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

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 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 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 CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;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>08-772-1H</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&amp;ndash;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/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/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/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

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

科研

遗传学

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

生物化学

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 ...

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

神经科学

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

使用荧光相关光谱检测蛋白质聚合

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

调查Coronin A在早期饥饿反应中的作用

摘要

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此视频中的章节

基于细菌选择和生长的盘状细胞基因工程

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

研究蛋白质的质量控制体系

对急性机械刺激的网柄反应的评估

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

使用荧光相关光谱检测蛋白质聚合