这项工作是支持由美国国立卫生研究院R01 NS042228妇幼保健奥林巴斯FV1000共聚焦与美国国立卫生研究院的共享仪器授予S10RR023717威斯康星大学动物学系（PI条例草案Bement）收购。 埃里卡Namrata Asuri，安德森，和马修粘土本文同等贡献。

1。大会注射的幻灯片和影像幻灯片注射用幻灯片： <ol><li>根据制造商的指示准备Sylgard有机硅弹性体。 </li><li>使用Sylgard债券三个标准玻璃显微镜幻灯片一起堆放其他两个幻灯片，这是安排在长边侧端的交集上一张幻灯片。顶部的幻灯片创建一个直角的角落或“墙”之间的顶部和底部的幻灯片。 </li><li>让Sylgard设置过夜。这些幻灯片是可重复使用的的。 </li></ol>影像幻灯片： <ol><li>我们用一个标准的玻璃显微镜幻灯片（75毫米x 25毫米x 1毫米）大小相同的矩形不锈钢切，使商会适合显微镜舞台上的幻灯片持有人。切一个1.5厘米的洞是在矩形的中心。 </li><li>贴上22毫米2盖玻片Sylgard金属矩形。使用刚好够Sylgard彻底封盖玻片。倒置显微镜上的形象，胚胎将被安装在此盖玻片，并从底部成像。 </li><li>让Sylgard设置过夜。这些幻灯片是可重复使用的的和70％之间使用乙醇清洗。 </li></ol> 2。制备的DNA结构为了表达组织特异性的方式在的基因，感兴趣的基因被克隆背后的细胞特异性启动子。我们使用的斑马鱼neurogenin1的基因（ - 3.1ngn1）的顺式调控元件 2或SOX10基因（- 4.9sox10）3驱动器中的感觉神经元或神经嵴细胞的表达，分别。细胞和膜的动态可视化，我们对此表示细胞质或本地化膜荧光团。形象F -肌动蛋白的分布，我们表示融合mCherry（UtrCH mCherry）utrophin调宁蛋白同源结构域的DNA编码。 UtrCH mCherry传感器探头允许在不干扰与肌动蛋白动力学1，F -肌动蛋白的可视化。我们生成这些DNA的结构重组基于克隆策略4，在斑马鱼Tol2kit 5向量使用Invitrogen公司的网关。 <ol><li>生成表达载体（S），使用多站点网关技术和向量从斑马鱼Tol2kit 5 。质粒含有pT2KXIGDin Tol2骨干。 </li><li> QIAfilter密准备质粒试剂盒（QIAGEN公司），并在无菌水洗脱纯化的质粒DNA。 DNA并不需要注射线性。 </li></ol> 3。注射DNA的结构<ol><li>稀释的DNA的10-50微克/毫升的DNA终浓度为0.2％酚红水（在注射过程中的可视化）。 </li><li>填充和校准针：回填约1μLDNA溶液一把拉住毛细管针插入持针注射器具（我们使用一个Picospritzer）。用细镊子，打破关闭针尖端张开，大约是10毫米直径的尖端。测量的矿物油与目镜测微计液滴的直径。调整压力持续时间设置，以获得约1 NL体积的液滴。 </li><li>在E3胚胎培养基（5mm的氯化钠，氯化钾0.17mM，0.33mM 氯化钙 ，0.33mM硫酸镁4 * 7H 2 O，pH值7.0），收集1 -细胞阶段的胚胎。 </li><li>约30-60胚胎转移到注射幻灯片在角落的顶部和底部的幻灯片之间形成合适的角度和位置胚胎。删除的大部分表面张力的幻灯片，使胚胎举行的E3。 </li><li>用解剖针弯曲旋转胚胎细胞，所以对注射幻灯片的墙上。 </li><li>使用显微操作针，通过蛋黄和成单细胞胚胎，通过胚胎绒毛膜引导。注入0.5-1 NL进入细胞内的DNA溶液。 </li><li>转移到培养皿在E3和孵化的胚胎在28.5 ° C。如果有必要，可以放慢发展，在较低温度下进行孵化的胚胎。 </li></ol> 4。成像安装胚胎<ol><li>约一个小时，用细镊子胚胎取出chorions前胚胎达到成像（受精后约14-17小时，我们的目的）所需的年龄。 </li><li>选择与使用解剖显微镜配备荧光标记的细胞胚胎。我们使用尼康AZ100，因为它的工作距离长和高放大倍率的组合范围的4倍的目标（40X放大倍率）。 </li><li>胚胎放入一个离心管中，约50μL的E3含0.2％Tricaine麻醉（10X浓度）。 </li><li> 10毫米HEPES（保持在42 ° C）稀释Tricaine终浓度为0.02％，添加500μL的1％，在E3低熔点琼脂糖。 </li><li>立即转移到琼脂糖下降的胚胎使用大口径玻璃吸管的成像幻灯片的盖玻片。 </li><li>琼脂糖变硬之前，胚胎的位置，所以对底部的盖玻片（神经嵴细胞和背侧脊髓感觉神经元下降的背下来）标记细胞的地区面临。 </li><li>组装成像室：外套一个塑料环与硅真空润滑脂，并加盖金属成像幻灯片（外直径2厘米，3毫米高）的一方。大衣环的另一端与油脂。 </li><li>填写E3含10毫米的HEPES和0.02％Tricaine室。顶端脂环表面贴上22毫米2盖玻片密封腔顶部。 </li></ol> 5。 4D成像IX81显微镜奥林巴斯FV1000共聚焦细胞行为图像采集的细节将取决于你的显微镜系统的细节。在这里，我们描述奥林巴斯FV1000共聚焦显微镜的收购过程中的时间推移。看到以前的朱庇特的时间推移收购协议蔡司LSM 510共聚焦系统6。 <ol><li>将幻灯片固定在显微镜舞台上的成像室，并专注于使用透射光下一个10X或20X的目标胚胎。 </li><li>切换到一个60X的目标（1.2​​或更高的NA）和重点标记的细胞与epifluorescence。我们使用的油浸（1.35 NA 60xO UPLSAPO）或用水浸泡（1.20 NA 60xW UPLSAPO）镜头。 </li><li>在FV软件（版本1.7c），单击在XY重复开始激光扫描。 </li><li>调整收购设置和图像采集控制激光输出，扫描速度，高压，增益和偏移水平，优化信号，同时保持在最低水平（25％或更低）激光功率，以防止组织损伤和减少漂白。 </li><li>为了获得一个Z系列，Z - Stack的上限和下限，随着步长。确保从事深度图标，XY扫描图标的XY和Z突出。通过点击的XYZ扫描图标，开始收购。 </li><li>为了获得一个时间推移系列，TimeScan标题下，在采集设置“窗口设置的时间间隔的时间点和数量。图像采集控制窗口中点击时间图标，使XY扫描图标的XY（Z）和T突出。按一下XY（Z）T扫描图标，开始收购。 </li><li>另外，TimeController可用于建立一个时间推移。此编程生成大型数据集时非常有用，因为它不超过内存分配。在“设备”下，打开TimeController窗口。创建一个新的成像任务。图像参数“窗口将打开。点击FV状态“按钮，更新采集设置。确保保存和删除终止标题下检查。设置输出文件的名称，并单击“确定”以保存设置并关闭窗口。使用循环功能设置所需的时间间隔和时间点。单击“准备”，并开始着手收购。暂停和Z型转向功能可用于重新调整，而成像。 </li></ol> 6。代表性的成果数字和电影描绘的膜有针对性的荧光基团或肌动蛋白的生物传感器探头标记的感觉神经元和神经嵴细胞的例子。 <img src="/files/ftp_upload/1726/1726fig1.jpg" alt="图1" /> 图1共聚焦显微镜图像一个TG（ngn1：GFP - caax）（Z -预测）：mCherry - caax DNA的转基因胚胎注入25 PG ngn1。 mCherry - CAAX标签的所有标记Rohon胡子感觉神经元的背景下一个神经元红 （ 一）绿色（B）C）的绿色和红色通道的合并图像。比例尺= 50微米。 <img src="/files/ftp_upload/1726/1726fig2.jpg" alt="图2" /> 图2共焦Z -〜20 PG ngn1注入胚胎中的神经元 ： 预测 。mCherry - UtrCH DNA）Rohon胡子在16.5 HPF胚胎感觉神经元。 F -肌动蛋白的信号强度在生长锥（箭头）和二）在20 HPF胚胎神经元表现出强大的F - actin的信号在支周围神经轴突（箭头）和较弱的信号在较为成熟的中央生长锥沿新形成的神经轴突的突起。轴突（箭头）。比例尺= 50微米。

<ol>
	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, 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=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li></ol>

注入的DNA的最佳浓度会有所不同规模和实力的推动者而兴建，并应凭经验确定。太多的DNA注射剂可能会导致不健康的胚胎具有广泛的细胞死亡，而太少会导致表达转基因注入胚胎的比例很小。 DNA的表达水平的荧光信号，从细胞到细胞的不同强度相关。在epifluorescence胚胎排序时，排除那些与表达非常高的水平。显得非常明亮的标记的细胞，通常也显示出明显的迹象，如细胞形态异常，标签分子misloc...

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<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5040-250G</td>
<td>&nbsp;</td>
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<td>Sylgard Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>184</td>
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<td>QIAfilter Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen Inc.</td>
<td>12243</td>
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<td>Low melting point agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-014</td>
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<td>Picospritzer</td>
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<td>Parker Instrumentation, General Valve Division</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

斑马鱼是一种理想的模型成像细胞在体内发育过程中的行为。斑马鱼的胚胎是外部受精，因而很容易在发展的各个阶段。此外，他们的光学清晰度，可在自然环境完整的胚胎细胞和分子动力学的高分辨率成像。我们使用的是一种实时成像方法，分析在神经嵴细胞迁移和神经轴突的生长和指导细胞的行为。 实时成像是了解调节细胞运动过程的机制，特别有用。要前伸活动和分子动力学，细胞运动的细节，如可视化，这是有利的标签单个细胞。在斑马鱼，质粒DNA注入产生一个短暂的马赛克的表达模式，并提供比其它细胞标记方法明显的好处。例如，转基因株系往往标签整个细胞群，从而有可能掩盖在一个单元格（或分子分布的变化）的罚款突起可视化。此外，在单细胞阶段的DNA注射，创伤小，更精确比染料注射在稍后阶段。 在这里，我们描述一个标签个别发展中国家的神经元或神经嵴细胞，其在体内的行为成像的方法。我们注入1 - 细胞期胚胎，结果马赛克转基因表达质粒DNA。载体含有细胞特异性启动子驱动的感觉神经元或神经嵴细胞的一个子集的兴趣的基因表达。我们提供膜有针对性的GFP标记的细胞或生物传感器探头，允许在活细胞1的F -肌动蛋白的可视化的例子。 埃里卡Namrata Asuri，安德森，和马修粘土贡献同样对这项工作。

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

本协议描述了生活的斑马鱼胚胎的单个神经元或神经嵴细胞成像。使用此方法来研究细胞行为和肌动蛋白定位使用荧光共焦时间推移显微镜。

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

live-imaging-cell-motility-actin-cytoskeleton-individual-neurons

Research

JoVE Journal

Biology

13.3K 次观看.  University of Wisconsin-Madison. 本协议描述了生活的斑马鱼胚胎的单个神经元或神经嵴细胞成像。使用此方法来研究细胞行为和肌动蛋白定位使用荧光共焦时间推移显微镜。

视频: Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...