毒盖玻片添加到一个新的6孔板。盖玻片，可增强细胞粘附涂层。 </li><li>转染细胞后，洗细胞两次，用PBS和收获最小的trysinization细胞，每孔加入约100μL胰蛋白酶溶液，简要搅拌充分大衣每口井的板，并立即吸胰蛋白酶。一旦细胞已经解除，加1.5毫升的新鲜传媒。 </li><li>此时，细胞混合或单独到无菌盖玻片的人口（控制）应镀。 </li><li>让细胞恢复了12个小时。 </li></ol> 2。细胞融合<ol><li>删除从细胞的培养基，用PBS洗两次。 </li><li>在这一点上，它是建议治疗与放线菌酮（100微克/毫升）的细胞，抑制蛋白质的合成。允许细胞孵育2小时，然后用PBS洗涤细胞两次。 </li><li>保险丝细胞暴露在室温为125秒，50％聚乙二醇1000（PEG 1000），在无血清DMEM溶液。 </li><li>用PBS清洗细胞，以消除聚乙二醇1000解决方案，并加入2 ml新鲜温暖和平衡的媒体。 </li><li>允许细胞孵育18 - 24小时。 </li></ol>荧光显微镜<ol><li>取出培养基，并在PBS洗细胞两次。 </li><li>加入1毫升4％多聚甲醛溶液（PBS）的每口井，修复细胞。轻轻搅拌15分钟。 </li><li>在PBS洗两次固定细胞。 </li><li>小心地从板盖玻片，灯芯多余的PBS。反转到显微镜玻片上装有安装介质的下降（90％甘油，100MM的Tris pH值7.5，2％的DABCO（1,4 - diazabicoyclo辛烷值）含有的DAPI（4&#39;,6 - diamidino - 2 -苯基吲哚）。 </li><li>从幻灯片中删除多余的安装介质，密封合作VER单用指甲油。 </li><li>通过共聚焦或epifluorescence显微镜可视化的细胞。 </li></ol> 3。代表性的成果图2显示了一个例子异核实验，使用的主要的成纤维细胞和H1299非小细胞肺癌细胞。在这个实验的兴趣蛋白（鸡贫血病毒VP3）epifluorescence显微镜观察显示本地化主要是细胞质的主要细胞类型，并在转化细胞类型的核本地化。作为一个融合（无论是绿色荧光蛋白的pEGFP - N1载体，Clontech公司）在主包皮成纤维细胞（PFF）或DsRed的H1299细胞（DsRed的N1载体，Clontech公司）的蛋白表达。控制样品涉及自我融合（前两行）显示多核细胞，在稳定状态下的本土化模式没有改变。这种变化，否则可能出现由于融合条件或syncitia形成的敏感性。异核体的融合（下排）证明GFP融合的主要细胞衍生的蛋白质和引进转化的细胞材料DsRed的融合蛋白的共定位核项目。显示的例子是一种比较罕见的只有两个单元，每个类型之一存在两个氟蛋白信号表明，异核体融合。除了检测本地化转换这些类型，核质穿梭活动，可以很容易被发现在两个细胞核的单荧光团的存在。这种类型的决心是明确的审查1:1细胞融合时，将取决于抑制翻译。 <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="图1" /> 图1。流程图异核体使用主成纤维细胞和非小细胞肺癌细胞H1299的方法 。感兴趣的基因被克隆产生帧融合两个荧光蛋白基因之一。要么构造，然后瞬时转染细胞系，并允许恢复。异核体的形成是诱发进一步孵化与聚乙二醇（PEG）进行了简短的治疗。最后，感兴趣的蛋白质的亚细胞定位是使用各种形式的荧光显微镜观察。 <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="图2" /> 图2。胞核贩运和鸡贫血病毒VP3蛋白的穿梭活动可视化的异核体的融合。首行：代表的图像，中间一排的自我融合H1299细胞表达DsRed的- VP3：小学包皮成纤维细胞的PEG融合后表达GFP - VP3（PFF）代表epifluorescence 图像底部行 ：PFF和H1299细胞的异核体的融合。黄色表示的绿色荧光蛋白DsRed的信号的共定位。细胞成像使用徕卡AF6000E荧光显微镜和Leica成像软件。

<ol>
	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

这里介绍的异核协议提供了一个高效和容易解释的方法对细胞的特定类型的本地化行为，以及核质穿梭活动表征。此方法采用荧光分析的灵敏度，以及图像的活细胞和执行蛋白质动力学的研究能力的优势。该技术是很容易适应许多不同类型的细胞，是适合生活和固定细胞成像。例如，倒置荧光显微镜可用于实时成像和时间的推移视频采集。相比之下，安装细胞可用于epifluorescence显微镜无论是稳态本地化或更详细的共焦成像离散本地化的决心。此外，如荧光漂白后恢复（FRAP），或在漂白荧光丢失（FLIP）的技术可用于确定本地化动力学 1 。协议的替代荧光团的法团，将允许的蛋白质相互作用的实验荧光共振能量转移（FRET） 将在演唱会2进行。 leptomycin B或显性负，如蜂窝贩运的各种抑制剂冉GTP酶的结构，可以用来建立对特定进口或出口的途径5,6,9的依赖。 <ul><li>在某些实验涉及穿梭活动中，必须小心，以避免由于新的蛋白质合成的本地化效果。在这种情况下，转换酶抑制剂或有限的时间点必须使用。然而，由于翻译抑制的文物仍然是一个可能性。含有不同的细胞类型的细胞融合是最想要的穿梭和本地化的行为明确的解释。优化细胞的数量和融合条件（时机和浓度）可能是必要的，以促进这种良好的1:1融合事件。 </li><li>重要的是要注意，在协议中的某些步骤的优化，将需要使用特定的细胞类型而定。在实验过程中，如融合效率，转染效率，融合后的生存能力方面会有很大的改变之间的细胞类型。尤其是主要的细胞类型可以是具有挑战性的管理，由于复杂的文化条件和转染效率低。龙沙Nucleofector设备，如技术的使用可以使细胞的快速处理，以及转染效率显着提高。此外，电的方法，使悬浮液中的细胞融合的后续步骤，便于转移。 </li></ul>

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3023602</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s modified Eagle&#x2019;s medium (DMEM)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH30243FS</td>
<td>High glucose</td>
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<td>paraformaldehyde</td>
<td>&nbsp;</td>
<td>FisherChemical</td>
<td>O4042-500</td>
<td>&nbsp;</td>
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<td>4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D9542-10MG</td>
<td>&nbsp;</td>
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<td>Hyclone Serum (fetal bovine)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH3008803HI</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 6-well plates</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-772-1B</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol 1000</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>528875-25GM</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545-85</td>
<td>&nbsp;</td>
<tr>
<td>DABCO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2522-25G</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector HDF kit</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPD-1001</td>
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heterokaryon technique for analysis of cell type-specific localization

大量的蛋白质，是受细胞内贩卖或nucleocytolasmic穿梭。这些蛋白质显示了包括核进口/出口RNA和蛋白质，转录调控和细胞凋亡的细胞功能的多样化。有趣的是，主要包括细胞分裂，分化和改造的细胞重组，往往涉及这类活动 3,4,8,10 。这些蛋白质和各自的监管机制的详细研究是具有挑战性的，这些本地化的变化可以难以捉摸的刺激，和运动本身可以是相当动态的，难以跟踪。涉及细胞癌变的研究，例如，继续受益于理解的途径和蛋白质的活动之间的正常原代细胞和转化细胞6,7,11,12不同。由于许多蛋白质​​在改变本地化改造或作为一个转型的结果，有效地描述这些蛋白质的方法和途径其所参与的立场，以改善癌变和靶向药物开辟新的领域的理解。 这里我们提出一个分析蛋白质贩运的方法，小学和转化哺乳动物细胞之间的穿梭活动。这种方法结合荧光显微镜的异核体融合的生成提供了一个灵活的协议，可用于检测的稳态或动态的蛋白质本地化。正如图1所示，两个独立的细胞类型，瞬时转染轴承氟蛋白基因连接到感兴趣的基因质粒结构。表达后，细胞融合使用聚乙二醇，蛋白质本地化可能使用多种方法进行成像。这里介绍的协议可能会增加专业技术的一个根本途径。

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

一个细胞类型特异性的蛋白定位和穿梭核质特性的灵活和有效的方法是描述。这异核体的方法是使用荧光标记的融合蛋白细胞融合后图像蛋白质本地化。该协议是经得起稳态本地化或活细胞成像的基础上更具活力的测定。

异核技术的细胞类型特异性定位分析

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

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16.3K 次观看.  Worcester Polytechnic Institute- WPI. 一个细胞类型特异性的蛋白定位和穿梭核质特性的灵活和有效的方法是描述。这异核体的方法是使用荧光标记的融合蛋白细胞融合后图像蛋白质本地化。该协议是经得起稳态本地化或活细胞成像的基础上更具活力的测定。

视频: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

斑马鱼镜头核定位与功能完整性的评价

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...