We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li>通过观察时间推移显微镜粘菌的早期饥饿反应。 <ol><li>生长DH1.10细胞或CORA缺陷细胞在Erlenmayer烧瓶含有HL-5培养基（对于1升:5克蛋白胨，5克thiotoneë胨10克葡萄糖，5克酵母提取物0.35克Na 2 HPO 4 * 7H 2 O，0.35克KH 2 PO 4，0.05g的双氢硫酸盐，pH值6.6）在22℃与160 rpm的旋转振荡培养箱。保持细胞以0.01×10 6个细胞/ ml和2×10 6个细胞/ ml之间的密度。 </li><li>检验细胞聚集，通过收获对数期生长DH1.10细胞21或CORA缺陷细胞在DH1.10背景生成10，在22℃用HL-5培养基振荡培养生长。这样做，取细胞（通常为10至50毫升）中，离心适量在400 xg离心3分钟，并在BSS（10毫摩尔NaCl，10毫氯化钾洗涤细胞两次，2.5mM的氯化钙 ，pH值6.5）。 </li><li>算使用血球细胞。接着，板细胞以（5，10，20，或40）的密度×10 4个细胞/ cm 2在24孔板中。让他们在22℃，在BSS坚持1小时。 </li><li> 10如前所述，拍摄图像的每135秒通过可视化时间推移显微镜聚集。使用活细胞成像设置配备5X物镜和由适当的软件自动化的电子倍增电荷耦合器件照相机（见材料数据表 ）。 </li></ol></li><li>饥饿时粘菌细胞内cAMP脉冲。 <ol><li>检查外部施加的cAMP脉冲上DH1.10细胞21或DH1.10 CORA缺陷细胞10的发展的影响，通过收获细胞。这样做，取细胞的适量（通常在10至50毫升）中，离心以400 xg离心3分钟，并在BSS洗涤两次。 </li><li>腠nt的细胞使用血细胞计数器和重悬细胞，以在BSS 1×10 7细胞/ ml的密度。施加脉冲之前摇动培养（160转）在​​22℃2小时。添加使用定时器控制蠕动泵的cAMP脉冲。编程泵历时5小时的递送5秒脉冲的15微升50纳米的cAMP（最终浓度）的每6.5分钟。 </li><li>算使用血球细胞。随后，镀细胞以（5，10，20，或40）的密度在24孔板×10 4个细胞/ cm 2。允许坚持在BSS 1小时。 </li><li>之后用5X客观亮场显微镜在22℃，16小时可视化聚集。 </li></ol></li><li>通过暴露于条件培养基粘菌发展的诱导。 <ol><li>描述22准备新鲜的条件培养基。收集日志相DH1.10细胞21或DH1.10 CORA缺陷细胞10，颤抖崇拜使用移液管，离心3分钟，在400 XG用HL-5培养基数目字在22℃和洗涤细胞三次PBM（0.02磷酸钾，10μM 氯化钙和1mM MgCl 2的，pH值6.1）。 </li><li>算使用血球细胞，重悬这些在PBM以1×10 7个细胞/ ml的密度，并在110转/ 22℃下摇动20小时。 </li><li>收集条件培养基，在400×g下 3分钟离心后，在8000×g下通过离心澄清在4℃15分钟。 </li><li>通过0.45微米的过滤器过滤条件培养基（见材料表 ）和PBM稀释三倍。 </li><li>算使用血球和板细胞以（5，10，20，或40）的密度×10 4个细胞/ cm 2在24孔板中。让他们在22℃下坚持1小时。 </li><li>与前面制备的条件培养基的细胞的交换上清液。 </li><li>可视化聚集的压脚提升16小时使用5X目标明视场观察。 </li></ol></li></ol>

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No conflicts of interest declared.

的coronin蛋白质在真核分支的最类群找到。 粘菌 coronin A，哺乳动物coronin 1的同源物，是参与早期饥饿反应，由于coronin A缺乏的细胞不能够在早期发育，以形成聚集的中心周期10。为了能够定量和准确地评估在菌株之间发展的延迟，在显微镜活细胞成像设置与自动阶段控制器是一个必不可少的工具。 D.菌在其生命周期1-4的几个阶段利用cAMP的作为必要的信号分子。一期间的cAMP是必需的检查点是从单个细胞到多细胞结构的形成的过渡。环磷酸腺苷波饥饿〜6小时是由细胞检测，并提供趋化信号朝聚集中心5移动后发病。这些环磷酸腺苷波可通过实验脉冲饥饿D.被模仿菌细胞的cAMP每6.5分钟一段5H 8,9。使用此过程来饿死细胞长达施加的cAMP脉冲，以便获得可靠的结果之前在2h是重要的。利用该实验装置，我们能够证明coronin A的作用cAMP信号的上游，因为这些外部施加脉冲救出CORA缺陷细胞的发展（ 图3和图 5）。这些结果还表明，尽管coronin A显示在感测早期饥饿响应于具有至关重要的作用，它是可有可无的，如趋化和细胞聚集本身 10处理。 该聚集试验本身是研究粘菌的早期发展一个可靠的方法。但是，也有可能影响这些电子的再现几个陷阱xperiments，并且因此重要的是几个因素都考虑在内:分析CORA缺陷细胞，当细胞的年龄起着重要的作用。这些细胞趋向从它们解冻点2周后约到的表型和重新聚集补偿。细胞的密度是一个重要的因素，以及，作为细胞，可展示信令缺陷，而不是一个趋缺陷可能会倾向于在较高的密度聚集由于早期饥饿因子的浓度增加。细胞在这些分析的精确计数和稀释的重要性怎么强调也不过分。对于不同的菌株，测定的多个变量，诸如细胞密度，粘附的时间，发展的时间的调节，是必需的。如这里描述的聚合实验是一个通用的技术可以被修改以适应不同的查询:可以将细胞饥饿，或发展可以刺激或抑制使用不同的化合物中，所有由于用于聚合的特定调制。在此处描述的设置还允许无偏方法与早期发育缺陷识别菌株，以及这对早期发育重要的基因或因子的鉴定。这也是该方法的主要缺点是:在聚集测定是完整发育周期的缩小版本。因此，它不适合于影响发育周期的后期阶段的表型的分析。的主要优点是采用聚集测定来大规模和自动化形式的可能性:在48孔板并用在开发过程中自动摄像记录的16小时后，将聚集测定证明是更多的时间和资源效率比上发展琼脂平板上。 在我们绕过早期的饥饿反应，利用外部施加的cAMP脉冲实验中，我们发现，coronin A是由&lt;参与分泌因子检测EM&gt;在饥饿盘基网柄细胞。 coronin A-介导的信号的早期饥饿反应中的确切机制尚不清楚。在这个阶段的发展所涉及的几个因素进行了研究，特别是条件培养基因子（CMF）25,26。然而，CMF是否作用在它自己的或者与辅因子尚不清楚其信号级联只有部分了解。使用粘菌细胞的聚集能力 下浸没条件此处描述为正读出的发展使得相比于琼脂表面聚集测定以时间有效的方式进行大规模筛选实验。然而，淹没条件下聚合试验确实有不履行完整的发育周期细胞会在初始阶段聚集被逮捕的缺点。因此，此处所描述的技术不适合于以后发育PROCE的检查小规模企业。总之，这里介绍的实验方法可以允许诱导coronin A-依赖进入早期饥饿反应潜在的早期饥饿因子（S）的进一步鉴定。

蛋白质的coronin家族是高度整个真核生物保守的。这些蛋白由氨基末端色氨酸天冬氨酸（WD）区域，随后由连接到羧基端卷曲螺旋结构域13,14独特区域含有重复-（ 图1）的存在为特征。 Coronins已牵涉多种细胞功能，包括细胞骨架调节和信号转导12。在哺乳动物中，多达六个短coronin分子（coronin 1-6）以及一个"串联"coronin 7，可共表达12,15。 Coronin 1是最广泛研究的家族成员，并且显示是参与病原体的破坏，T细胞存活和神经信号。怎么样，究竟coronin 1进行这些活动仍不清楚。而coronin 1所示调节的 Ca 2+和信令依赖cAMP的-以及F-肌动蛋白细胞骨架调制16-18中，潜在的共同在哺乳动物中最多7家族成员的-expression使得它具有挑战性的研究coronins的分子功能在这些系统中，由于潜在的冗余。不同于哺乳动物的有机体中，低等真核生物粘菌表示只有两个coronin家庭成员（coronin A，哺乳动物coronin 1和coronin B的同源基因，哺乳动物coronin 7的直系同源基因）显然与非冗余功能15,19,20。这一事实使粘菌一种强效的模型来研究coronins的功能。 研究coronin A在粘菌中的作用，我们通过在含有用野生型的细胞或细胞缺乏coronin 10平衡盐溶液（BSS）缓冲组织培养板饥饿诱导的发展周期。我们发现，缺乏coronin A细胞无法形成在饥饿的多细胞聚集体。对于这种表型的的准确定量评估在本协议中所述的自动化活细胞成像是一个重要的工具。在缺乏coronin A细胞早期饥饿反应开始时的缺陷可以通过提供的cAMP脉冲，表明coronin内cAMP级联的上游A行为解救。 cAMP的脉冲的外源应用到模拟发展的起始在过去8,9被利用由几个实验室。然而，这个过程也被称为是高度依赖于细胞密度和定时。因此，这里描述的方案的目的是减少以保证高度的重现性，这些变异。两者合计，在这些研究中所使用的技术提供了强大的工具中粘菌的发育周期的早期阶段，以调查蛋白质的功能和必须识别向上以及coronin函数的下游效应的潜力。

<table>
	<tbody>
		<tr>
			<td>HL-5 media (for 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiotone E peptone</td>
			<td>BD Bioscience</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>AppliChem</td>
			<td>A3666</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>AppliChem</td>
			<td>A1043</td>
			<td></td>
		</tr>
		<tr>
			<td>dihydrostreptomycin-sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>D1954000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBM (0.02 M potassium phosphate, 10 &#956;M CaCl2, and l mM MgCl2, pH 6.1)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;m Filtropure S filter</td>
			<td>Sarstedt</td>
			<td>83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellobserver microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>custom built</td>
		</tr>
		<tr>
			<td>AxioVision software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPC Microprocessor&#8211;controlled dispensing pump</td>
			<td>ISMATEC</td>
			<td>ISM 931</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 135M microscope</td>
			<td>Zeiss</td>
			<td>491237-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation Shaker</td>
			<td>Inforst HT Minitron</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

粘菌变形虫发现于土壤中，对细菌为食。当食物来源变得稀少，它们分泌的因素来启动多发展计划，在此期间，单个细胞对聚集中心，趋化1-4。这个过程是依赖于环磷酸腺苷（cAMP）的5的释放。 Camp在波通过腺苷酸环化酶和磷酸二酯酶的一致行动产生的，结合G蛋白偶联受体的cAMP 6,7。一种广泛使用的测定法来分析所涉及的低等真核生物粘菌的发育周期的机制是基于细胞聚集在浸没条件8,9的观察。这个协议描述coronin A在发育周期的作用的分析通过饥饿在平衡盐溶液（BSS）10浸没组织培养板中。 Coronin A是广泛保守的PR的一员otein家人已经在各种各样的活动11,12牵连coronins的。缺乏coronin一盘基网柄细胞不能形成多聚集，而这个缺陷可以通过提供的cAMP脉冲，这被救出的coronin上游A行为cAMP的级联10。在这些研究中所描述的技术提供了强大的工具中粘菌的cAMP的级联的上游发育周期的初始阶段进行调查的蛋白质的功能。因此，利用这种聚集试验可允许coronin函数的进一步研究和推进我们coronin生物学的理解。

细胞在coronin一位选秀节目的缺陷在早期发育缺陷（ 图2）。在不存在coronin A细胞都无法形成多细胞聚集体，这是粘菌的发育周期期间的最初步骤。因此，coronin A显示早期饥饿反应和/或cAMP信号中发挥作用。事实上，由于缺乏在没有coronin A的多细胞聚集形成是伴随着减少cAMP信号10。然而，外源的cAMP脉冲的施加充分恢复野生型表型（ 图3）。这暗示该coronin A，而不是被直接参与cAMP信号，cAMP的级联的上游功能（ 图5）。当盘基网柄细胞暴露在饥饿条件下，它们导致早期饥饿的分泌因子22,23。一个最好的甜心涉及早期饥饿反应cterized因素是条件培养基因子（CMF）22。根据我们的结果，我们推测coronin一个或者调节这些早期的饥饿因子的分泌或参与由这些因素引起的信号转导。为了能够这两种效应，我们来进行条件培养基实验之间进行区分。的野生型或CORA缺陷型细胞暴露于从任饥饿野生型或CORA缺陷细胞10（ 图4）的上清液。这些上清液是多细胞聚集体形成的有效的引发剂，因此，含有该启动下游发育信号通路，包括cAMP信号的因素。我们的结果表明，从CORA缺陷细胞上清液同样能够诱导发展是从野生型细胞上清液的情况下（ 图4）。这暗示了CORA -缺陷细胞能够生产与在相似的水平，以它们的野生型对照分泌早期饥饿的因素。然而，CORA缺陷细胞既不能够给自己的上清液也不从野生型细胞（ 图4）提供的上清反应。因此，coronin A显示要参与信号早期饥饿因子诱导的途径，而不是他们的表达/分泌10（ 图5）。 在一般情况下，能够进一步调查由饥饿粘菌细胞诱导发育周期分泌的信号，淹水条件下的聚集测定是有效的读出。细胞以更高的密度将种子本能地聚合在一起，由于丰富的所有必需的信号。然而，在较低的密度仅加入条件培养基将诱导细胞聚集（ 图4）22,24 </suP&gt;。因此，此过程可以用于分级分离的条件培养基和早期的饥饿反应中研究了它们的活性不同的组件。 <img alt="图1" src="/files/ftp_upload/53972/53972fig1.jpg" /> 图1:存在于哺乳动物和 粘菌 Coronin蛋白的coronin家族的特征在于N-末端WD重复区域的存在，随后由唯一的结构域和C末端的卷曲螺旋区。虽然哺乳动物表达7 coronin蛋白（A）， 粘菌只有两个家族成员（B），研究它们的功能时，使冗余不太可能的风险。 Coronin A是哺乳动物coronin 1和coronin B的直向同源物显示了类似的结构域结构coronin 7.缩写:CC，卷曲螺旋结构域; UD，独特的领域; WD重复，色氨酸<html>天冬氨酸重复。长度，给出了在氨基酸（AA）小鼠序列（A）中。从皮特斯等人 ，2013 12修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53972/53972fig2.jpg" /> 图 2:CORA - 粘菌 无法早期饥饿反应过程中聚集的野生型（A）或CORA缺陷型（B）的粘菌细胞接种到多孔板以2×10 5个细胞/ cm 2的密度，在饿死BSS和成像历时20小时。比例尺= 100微米。.JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53972/53972fig3.jpg" /> 图3:从外部施加的cAMP脉冲营救 CORA 的早期发育缺陷 - 粘菌营养野生型（A和B）和科拉缺陷细胞（C和D）与正在脉冲之前进行洗涤和饥饿2小时（B。和D）或无（A和C），50纳米的cAMP 5小时每隔6.5分钟的暂停期间。然后洗涤细胞，再悬浮于BSS，并在10×10 4个细胞/ cm 2的密度置于培养皿。图像细胞播种后采取20小时。比例尺= 100<html>微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53972/53972fig4.jpg" /> 图4:CORA 缺陷型 粘菌 能够生产对发育感应所有必需的因素，但无法应对它们指数生长的野生型和CORA缺陷细胞中的PBM被洗涤并在的密度接种到多孔板（。 5，10，20，或40）×10 4个细胞/ cm 2时，在从野生型（A）或CORA缺陷型饥饿细胞（B）中得到的条件培养基温育。图像被接种细胞后取16小时。比例尺= 100微米。tp_upload / 53972 / 53972fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53972/53972fig5.jpg" /> 图 5:Coronin的当前模型中 粘菌 函数 Coronin A被参与早期饥饿反应导致的cAMP级联的诱导，并通过发育周期进展期间分泌因子（多个）的信号转导。从Vinet是等修改2014年10。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

调查Coronin A在早期饥饿反应中的作用<em&gt;粘菌</em&gt;通过聚集测定

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

investigating-function-coronin-early-starvation-response

Nanyang Technological University

Research

JoVE Journal

Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K 次观看.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

视频: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法:另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

可视化吞噬的早期阶段

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...