我们的工作是支持的生物技术和生物科学研究理事会。我们想感谢我们恳请提供CFP-LC3的表达质粒吉森TAMOTSU教授。

1。细胞的制备<ol><li>种子的低传代次数的HEK-293T细胞稳定表达GFP-DFCP1对22毫米的圆形盖玻片的Dulbecco修饰老鹰的培养基（DMEM）中培养细胞过夜，至30-40％汇合（大致目标为80％汇合后2天 - 一天的活细胞成像）。 </li></ol> 2。细胞转染<ol><li>准备转染复杂的混合，每块板，含100微升的OptiMEM的我减少血清培养基，3微升X-tremeGENE的DNA转染试剂和0.5微克pECFP-LC3质粒DNA。上下吹打，轻轻混匀，在室温下孵育15分钟。 [注：我们已经多次发现其他脂质体2000转染试剂，比如，有很多毒性，此外，他们还与许多显微镜技术对自己的干扰产生的荧光颗粒。 </li><li>吸介质板和补充新鲜DMEM预温在37℃ </li><li>转录sfection复杂的细胞通过移液，孵育细胞24小时。 </li></ol> 3。细胞孵育的细胞器标记（可选） <ol><li>添加Mitotracker有/ lysotracker的DMEM培养液的最终浓度为75 nM，并保持在冰上，用铝箔覆盖在隼管，沿整个实验天，以避免曝光和重复的冷冻和解冻循环。 </li><li>取出2毫升的Mitotracker有/ lysotracker的含培养基的等分试样，并在37℃下预热，从转染细胞中吸出培养基，更换Mitotracker有/ lysotracker的含培养基孵育细胞30 - 60分钟。 </li></ol> 4。制备的活细胞成像的孵化室（图1） <ol><li>清洁金属和塑料O型圈，用75％乙醇和应用硅润滑脂，金属O型圈的边缘上。 </li><li>使用镊子除去盖玻片从板干燥介质从盖玻片的底侧的多余的避免介质与油脂混合太多，因为这会增加泄漏的可能性。 </li><li>离开盖玻片塑料O型圈的窗台上休息，适合金属O型圈顶部的塑料O型圈，与盖玻片夹在中间，以创建一个封闭的腔。 </li><li>从这点上，无缓冲剂，避免长时间培养的细胞在DMEM培养基中，以防止pH值的变化，以发生，DMEM中的碳酸氢盐缓冲系统需要人工的CO 2浓度顶起来的腔室与从板的介质;为5-10％，环境空气中的CO 2浓度要低得多。 </li></ol> 5。饥饿的细胞<ol><li>将孵化室显微镜舞台上。 </li><li>完全培养基吸出洗涤用2ml饥饿培养基中的3倍，以确保没有氨基酸的DMEM培养液，最终抑制自噬反应仍然设置计时器。 </li></ol> 6。显微镜<ol><li>将需要一个适当的成像系统，其被配置在活细胞的宽视场落射荧光。这通常包括研究级倒置显微镜框架，激烈的广谱光源，反射镜和过滤特定的荧光蛋白（S）/染料（S）的利益，一个高品质的物镜，一个敏感的CCD / SCMOS相机和孵化室。所有主要的显微镜制造商提供完整的宽视场系统适合活细胞成像，但它也有可能以家庭建立一个系统，使用各种厂家的组件和控制它使用开放源码软件如微经理（ <a href="http://valelab.ucsf.edu/~MM/MMwiki/" target="_blank">HTTP： / / valelab.ucsf.edu /月/ / MMwiki</a> ）。在我们看来，最重要的方面是使用高灵敏度的系统，这样的表达水平的荧光记者可以保持在最低限度。 </li><li> SEL阿拉斯适当的细胞图像。 <ol><li>选择大而扁平的细胞，将允许更多的自噬体形成事件被捕获。此外，已经开始生产更大数量的omegasomes的的选择的细胞。 </li><li> 30分钟后开始视频捕获或到自噬响应，以争取更大的自噬体形成比例，每部影片的事件。 </li></ol></li><li>成像。 <ol><li>使用高倍率镜头（100X 1.4 NA）。 </li><li>调整的激发光的强度最大的10-20％，以防止光漂白。 </li><li>设置相机（滨松ORCA ER，像素大小为6.45微米）到100-500毫秒曝光，2x2的分级和100的增益。 </li><li>设置每10秒1帧图像采集率。 </li></ol></li></ol> 7。自噬体形成ImageJ的活动创建蒙太奇 [这是可以做到在一个非系统的方式，通过简单地扫描为电子合并后的视频通气孔的兴趣，但它也可以是系统化的，如下面所述。] <ol><li>打开图像栈3（或2）捕获ImageJ的/斐济的渠道。 </li><li>应用绿色，红色和蓝色的LUT（查找表），相应的通道;合并3种颜色，并保存。 </li><li>从“分析”选项卡中，选择“工具”&gt;“ROI经理... &gt;设置，然后选择一个随机区域定义大小的堆栈中的第一张图像。 </li><li>从“图片”选项卡，选择“复制”，以便复制所选区域的堆栈。 </li><li>从“图片”选项卡，选择“堆栈”&gt;蒙太奇...创建一个试探性的蒙太奇包含的所有帧抓获。扫描所有帧中的蒙太奇，一个完整的自噬体形成事件的第一个和最后一个帧事件的记录。 </li><li>从“分析”选项卡中，选择“工具”&gt;“ROI经理...选择在第一幅图像的堆栈对其他2种颜色，以及合并后的颜色图像和同一地区重复堆叠。 </li><li>从T他文件“选项卡上，选择”新建“&gt;”图像“，并设置宽度根据初步选定像素使用ROI经理，设置高度的数量4倍的高度设置在ROI经理加上3个像素之间的颜色和空间合并，并设置在每个堆栈的帧的数量的片。 </li><li>从“图片”选项卡，选择“堆栈”&gt;“工具”&gt;“插入...，然后插入一个在另一个之上，每个子协议栈之间留下1个像素的空间。 </li><li>从“图片”选项卡，选择“堆栈”&gt;蒙太奇...创建一个蒙太奇的起点和终点与第一个和最后一个帧的自噬体形成捕获的事件。 </li></ol>

<ol>
	<li>Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+process+and+function.">Autophagy: process and function.</a> Genes Dev. 21, 2861-2873 (2007).</li><li>Mizushima, N., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Atg+proteins+in+autophagosome+formation.">The role of Atg proteins in autophagosome formation.</a> Annual review of cell and developmental biology. 27, 107-132 (2011).</li><li>Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+from+phenomenology+to+molecular+understanding+in+less+than+a+decade.">Autophagy: from phenomenology to molecular understanding in less than a decade.</a> Nat. Rev. Mol. Cell Biol. 8, 931-937 (2007).</li><li>Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+revisited:+a+conversation+with+Christian+de+Duve.">Autophagy revisited: a conversation with Christian de Duve.</a> Autophagy. 4, 740-743 (2008).</li><li>Yla-Anttilba, P., Vihinen, H., Jokitalo, E., Eskelinen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tomography+reveals+connections+between+the+phagophore+and+endoplasmic+reticulum.">3D tomography reveals connections between the phagophore and endoplasmic reticulum.</a> Autophagy. 5, 1180-1185 (2009).</li><li>Hayashi-Nishino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subdomain+of+the+endoplasmic+reticulum+forms+a+cradle+for+autophagosome+formation.">A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.</a> Nat. Cell Biol. 11, 1433-1437 (2009).</li><li>Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+in+vivo+analyses+of+cell+function+using+fluorescence+imaging+(*).">Emerging in vivo analyses of cell function using fluorescence imaging (*).</a> Annu. Rev. Biochem. 80, 327-332 (2011).</li><li>Mizushima, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+autophagosome+formation+using+Apg5-deficient+mouse+embryonic+stem+cells.">Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.</a> The Journal of Cell Biology. 152, 657-668 (2001).</li><li>Itakura, E., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+autophagosome+formation+site+by+a+hierarchical+analysis+of+mammalian+Atg+proteins.">Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins.</a> Autophagy. 6, 764-776 (2010).</li><li>Axe, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosome+formation+from+membrane+compartments+enriched+in+phosphatidylinositol+3-phosphate+and+dynamically+connected+to+the+endoplasmic+reticulum.">Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.</a> J Cell Biol. 182, 685-701 (2008).</li><li>Walker, S., Chandra, P., Manifava, M., Axe, E., Ktistakis, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+autophagosomes:+localized+synthesis+of+phosphatidylinositol+3-phosphate+holds+the+clue.">Making autophagosomes: localized synthesis of phosphatidylinositol 3-phosphate holds the clue.</a> Autophagy. 4, 1093-1096 (2008).</li></ol>

没有利益冲突的声明。

在这个协议中所描述的方法允许自噬体的形成过程中的蛋白质的定位的可视化。我们已经尝试了各种方法，可视化描述的事件，包括点扫描共聚焦，旋转盘共聚焦和全内反射荧光显微镜（TIRF）。我们发现，一般用途的标准宽视场落射荧光灵敏度和分辨率之间的最佳折衷。这可确保良好的信号噪声，最小photo-bleaching/photo-toxicity和快速收购。光学切片的缺乏不是一个问题，如果被选择的单元格的适当...

自噬是一个高度动态的过程，这就需要大量的蛋白质，协调的最终结果自噬体形成1-3。显微镜的技术可能是最常用的应用为研究自噬4。大多数自噬蛋白的本地化已被广泛地研究了在固定的细胞，通过免疫染色的内源性蛋白和荧光标记的外源蛋白质的表达。此外，电子显微镜（EM），单独和组合使用免疫金标，精致的细节描 ​​述了这些结构5,6。尽管这些技术已经建立了一个事实，即我们了解自噬体形成的3个维度空间，他们并没有提供足量的4 个维度的信息-时间。活细胞成像克服了这个障碍，因为它允许自噬体的形成可能接近ble到实时7。这种技术最早是研究自噬吉森和同事8，并已被越来越多地用于今后。 时间推移显微镜捕获的POI在活细胞中，并经过一段时间的本土化。通过比较这些信息与一个特征的自噬和/或细胞器标记活细胞成像分析，可以把兴趣点，在更大的空间和时间范围内自噬体的形成。活细胞成像分析是基于POI本地化沿自噬体的形成的所有步骤的重复捕获，而固定细胞成像是基于一个单一的捕获。因此，活细胞成像可以证明在自噬体形成的具体步骤的POI的贡献，而固定细胞成像，只能承担角色的POI，其平均定位的基础上同时捕获许多自噬在不同阶段其lifecyCLE。 虽然活细胞成像分析能力高的方法，它具有一些固有的局限性，这应该加以考虑。首先，活细胞成像的要求中的一个或多个外源性荧光标记蛋白的表达。荧光标记往往是体积大，它们有时可以改变一种蛋白质的行为，由于空间位阻的原因。这种情况加剧膜蛋白的，因为他们需要在有限的空间发挥作用的2维膜。值得注意的是，自噬体膜的结构，因此它们的形成需要大量的膜相关蛋白。 另一些问题是连接到该POI的表达水平。原则上，应表达的外源蛋白质与内源性蛋白的水平。这确保了其亚细胞定位的重要调节器将不饱和的，和日E解析生物相关。此外，应该避免的，自噬蛋白的过度表达时，他们都表达了以上的内源性水平，他们往往会抑制自噬反应9。相反，因为POI的表达水平应该是很高的，足以让一个很好的一段时间后，其本土化没有光漂白，要达到一个妥协。实现最佳的外源蛋白质在哺乳动物细胞中的表达水平，需要微调的特定的，但它是可行的，通过建立和筛选稳定表达细胞系的不同水平的POI。 即，可以实现与标准的荧光显微镜的空间分辨率是另一个限制因素。分辨率可能被限制为一定数量的原因，但在最好的情况下，将横向分辨率在250 nm附近。这意味着，比这更小的距离分隔的任何对象会出现连接（或作为一个单一的将代表大于他们实际上是在图像对象），小于250纳米的物体。因此，图像应始终被解释考虑到这一点和互补的技术，如EM，将需要解决精美的超结构的细节。 最后，活细胞成像的本质要求露出光电池，有可能在很长的一段时间。这可能会改变细胞的生理反应，这种现象被称为光毒性。 我们已成功地用于活细胞成像的PI3P结合蛋白DFCP1的第一次来形容，自噬来源于富含PI3P环状结构称为omegasomes，这是密切相关与ER股10,11。我们已经清楚地表明，LC3阳性结构开始形成在与omegasomes密切相关。在这里，我们建议，用人的细胞系稳定表达GFP-DFCP1活细胞成像的感兴趣的蛋白质，建立了一个强大的空间和时间的帧的表征，它的作用在自噬体的形成。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>OptiMEM I</td>
			<td>Invitrogen</td>
			<td>31985-062</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MitoTracker Red FM</td>
			<td>Invitrogen</td>
			<td>M22425</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LysoTracker Red DND-99</td>
			<td>Invitrogen</td>
			<td>L-7528</td>
			<td></td>
		 </tr>
		 <tr>
			<td>X-tremeGENE 9 DNA Transfection Reagent</td>
			<td>Roche Applied Science</td>
			<td>6365787001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>22 mm coverslips</td>
			<td>VWR</td>
			<td>631-0159</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm plates</td>
			<td>Fisher NUNC</td>
			<td>153066</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Silicon grease</td>
			<td>RS Components Ltd. </td>
			<td>RS 494-124</td>
			<td></td>
		 </tr>
		 <tr>
			<td>O-rings</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		 </tr>
		 <tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td>Suggested alternative to custom-made O-rings</td>
		 </tr>
		 <tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>Inverted microscope</td>
		 </tr>
		 <tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UPLSAPO 100XO</td>
			<td>N.A. 1.4, W.D. 0.13, FN 26.5</td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600 10B</td>
			<td>Progressive scan interline CCD</td>
		 </tr>
		 <tr>
			<td>Illuminator</td>
			<td>TILL Photonics</td>
			<td>Polychrome V</td>
			<td>Ultrafast monochromator</td>
		 </tr>
		 <tr>
			<td>Incubation chamber</td>
			<td>Solent Scientific</td>
			<td>Cell^R IX81</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software</td>
			<td>Olympus</td>
			<td>SIS xcellence</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

live cell imaging of early autophagy events: omegasomes and beyond

自噬细胞反应缺乏的营养物质，特别是氨基酸的情况下触发。自噬是指由形成的双层膜结构，称为自噬体，即隔离细胞质中，寿命长的蛋白质和蛋白质聚集体，有缺陷的细胞器，甚至是病毒或细菌。自噬体最终导致大量降解它们的内容与溶酶体融合，所产生的营养成分被再循环返回到细胞质。因此，细胞自噬是细胞的动态平衡至关重要，自噬失调可导致疾病，最显着的神经退行性疾病，衰老和癌症。 自噬体的形成是一个非常复杂的过程，其的细胞已分配一组特定的蛋白质，被称为核心自噬机械。是该核心自噬机械的功能辅以不同的细胞过程中所涉及的额外的蛋白质， 如在MEMBRANê贩运，线粒体和溶酶体生物学中。自噬体的形成和降解的协调，这些蛋白质构成的高度动态和复杂的响应的自噬。活细胞成像允许按照每个自噬相关蛋白的分子贡献水平的自噬体形成一个单一的事件，并实时的，因此该技术提供了一个高时空分辨率。 在这里，我们使用的细胞系稳定表达GFP-DFCP1的，我们的分析建立了空间和时间的情况下。 DFCP1的痕迹omegasomes，易制毒化学结构导致自噬体的形成。可以打上一个红色或青色荧光标记蛋白质的兴趣点（POI）。不同的细胞器，如急诊室，线粒体和溶酶体，都参与自噬体的形成在不同的步骤，并且可以使用一个特定的跟踪染料标记。时间推移显微镜AUTOPhagy在这个试验性设置，使得要被提取的信息有关的第四维， 即时间。因此，我们可以按照自噬在空间和时间上的POI的贡献。

描述的协议，我们已经按照本地化CFP标记LC3稳定的细胞系表达GFP标记DFCP1的自噬诱导条件下，使用时间推移显微镜。这个实验的结果是2系列或成堆的图像，从绿色和蓝色通道之一，对应GFP-DFCP1和CFP-LC3的捕获。我们已进一步分析这些视频使用ImageJ，在以对应于单一的自噬体形成的协议部分中所描述的事件，创建蒙太奇。这种分析使我们能够证明LC3阳性的自噬体来源于一个阳性DFCP1的omegasome。在蒙太?...

Watch this Scientific Journal Video about Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond at JoVE.com

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

时间推移显微镜荧光标记的细胞自噬标记允许自噬反应具有高时空分辨率的动态监测。使用特定的自噬和细胞器标记在3种不同颜色的组合，我们可以按照自噬体形成在一个强大的空间和时间上下文的蛋白质的贡献。

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

live-cell-imaging-of-early-autophagy-events-omegasomes-and-beyond

Research

JoVE Journal

Biology

18.4K 次观看.  The Babraham Institute. 时间推移显微镜荧光标记的细胞自噬标记允许自噬反应具有高时空分辨率的动态监测。使用特定的自噬和细胞器标记在3种不同颜色的组合，我们可以按照自噬体形成在一个强大的空间和时间上下文的蛋白质的贡献。

视频: Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

科研

生物学

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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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  ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...