韩国国家研究基金会（NRF-2015R1A2A1A10053576），这项研究得到支持，从下一代BioGreen 21计划的资助（SSAC，授予PJ01137901），农村振兴厅，韩国。 RK，WS，ABB和DK经脑韩国21 Plus计划（BK21 +）的支持。

1突变体筛选与改变向光性和向重力响应<ol><li>制备1×Murashige和Skoog（MS）培养基，pH值5.7，用0.8％的琼脂一天实验之前。 <ol><li>加800毫升双蒸水至一个2升的锥形烧瓶中，并用磁力棒搅拌。 </li><li> 4.4克MS盐添加到锥形瓶中。 </li><li>加入0.5克2-（N-吗啉代）乙烷磺酸（MES），搅拌直到所有的盐的完全溶解。 </li><li>调整培养基至5.7用1M KOH的pH。 </li><li>介质传送到海量缸和使终体积到1L的DDH 2 O </li><li>倒回介质到2升锥形瓶中，加入8克植物琼脂。 </li><li>用铝箔紧紧包裹锥形烧瓶口。 </li><li>高压灭菌的MS培养基和DDH 2 O的15分钟，在121℃，15磅，和高压灭菌后，保持介质，在60℃培养箱和DDH 2 O的室温。 </li><li>冷却至60℃之后，倒入介质成125×125×20mm的3方板的层流长凳上（50毫升每块板）。 </li><li>允许琼脂通过保持所述板部分打开在RT固化约1小时。如果板被不立即使用，通过在冰箱用保鲜膜和储存在4℃下密封。 </li></ol></li><li>表面消毒用1.1％的次氯酸钠的种子点在1.1节制备的1倍MS琼脂平板上的种子。 注:在这里， 拟南芥 {Col-0中的种子，dsGSL8（Col-0中 ）} 15用于热带响应，胼胝质染色和HPTS装载拟南芥 {Col-0中和dsGSL8（Col-0中 ）EMS突变}种子（。未公布的）被用于在图2中嗜响应。 <ol><li>高压灭菌300毫升的DDH 2 O的瓶子。 </li><li>添加大约为一百种子为每个背景， 即突变体/过表达系与野生型（Col-0中 ）种子，到1.5ml微量离心管中。 </li><li>取出从4℃的冰箱1倍的MS琼脂平板上，在部分打开位置晾干层流工作台上。 </li><li>通过加入1ml的0.2×漂白溶液（1.1％次氯酸钠）的表面消毒的种子，并保持在微量离心管在摇床上以250rpm进行5分钟。 </li><li>让种子通过重力沉在底部，然后取出用微量漂白粉溶液。 </li><li>加入1毫升的新鲜的DDH 2 O（灭菌）的种子并颠倒离心管数次拌匀。让种子再次沉降，然后除去水分。 </li><li>重复步骤1.2.6 5-6倍。 </li><li>水双倍量加入到最终洗涤之后被留下的种子的体积。 </li><li>点上使用含有200微升的种子1毫升微量移液器尖端的半干燥1倍的MS琼脂平板上的消毒的种子（在cluding水）。 <ol><li>保持两个种子和​​至少两种行之间1.8厘米之间约0.1厘米的距离。圆点每平方板（125×125×20mm的3）五横行的种子。让水将盖到前盘稍干。 </li></ol></li><li>密封采用外科胶带板，堆栈中的所有板垂直，并用铝箔包裹。 </li><li>转移包裹板，以暗箱（没有进入光）。然后，保持暗箱在4℃的室温/冰箱3天。 </li><li>从该步骤开始，保持板不变的方向。经过冷处理3天，保持在22℃，植物培养室在接下来的3天暗箱。 3天后，检查在黑暗的房间在绿灯种子的发芽状态。 注:通常3日龄的Col-0黄化苗可长到1.6厘米长。 </li></ol></li><li>分析改变向光性一个在突变体或过度表达的特定基因的线十二次向地响应。在黑暗的房间中选择下绿灯只有同样大小和垂直生长的直苗。传送所选苗使用杀菌牙签的前端的新鲜的1X MS琼脂板上。 <ol><li>轻轻地选择自己的下胚轴的最低部分秧苗时不接触任何其他部分。确保所有的苗木具有相同的子叶/挂钩方向，转让后，即苗挂钩的方向保持不变。使用最少20苗​​每行中的每个背景和用于进一步分析每个时间点至少两个板组成。 注意:四种不同的背景可以在每个板（4行）进行比较。 可选:在使用地塞米松（DEX）诱导型的RNAi /过表情纹，上面的苗转移到1X MS与+ DEX MS板块沿（含有1x MS板，0.8％琼脂培养基中添加20μMDEX）FOR 3小时，并在暗箱保持垂直板。 </li><li>要检查趋光反应，垂直转移上述板到黑匣子只开了一边。包含板的黑盒单侧​​白光转移到植物生长室。以确保单侧光，将朝向光源（未前侧）的板的边缘。指设置在图1中。 </li><li>同样，检查向地反应，保持与平板转移苗垂直，并用铝箔覆盖。其次，改变垂直板方向90度，并保持它从四面八方覆盖的黑盒子里面。指设置在图1中。 </li><li>在给定的时间点后，（通常为1.5小时，3小时，6小时和12小时）扫描所述板用扫描器，并保存在JPEG格式的图片。 </li><li>测量使用ImageJ软件（http://imagej.nih.gov/ij/index.html）幼苗的弯曲角度（ 图URE 2）。 <ol><li>启动通过双击（ 图2A）的ImageJ的程序。 注意:在角度测量中使用的主要工具是图2B所示。 </li><li>点击"文件"选项后面的子选项"打开"ImageJ的工具栏，并保存在JPEG文件格式来衡量热带弯曲角度（ 图2C）打开图片。 </li><li>点击放大镜工具，通过点击鼠标左右键，分别如图2B所示放大或缩小图片。 </li><li>点击滚动工具拖动和放置图像（ 图2D）。接下来，单击角度工具来测量弯曲（ 图2E）的角度。 <ol><li>使原始胚轴生长方向点&#39;A&#39;，拖动鼠标弯曲起始点&#39;B&#39;（ 图2F），然后单击左屁股左键双击上绘制的第一行（ 图2G）。 </li><li>从b点鼠标拖动到弯曲结束点"C"，并单击左键绘制第二行（ 图2H）和形成固定<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A.注意:真正的弯曲角<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B，它等于180° - <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A.（ 图2F）。 </li><li>测量和记录<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />通过按下键盘上的&#39;M&#39;A。 如图2I结果文件将单独打开。测量和一个记录所有苗木之一。例如，第一测量对于所有从Col-0中的下胚轴的弯曲角度，然后为突变品系。 </li></ol></li></ol></li><li>从ImageJ的转移数据集电子表格文件，使AV弯曲每个后台的角度erage。 </li><li>进行定量分析，使用统计测试结果，包括标准误差棒和概率的值（参见图2）的柱状图图。 <ol><li>计算真实弯曲<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B，使用电子表格设计一个公式， 即: <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B = 180° - <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />一张纸条: <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A是一个从ImageJ的分析得出的值。 </li><li>测量平均弯曲角度<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B中各使用后台电子表格工具（ 图2J）。 </li><li>分析使用电子表格"公式"的工具（ 图2J）每次测量重复之间的标准偏差。 </li><li>测试SIG结果通过施加t-试验对平均弯曲角度和上述计算出的标准偏差值的着性。 </li><li>使用上述值来使用的电子表格"插入"工具（ 图2K）以图形方式表示在两个不同的背景之间的弯曲角度的定量差绘制一个直条图。 注:强有力的候选基因突变/过度表达线将显示方面明显的差异野生型，如无弯曲，弯曲快或慢弯曲。 </li></ol></li></ol></li></ol> 2.屏幕株系有缺陷的热带响应由于PD SEL变化具有改变PD胼胝质水平<ol><li> 8羟基芘- -1,3,6-三对顶部幼苗与HPTS琼脂糖块酸（HPTS）染料加载执行下胚轴加载分析，比较染料的突变体之间的运动/过表达线和Col-0中 。 <ol><li>准备HPTS琼胶SE块胚轴加载试验。 <ol><li>取10毫升DDH 2 O的在50ml锥形瓶中，并添加0.1克琼脂糖以制备1％琼脂糖凝胶。煮沸琼脂糖在微波炉1-2分钟间歇摇动，并让它冷却5分钟。 </li><li> 50毫克HPTS的可加10毫升1％琼脂糖溶液，搅拌均匀，直到溶液变为绿色。 </li><li>倒入上述混合物至10毫米培养皿，并允许它固化15分钟。 </li><li>用锋利的刀片解剖剪琼脂糖凝固HPTS做小琼脂糖块（0.5×2 平方厘米）。 </li></ol></li><li>准备植物样品的HPTS染料加载试验。 <ol><li>设置了HPTS染料负载测定的平台;上放置一个新的MS培养基平板的显微镜盖玻片（24×50毫米2）， 如图3A所示 。 </li><li>从钩的基地使用锋利手术剪消费为期3天的老黄化苗。 </li><li>立即传送上述秧苗（从每个背景最少5苗），以微观盖玻璃的表面中，使得只有0.5厘米处的切除位置胚轴的应保留在盖玻片上的方式，和胚轴的其余部分应该接触如在图3B中所示的介质。 </li><li>放置在胚轴的顶部HPTS琼脂糖块（带有一个切钩）中5分钟以这样的方式使切下的区域接触HPTS琼脂糖块。 </li><li> 5分钟装载后，从胚轴表面去除琼脂糖块中，下胚轴转移到含有DDH 2 O的10毫米的培养皿并在DDH 2 O洗胚轴与连续摇动15分钟。 </li></ol></li><li>准备共聚焦显微镜的样品。 <ol><li>从激光扫描共聚焦显微镜下的每个背景观察选择至少5苗。 </li><li>在激光信道，波长设置为特定EXC 488纳米itation和500至550nm为带通发射。 </li><li>在2台传送秧苗（一个的Col-0 +一个突变）到一个新的显微镜载玻片用的DDH 2 O洗涤后添加100-150微升双蒸水至滑动，并用盖玻片覆盖。移幻灯片以共焦显微镜的阶段。 </li><li>采用亮场（白光）照明聚焦下胚轴区域（具有20X 40X或物镜）。然后，扫描荧光的幻灯片。 </li><li>拍摄图像最少10套，并通过从装货点检查染料移动的范围的两个背景进行比较。 注意:突变/过表达系具有改变的共质体的运动将表现出与野生型相比Col-0中染料载荷不同的模式。 </li></ol></li></ol></li><li>分析胼胝质水平突变/过度表达线条显示修改过的向光性和向重力响应和显示鲜明HPTS染料运动&lt;br/&gt;比较教育署COL-0。 <ol><li>准备苯胺蓝染色胼胝质染色。 <ol><li>使1M甘氨酸，pH为9.5和0.1％（W / V）的苯胺蓝在高压灭菌的DDH 2 O </li><li> 3体积比:通过混合0.1％苯胺蓝和2 1M甘氨酸使染色缓冲液。例如，对于50毫升染色缓冲液中，取20毫升0.1N％苯胺蓝和30毫升的1M甘氨酸。注意:请染色缓冲最低使用和储存在黑暗中前48小时。 </li></ol></li><li>染色弯曲或无弯曲与特定的胼胝质苯胺蓝染色苗。 <ol><li>以在染色过程中抑制从头胼胝质合成，1.5mM的2-脱氧-D-葡萄糖（DDG）的最终体积加至50ml染色缓冲液中。 </li><li>就拿胼胝质染色（有或无响应热带）3日龄的黄化苗。 </li><li>切薄用手术剪从他们的钩地区的苗。 </li><li>转移苗小培养皿中含有ING用牙签的尖端2毫升染色缓冲液中。 </li><li>孵化用染色缓冲幼苗在黑暗中2小时连续摇动以30rpm。 </li><li>染色后，清洗与DDH 2 O的幼苗2分钟以除去过量的染色缓冲液中。 </li><li>选择一个最小的5苗的激光扫描共聚焦显微镜下每个工厂背景观察。 </li></ol></li><li>准备共聚焦显微镜的样品: <ol><li>转移苗组（单的Col-0 +一个突变）清洁显微镜的DDH 2 O.洗涤后滑</li><li>添加DDH 2 O的100-150微升，并用盖滑盖。移幻灯片以共焦显微镜的阶段。 </li><li>在激光信道，波长设置为特定激发405nm和500至550nm发射苯胺蓝荧光的成像。 </li><li>与10X和后期带来胚轴区成为关注的焦点，第一R，其中采用亮场（白光）照明20X 40X或物镜。 </li><li>然后，打开过滤光线，扫描幻灯片荧光，并保存图像。 </li><li>衡量ImageJ的软件（http://imagej.nih.gov/ij/index.html）胼胝质信号强度（ 见图4）。 <ol><li>改变从奥林巴斯图像二进制格式（* .oib）到JPEG文件格式共聚焦显微镜图像的格式。注意:只有JPEG格式是由ImageJ的软件支持。 </li><li>启动通过双击（ 图4A）的ImageJ的程序。注:箭头指示矩形工具在胼胝质强度测量（ 图4B）被使用。 </li><li>点击"文件"选项后面的子选项"打开"ImageJ的工具栏，然后打开显微镜照片中保存的JPEG文件格式来测量胼胝强度（ 图4C）。 </li><li>点击伊玛矩形图标GEJ工具栏（ 图4D）。 </li><li>分析信号强度，拖动光标在画面和选择区域进行检查（ 图4E） </li><li>测量，按"M"键盘（ 图4E）上录制的信号强度的数值。注:结果文件将开立单独的（ 图4F）。 </li><li>测量和记录的强度值逐一对所有从每个工厂背景的苗木。 注意:在ImageJ的，结果文件"平均"表示所选择的"区域"的信号强度。 </li></ol></li><li>平均值从ImageJ的结果文件的拷贝，并在定量分析的电子表格文件粘贴。 </li><li>进行定量分析，使用统计检验，包括标准误差棒和概率值（ 图4）的柱状图图。 <ol><li>测量的平均信号强度（从ImageJ的意思）使用电子表格"公式"工具（ 图4G）每个后台。 </li><li>分析使用电子表格"公式"的工具（ 图4G）用于每次测量重复之间的标准偏差。 </li><li>计算使用thespreadsheet"公式"工具的标准误差（SE）。 注意:SE = S /√n，其中（s）是总体的标准偏差，和（n）是该样品的尺寸（观测数）。 </li><li>通过施加t检验到的平均信号强度，并且如上所述计算出的标准误差值测试结果的意义。 </li><li>使用上述值来使用的电子表格"插入"工具（ 图4H）以图形方式表示在两个不同的背景之间的胼胝质水平的定量差绘制的柱状图图。 注:强有力的候选人的突变/过度表达线将小号与野生型胼胝质的水平，如无胼胝质，胼胝质很少或超PD胼胝质的积累有什么不同。 </li></ol></li></ol></li></ol></li></ol>

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P., Nielsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+auxin+transport+by+the+AUX1+influx+carrier+protein.">High-affinity auxin transport by the AUX1 influx carrier protein.</a> Curr. Biol. 16 (11), 1123-1127 (2006).</li><li>Geisler, M., Murphy, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+of+auxin+transport:+the+role+of+p-glycoproteins+in+plant+development.">The ABC of auxin transport: the role of p-glycoproteins in plant development.</a> FEBS Lett. 580 (4), 1094-1102 (2006).</li><li>Band, L. R., 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=Root+gravitropism+is+regulated+by+a+transient+lateral+auxin+gradient+controlled+by+a+tipping+point+mechanism.">Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping point mechanism.</a> Proc. Natl. Acad. Sci.USA. 109 (12), 4668-4673 (2012).</li><li>Vanneste, S., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin:+a+trigger+for+change+in+plant+development.">Auxin: a trigger for change in plant development.</a> Cell. 136 (6), 1005-1016 (2009).</li><li>Han, X., 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=Auxin-callose-mediated+plasmodesmal+gating+is+essential+for+tropic+auxin+gradient+formation+and+signaling.">Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling.</a> Dev. Cell. 28 (2), 132-146 (2014).</li><li>Kumar, R., Kumar, D., Hyun, T. K., Kim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Players+at+plasmodesmal+nano-channels.">Players at plasmodesmal nano-channels.</a> J. Plant Biol. 58, 75-86 (2015).</li><li>Chen, X. Y., 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=The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning.">The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.</a> Plant Physiol. 150, 105-113 (2009).</li></ol>

作者没有披露。

在此手稿，一个策略来筛选由于改变的PD胼胝质，因此，PD SEL中详细描述的突变体/过表达系是在向光性和向地响应缺陷的。 PD胼胝质合成和降解主要由胼胝质合成酶和β-1,3-葡聚糖酶来实现，但这些酶的调节是通过许多上游因素控制。要搜索这样的上游因素或直接参与PD调控的候选人，我们已建立了筛选此方法。这种设置有一些限制，调节PD胼胝质合成（gsl8）的一些关键酶的突变体是致命的15和理清这些突变将这个设置限制。此外，如果候选基因示出更快的或具有开放或封闭的PD沿慢嗜响应，但胼胝质水平保持不变，则需要详细分析，以进一步表征这些候选人。关键因素和故障排除PROCE在每一步小规模企业也详细阐述。首先，在介质制剂，可以影响植物生长的关键因素是媒体，其范围应为5.7-5.8的pH。第二，琼脂平板的适当的表面的干燥是重要的;这将是难以解除3天龄幼苗不与水含量高的琼脂平板的任何损害。但是，琼脂平板的过分干燥可以孵育期间引起媒体一些裂缝并导致非对称幼苗生长。在使用次氯酸钠溶液种子表面消毒，必须小心通过反复洗涤蒸压DDH 2 O的彻底清除次氯酸钠，因为它是一个强大的漂白剂。蒸压DDH 2 O的应新鲜配制和无菌处理，因为老股可能会导致MS板真菌污染。灭菌后，种子由冷处理分层同步发芽。可以通过转任一执行该过程摹消毒湿种子直接到寒冷的房间在黑暗条件下或先点睛种子到MS板，然后将板转移到寒冷的房间在黑暗条件下。然而，我们优选后者，因为它给苗更均匀的生长。而打点种子，两个相邻的种子之间的距离应在为0.1cm至少因为很密切点缀种子会导致重叠苗，这是非常困难的在随后的实验转移的生长。 为了正确向光性和向地反应，苗不应该被曝光并应保持完好。为了避免白光的背景效果，慎重选择在黑暗的房间在一个绿色光源幼苗为植物不绿光回应。可以通过使用透明绿色乙烯板覆盖所述白色光源来产生绿色光源。对于选择种苗，消毒的牙签是最好的选择。触摸日e为牙签胚轴的非常下部解除幼苗，使它们可以很容易地转移到新的板上。所有的幼苗在尺寸上应与钩方向相似。在我们的实验中，我们看到，如果钩子方向是在光源的相反侧，植物会表现出比具有相同的钩子方向作为光源植物更快嗜响应。通常情况下，在向光性的情况下，人们可以看到3小时内于真正的候选和Col-0之间的弯曲角的明显的差异，而在向地的情况下，通常需要6-12小时。最后，有统计学上相关的数据，最少三个生物学重复和20苗每次都需要。 测试不同的植物背景之间共质体运动的程度，HPTS染料加载是一种方便的技术，因为它不需要复杂的工具和仪器。为HPTS装载测定中，克的百分比埃尔在HPTS琼脂糖块是非常重要的，因为一个非常脆的或硬的凝胶不会给最佳的结果。最好是使用50毫升的锥形瓶中以松散橡胶和不使用大容量的烧瓶中，用10ml的水琼脂糖沸腾。 HPTS琼脂糖块可用于一周制备之后，如果保持在4℃下用铝箔包裹。在HPTS装载测定另一关键因素是胚轴钩区域的切除。切除应锐性剥离的剪刀，在所有苗木类似的立场进行。切除钝的剪刀可能会导致不必要的幼苗损失和可能阻碍HPTS加载。此外，幼苗生长条件和发育阶段可能在染料移动了至关重要的作用，因为胼胝质堆积是更容易发生这种变化，最终占染料运动障碍作出响应。因此，在最佳条件下生长的幼苗是必要的。胼胝质在PD起着的关键作用通过调节PD SEL，这是生长素梯度形成15重要黄化苗的热带响应。胼胝质的水平可通过与苯胺蓝或免疫标记染色来检测。与苯胺蓝染色是用于检测胼胝质水平的一个简单而快速的方法。因为胚轴细胞具有高膨压和外延角质层层，这是不容易的染料渗透到细胞中。因此，我们开发了切口染色方法，以允许苯胺蓝染料容易渗入。然而，有新的胼胝质，可以影响染色的结果，这是正常的胼胝质染色方法的一个限制的合成的危险。为了避免这种影响，染色协议通过添加胼胝质合成酶抑制剂2-D-脱氧葡萄糖（DDG）到染色缓冲液进行修改。因此，该协议包括预先存在的胼胝质只在这正是切割部位下方的胚轴的下部区域的测量。避免使用新鲜prepar的ED苯胺蓝，并保持在室温苯胺蓝溶液最少使用前48小时。我们注意到，新鲜苯胺蓝染色解决方案不提供最佳的胼胝质染色的结果。 总体而言，我们已经提出了一组可用于突变体/过表达系的快速筛选以通过直接或间接地调节PD胼胝质改变PD SEL有缺陷的或增强的嗜应对策略。关于热带响应一个较早事实被majorly限于生长素载体和信令球员，如PINIOD 2-14的作用。此外，我们还建立在维持胚轴系统15生长素梯度生长素共质体运动的一个重要的作用。这种方法的简单性和通用性无疑加强调查候选基因的PD SEL的调节，从而起到在植物发育中的重要作用，包括热带响应其效用。

植物，如固着活的生物体，已经开发了细胞 - 细胞信号传导的高度复杂的网络，以解决各种环境刺激。热带反应是由植物对环境刺激作出反应的现象之一。植物显示出两个主要的热带响应，向光性和向地。光合植物朝向光源通过弯曲向光性收获最大的能量。同样地，向重力使植物朝向重心生长。导致这些热带反应的基本机制涉及到植物激素生长素1的不对称形成梯度。当地生长素梯度的形成的行为被很好地表征;所涉及的这种机制的基因提供激素作用2-8的路线图。生长素流出载体，如个人识别码形成的（PIN）和P糖蛋白的特定位置，从细胞质到供体细胞9,10的细胞壁执行生长素的运动。 Furthermore，通过将活性的 H + / IAA同向转运生长素涌入载体，如AUX1 / LAX家族蛋白的活性，生长素最终递送到邻近的接收器单元2,11,12。生长素的这个方向的运动被称为极性生长素输送（PAT）。 PAT中各个发育阶段以及响应于不同的环境刺激13,14导致差分生长素分布。此外，在任何这些生长素流入或流出的载体的定位或表达的破坏导致的PAT严重改变，这会导致生长素梯度的破坏，导致发育缺陷。近日，韩寒等人 。报告说plasmodesmal调节也有必要保持生长素梯度15。迄今为止，已有30 plasmodesmal蛋白质已经确定了16个 。在这些蛋白质，AtGSL8已被报告为在胞间连丝（PD），用于胼胝质合成的关键酶，因此起着MAINT至关重要的作用癌宁对PD尺寸排阻极限（SEL）。压抑AtGSL8表达导致扭曲的生长素梯度图案导致在对比野生型幼苗15没有嗜响应。 在这份手稿，方法，探索参与PD调控提供了新的候选基因。 AtGSL8用作模型蛋白来测试这些方法，因为它是促进PD胼胝质的生物合成的关键酶。由于gsl8敲除突变体17秧苗杀伤力，地塞米松（DEX）诱导型的RNAi线按照使用了与以前公布的报告15。这里提供的策略可适于筛选了在用PD SEL受控胚轴嗜响应牵连的基因。

<table border="0" cellpadding="0" cellspacing="0" width="890">
	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:223px;">HPTS (8-Hydroxypyrene -1,3,6-trisulfonic acid trisodium salt)</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">H1529-1G</td>
			<td style="width:335px;">Fluorescent dye as symplasmic tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LE Agarose</td>
			<td style="width:157px;">Dongin-Genomic</td>
			<td style="width:175px;">GEL001-500G</td>
			<td style="width:335px;">Used for HPTS agarose block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microwave oven</td>
			<td style="width:157px;">LG-Goldstar</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">Machine for boiling agarose gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">100 mL glass conical flask</td>
			<td style="width:157px;">Dong Kwang</td>
			<td style="width:175px;">A0205</td>
			<td style="width:335px;">Used to boil HPTS agarose gel</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Petri Dish (35x10 mm)</td>
			<td style="width:157px;">SPL life sciences</td>
			<td style="width:175px;">SPL10035</td>
			<td style="width:335px;">Used to make HPTS agarose blocks and wash plant samples&#160;</td>
		</tr>
		<tr height="79">
			<td height="79" style="height:79px;width:223px;">Microscope cover slides and glass slides (24 x 50 mm)</td>
			<td style="width:157px;">Marienfeld Laboratory Glassware</td>
			<td style="width:175px;">101222</td>
			<td style="width:335px;">Used for HPTS agarose blocks and microscopic sample preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">MS medium plates 125 x 125 x 20 mm</td>
			<td style="width:157px;">SPL life sciences</td>
			<td style="width:175px;">SPL11125</td>
			<td style="width:335px;">Plates to make MS agar medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scissors</td>
			<td style="width:157px;">Germany Stainless</td>
			<td style="width:175px;">HSB 942-11</td>
			<td style="width:335px;">Used to excise hook region of plant samples</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Murashige and Skoog (MS) basal salt mixture</td>
			<td style="width:157px;">Duchefa</td>
			<td style="width:175px;">P10453.01</td>
			<td style="width:335px;">MS medium including vitamins.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">(N-morpholino) ethanesulfonic acid (MES) monohydrate</td>
			<td style="width:157px;">Bioshop</td>
			<td style="width:175px;">3G30212</td>
			<td style="width:335px;">To make MS media.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Plant agar</td>
			<td style="width:157px;">Duchefa</td>
			<td style="width:175px;">P1001.1000</td>
			<td style="width:335px;">To solidify MS media.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Autoclave</td>
			<td style="width:157px;">ALP</td>
			<td style="width:175px;">CL-40L</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:223px;">Shaker</td>
			<td style="width:157px;">Wise Mix</td>
			<td style="width:175px;">SHO-1D</td>
			<td style="width:335px;">To wash off the aniline blue staining buffer and HPTS dye in a placid way.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1 ml Blue tips</td>
			<td style="width:157px;">Sorenson</td>
			<td style="width:175px;">10040</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1 ml pipette</td>
			<td style="width:157px;">BioPette</td>
			<td style="width:175px;">L-1101-2</td>
			<td style="width:335px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Surgical tape</td>
			<td style="width:157px;">MIcropore</td>
			<td style="width:175px;">1530-0</td>
			<td style="width:335px;">To seal the MS plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Aniline blue (Methyl blue)</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">M5528-25G</td>
			<td style="width:335px;">Used to prepare aniline blue staining buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glycine</td>
			<td style="width:157px;">Bioshop</td>
			<td style="width:175px;">GLN001</td>
			<td style="width:335px;">Used to prepare aniline blue staining buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DDG</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:175px;">D8375-1G</td>
			<td style="width:335px;">Used for the inhibition of callose synthases.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Confocal microscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:175px;">FV1000MPE SIM</td>
			<td style="width:335px;">To check aniline blue staining and HPTS dye loading result.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Stirrer&#160;</td>
			<td style="width:157px;">I lab&#160;</td>
			<td style="width:175px;">K400</td>
			<td style="width:335px;">To mix media solution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Aluminium foil</td>
			<td style="width:157px;">SW cooking foil</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">To wrap plates in a dark condition.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Sodium hypochlorite</td>
			<td style="width:157px;">Samjin Industry</td>
			<td style="width:175px;"></td>
			<td style="width:335px;">To surface-sterilized seeds</td>
		</tr>
	</tbody>
</table>

a strategy to validate the role of callose-mediated plasmodesmal gating in the tropic response

植物激素生长素起着许多生长和发育过程，包括对光和重力热带反应中起重要作用。生长素梯度的建立是导致向光性和向地关键事件。先前，生长素极性运输（PAT）的结果表明在植物中的不同蜂窝结构域建立生长素梯度。然而，韩等人最近证明为正确生长素梯度的形成，在相邻小区之间plasmodesmal胼胝质介导的共质体连通也是一个关键的因素。在此手稿，策略阐明特定基因，其中可通过改变通过调制plasmodesmal胼胝质合成的共质体连通影响向光性和向地的作用，进行了讨论。第一步是筛选从突变体或基因的过表达的行3天龄的黄化苗异常嗜应答与野生型一起。这初步筛选可导致一系列基因PAT功能或控制共质体连通的识别。第二次筛选涉及候选人，显示通过影响共质体连通改变热带响应的排序。为了解决这样的候选人，一个共质体示踪剂的运动和plasmodesmal胼胝质的沉积进行检测。探索新的候选基因可以在热带的反应和其他发育过程直接或间接调节共质体连通这一战略将是有益的。

在当前的设置，AtGSL8的地塞米松（DEX）诱导型的RNAi线[以下dsGSL8（+ DEX / -dex）]中使用，作为纯合子gsl8 T-DNA插入突变体幼苗致死18。 dsGSL8和野生型苗±DEX三日龄的黄化苗暴露于向光性和向地刺激。我们发现，dsGSL8（+ DEX）幼苗中向光性和向地15有缺陷。 图5清楚地表明，dsGSL8（+ DEX）呈现既向光性和向地的影响下没有弯曲。此外，改变在dsGSL8（+ DEX）和dsGSL8（-dex）或Col-0中共质体运动是由一个HPTS加载试验分析。与我们以前的发现是一致的，在dsGSL8的HPTS染料运动（+ DEX）是大大高于dsGSL8（-dex）或Col-0中更广泛， </em&gt; 如图6胼胝质为PD SEL的关键调节因子之一，并且AtGSL8称为PD胼胝质合成酶16。此外，胼胝质苯胺蓝染色进行，dsGSL8（+ DEX）之前和之后的嗜响应已持续所示的低PD胼胝质的水平， 如图7。 <img alt="图1" src="/files/ftp_upload/53513/53513fig1.jpg" /> 图1中所涉及的向光性和向地应答的刺激的步骤的流程图（A）转移垂直生长幼苗从不同的植物背景的一块板，但在不同的行。 （B）对于向光性，保持在该被打开的一侧暗箱朝单方面白色光朝向所述板。对于向地，首套用铝箔板，旋转90°，并转移到一个暗箱（covereð 来自四面八方的）。 （c）使用一个扫描器，不同的时间间隔，例如1.5小时，3小时，6小时和12小时后进行扫描板。 （D和E）这些面板使用的电子表格文件从ImageJ的数据分析显示数据产生，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig1large.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/53513/53513fig2.jpg" /> 图2.测量由ImageJ的弯曲角度。（A）查看通过双击的Image J程序。 （B）在角度测量中使用的主要工具。 （C）打开"文件"选项，后跟子选项"打开"的Image J工具栏打开被保存为JPEG文件格式显微图像，。 （D </s仲&gt;）通过点击鼠标左右键，分别使用放大镜工具来放大或缩小图片上。 （E）使用角度工具来测量弯曲的角度。 （F）的角度测量概要: <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> ABC定义了一个固定的<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A，以及真正的弯曲角被计算为<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B，它等于180° - <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />代表从A点到B点的距离A.（G）线。行（H）连续性AB到C点制作<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />答:（I）的Image J表现的结果文件<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />的值。 （J）代表电子表格文件显示，平均弯曲角度的测量，STAN准偏差，标准误差和t检验计算。 （K）条形图显示图两种不同的植物背景的Col-0和dsGSL8 EMS突变体之间的弯曲角度的测量。误差棒是SEM（平均值+标准误差）和***表示通过T检验（p值&gt; 0.01）的意义。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53513/53513fig3.jpg" /> 图3 概要中所涉及的HPTS装载测定的步骤。（A）中示出HPTS琼脂糖块的制备面板。 （B）的小组表示切除和幼苗转移和HPTS琼脂糖块的加载。 （C）的面板显示中涉及样品中的步骤对于共焦成像的准备。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig3large.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/53513/53513fig4.jpg" /> 图4.测量由ImageJ的胼胝质的强度。（A）看双击ImageJ的程序。 （B）在胼胝质强度测量中使用的主要工具。 （C）打开"文件"选项，后跟子选项"打开"ImageJ的工具栏打开被保存为JPEG文件格式显微图像，。 （D）的ImageJ工具栏上的矩形图标。 （E），用于分析的胼胝质强度图像的选中区域。 （F）的ImageJ结果文件显示胼胝质的强度值，因为"中庸"。 （G）代表电子表格文件显示平均胼胝质强度测量，标准偏差，标准误差和t检验计算。代表两个工厂背景之间的胼胝质的强度差（H）条形图图。误差棒是SEM（平均值+标准误差）和***表示通过T检验（p值&gt; 0.01）的意义。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53513/53513fig5.jpg" /> 图5. AtGSL8 表达 的减少 导致有缺陷的热带反应。（A）趋光反应显示出由dsGSL8（±DEX）以及Col-0中。（B）由dsGSL8（±DEX）所示重力性反应人<html>翁与Col-0中 。dsGSL8（+ DEX）没有显示出向光性或向地性反应。dsGSL8（+ DEX）和dsGSL8（-dex）象征地塞米松处理和未处理dsGSL8诱导型的RNAi线，分别为。比例尺代表0.2厘米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53513/53513fig6.jpg" /> 图6.共质体运动 HPTS加载后被送往dsGSL8±DEX胚轴切割面与野生型Col-0中一起AtGSL8。荧光图像的抑制后增加 。dsGSL8（+ DEX）显示HPTS染料的更广泛的运动，这表明增加共质体运动。比例尺条为0.2mm左右。COM /文件/ ftp_upload / 53513 / 53513fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/53513/53513fig7.jpg" /> 图7. AtGSL8 压制线呈下胚轴照明和阴影双方对称PD胼胝质的分布 。 （A）荧光图像显示为0小时的dsGSL8（-dex）苯胺蓝染色胼胝质。 （B）荧光图像显示向光性后的6小时的dsGSL8（-dex）苯胺蓝染色胼胝质。 （C）荧光图像显示向光性后的6小时的dsGSL8（+ DEX）苯胺蓝染色胼胝质。黄色箭头表示对下胚轴的阴影区域的胼胝质的积累。比例尺代表50微米。 （D）代表上午条形图图在dsGSL8累计（±DEX）PD胚轴胼胝质前后向光性的3小时和6小时的&#39;mount。荧光灶强度从十个独立胚轴测定（数据是平均值±标准差;学生t检验，* P &lt;0.01）。这个数字已经从修改（Han等，2014年），15。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53513/53513fig7large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response at JoVE.com

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

本文介绍了筛选基因控制的热带响应期间plasmodesmal渗透性，因此生长素梯度的方法。这包括在胚轴热带反应程度的测量 拟南芥和8羟基芘-1,3,6-三磺酸（HPTS）装载终于胼胝质水平评估检查plasmodesmal渗透性。

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

a-strategy-to-validate-role-callose-mediated-plasmodesmal-gating

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K 次观看.  Gyeongsang National University. 本文介绍了筛选基因控制的热带响应期间plasmodesmal渗透性，因此生长素梯度的方法。这包括在胚轴热带反应程度的测量 拟南芥和8羟基芘-1,3,6-三磺酸（HPTS）装载终于胼胝质水平评估检查plasmodesmal渗透性。 

视频: A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

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 ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

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 ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

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

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

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高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用