作者承认来自 NIH/研究院 (R01GM084135 至 HJN) 和弘水谷基金会 Glycoscience (授予 #110071 HJN) 的支持, 并感谢汤姆五. 李的讨论和建议的化验, 斯比洛斯 Artavanis, Tsakonas, 雨果 Bellen,罗伯特弗莱明, 肯欧文并且果蝇基因组学资源中心 (DGRC) 为质粒和细胞线。

1. 为欺诈击倒的双绞 RNA (dsRNA) 的制备
<ol>
	<li>产品的 PCR 扩增
	<ol>
		<li>使用野生类型的黄白色(y w) 基因组 DNA 和pAc5.1-EGFP作为模板和下面的入门对来放大 dsRNA 合成中使用的 DNA 片段。使用以下 PCR 热剖面: 变性 (95 ° c, 三十年代), 退火 (58 ° c, 三十年代) 和延长 (72 ° c, 1 min)。 
		增强型绿色荧光蛋白(EGFP) dsRNA 底漆 (5 "-3")- 正向引物-GAAATTAATACGACTCACTATAGGGGGTGAGCAAGGGCGAGGAG 反向底漆-GAAATTAATACGACTCACTATAGGGGGTCTTTGCTCAGGGCGG 
		dsRNA 底漆 (5 "-3")- 正向引物-TTAATACGACTCACTATAGGGGAGATGCTGTATGTGGACACGGAT 反向底漆-TTAATACGACTCACTATAGGGGAGATCCGTGGATAACCTTAACGA 注意: EGFP dsRNA 用作负控制。</li>
	</ol>
	</li>
	<li>根据制造商的协议, 用商业凝胶纯化试剂盒凝胶纯化聚合酶链反应 (PCR) 产品。</li>
	<li>根据制造商的协议, 使用能够从 T7 启动子转录的商用产品进行体外转录。</li>
	<li>根据制造商的协议和存储在-80 ° c, 纯化 dsRNA 使用 RNA 提纯试剂盒。</li>
	<li>使用以下步骤评估欺诈击击的效率 (1.5. 1-1. 5.5). 
	<ol>
		<li>使用例和板 5 x 105 S2 单元, 在1毫升/施耐德培养基中的6个井板的每一个井中, 加上10% 胎牛血清 (FBS) 和100的 U/毫升青霉素-链霉素, 对这些细胞进行手工计数。</li>
		<li>添加7.5 µg 的EGFP-或欺诈 dsRNA 每个井和孵化在25° c 24 小时. </li>
		<li>获取控制 (EGFP dsRNA 处理) 和欺诈 dsRNA 处理的 S2 细胞由温和的移, 然后在 1000 x g 的离心化, 在25° c 5 分钟, 并通过 rna 提取试剂盒的 rna 分离过程, 根据制造商的协议。</li>
		<li>利用 qPCR 试剂和底漆/探针和以下 pcr 热剖面 (95 ° c, 十五年代) 和退火/延长 (60 ° c, 1 分钟), 用分光光度计和过程100的 rna 对 rna 进行量化, 用于1步 qRT pcr。 注: 请参见材料表, 以获取有关入门/探头集和用于欺诈的仪器的信息, 并在这些研究中控制 qRT PCR 实验. </li>
		<li>使用 2 -ΔΔCT方法16计算相对欺诈 mRNA 级别。</li>
	</ol>
	</li>
</ol>
2. 评价切口受体与反式配体之间的结合
<ol>
	<li>准备信号接收细胞 (S2 细胞表达缺口受体)。
	<ol>
		<li>使用例和板 5 x 105 S2 和稳定的 S2-Notch 单元, 在1毫升/好的施耐德培养基中加上 10% FBS 和 100 U/毫升青霉素-链霉素, 以手动计数单元格。 注: 对于 S2-Notch 细胞, 可从果蝇基因组资源中心 (DGRC), 添加 200 nM 甲氨蝶呤在媒体。准备一个0.5 毫米的甲氨蝶呤库存解决方案, 首先准备20毫米甲氨蝶呤溶液250µL 1 米氢氧化钠。其次, 稀释此溶液为0.5 毫米在1米磷酸盐缓冲盐水和商店在-20 ° c。在 S2-Notch 细胞中, 缺口蛋白的表达在pMT向量中由果蝇金属硫蛋白启动子控制。</li>
		<li>每井加7.5 µg dsRNA, 在25° c 下孵育24小时。</li>
		<li>添加 0.7 mM 丘索4以诱导凹槽的表达, 并在25° c 下孵育3天。 注: 丘索4用于诱导金属硫蛋白启动子控制的蛋白质表达。</li>
	</ol>
	</li>
	<li>准备信号发送细胞 (S2 细胞表达三角洲或有锯齿配体)。
	<ol>
		<li>使用例和板〜 5 x 106稳定 S2-Delta 或 S2-SerrateTom单元格, 在1毫升/好的施耐德培养基中补充 10% FBS 和 100 U/毫升青霉素-链霉素, 以手动计数单元格。 注: 添加 200 nM 甲氨蝶呤为 S2-Delta 细胞和100µg/毫升霉 S2-Serrate汤姆细胞在媒体。三角洲和锯齿的表达式汤姆-一个番茄标记, 功能版本有锯齿的12-在pMT向量中的果蝇金属硫蛋白启动子下。</li>
		<li>添加 0.7 mM 丘索4以诱导配体的表达, 并在25° c 下孵育3小时。</li>
	</ol>
	</li>
	<li>执行信号发送和信号接收单元之间的聚合。
	<ol>
		<li>用温和的移和平板 2.5 x 105细胞 dsRNA 处理的 S2 (控制) 和 S2-Notch 细胞, 在24井板上通过手工计数例。</li>
		<li>将 5 x 105稳定 S2-Delta 或 S2-SerrateTom单元格添加到施耐德培养基的200µL 的总容量中 (辅以 10% FBS 和100的 U/毫升青霉素-链霉素)。 注: 所有 S2 细胞处理 (包括电镀、诱导和 dsRNA 处理) 在生物安全柜下进行。</li>
		<li>将盘放在 150 rpm (942.48 rad/分钟) 的轨道振动筛上。</li>
		<li>1分钟后, 将每一个井的内容混合, 取20µL, 计算骨料数。<ol>
			<li>同时采用10x 放大 (PLL 10/0.25 目标) 在倒置复合显微镜下进行具有代表性的图像。</li>
			<li>使用例手动计数聚合 (和 #62; 6 单元格)。</li>
		</ol>
		</li>
		<li>把盘子放在振动筛上。</li>
		<li>重复图像采集和计数后5分钟和15分钟的聚合。</li>
	</ol>
	</li>
	<li>执行反绑定的量化。
	<ol>
		<li>计算 S2 单元格和 S2-Delta 或 S2-SerrateTom单元格之间的每个 mL 的聚合数, 作为后台控件。</li>
		<li>计算 S2-Notch 和 S2-Delta 或 S2-SerrateTom单元格之间每毫升的聚合数 (跨绑定的大小)。</li>
	</ol>
	</li>
</ol>
3。由顺式配体对缺口与反式配体结合的抑制作用的评价
<ol>
	<li>准备信号接收单元。
	<ol>
		<li>板 5 x 105 S2 细胞在6井板的每井1毫升/好的施耐德培养基补充 10% FBS 和 100 U/毫升青霉素-链霉素。</li>
		<li>每井加7.5 µg dsRNA, 在25° c 下孵育24小时。</li>
		<li>用pBluescript和pmt-缺口11 (DGRC) 单独或与pmt-凹槽和pmt-增量11 (DGRC) 或pmt-锯齿 17共用 dsRNA 处理过的单元格根据制造商的协议, 使用商业转染试剂, 2 µg/井的 DNA 浓度。 注意: pBluescript用作控件。co-transfected 细胞将被称为 S2-Notch 和 #38;D elta瞬态或 S2-Notch 和 #38; 有锯齿的瞬态单元格。</li>
		<li>在25° c 下孵育瞬时转染的 S2 细胞24小时。</li>
		<li>添加 0.7 mM 丘索4以诱导切口和配体的表达, 并在25° c 下孵育3天。</li>
	</ol>
	</li>
	<li>准备信号发送单元, 如2.2 所述。</li>
	<li>如2.3 节所述, 执行信号发送和信号接收单元之间的聚合。</li>
	<li>通过cis配体对反式绑定进行量化抑制。
	<ol>
		<li>在 S2 单元格和 S2-Delta 或 S2-Serrate 单元格之间计算每个 mL 的聚合数作为背景控件。</li>
		<li>通过以下方式量化cis-配体的抑制程度。<ol>
			<li>将 A定义为 S2-Notch瞬态和 S2-Delta 单元格之间的聚合数。</li>
			<li>将B定义为 S2 单元格之间的聚合数, 瞬时 co-transfected 与凹槽和 Delta (S2-Notch 和 #38;D elta瞬态) 和 S2-Delta 单元格之间。</li>
			<li>计算相对聚合C = (B x 100)/A</li>
			<li>用cis-配体 (δ或有锯齿) 作为 100- C计算抑制的大小。 注意: 举个例子, 如果相对聚合为 45%, 则cis-配体被认为是抑制跨绑定的55%。</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>de Celis, J. F., Bray, S., Garcia-Bellido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+regulates+veinlet+expression+and+establishes+boundaries+between+veins+and+interveins+in+the+Drosophila+wing.">Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing.</a> Development. 124 (10), 1919-1928 (1997).</li><li>Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling:+the+core+pathway+and+its+posttranslational+regulation.">Notch signaling: the core pathway and its posttranslational regulation.</a> Dev Cell. 16 (5), 633-647 (2009).</li><li>del Alamo, D., Rouault, H., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+significance+of+cis-inhibition+in+Notch+signalling.">Mechanism and significance of cis-inhibition in Notch signalling.</a> Curr Biol. 21 (1), R40-R47 (2011).</li><li>Sprinzak, D., 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=Cis-interactions+between+Notch+and+Delta+generate+mutually+exclusive+signalling+states.">Cis-interactions between Notch and Delta generate mutually exclusive signalling states.</a> Nature. 465 (7294), 86-90 (2010).</li><li>Kopan, R., Ilagan, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+canonical+Notch+signaling+pathway:+unfolding+the+activation+mechanism.">The canonical Notch signaling pathway: unfolding the activation mechanism.</a> Cell. 137 (2), 216-233 (2009).</li><li>Huppert, S. S., Jacobsen, T. L., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+regulation+is+central+to+Delta-Notch+signalling+required+for+Drosophila+wing+vein+morphogenesis.">Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis.</a> Development. 124 (17), 3283-3291 (1997).</li><li>Lee, T. V., 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=Negative+regulation+of+notch+signaling+by+xylose.">Negative regulation of notch signaling by xylose.</a> PLoS Genet. 9 (6), e1003547 (2013).</li><li>Lee, T. V., Pandey, A., Jafar-Nejad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylosylation+of+the+Notch+receptor+preserves+the+balance+between+its+activation+by+trans-Delta+and+inhibition+by+cis-ligands+in+Drosophila.">Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila.</a> PLoS Genet. 13 (4), e1006723 (2017).</li><li>Jacobsen, T. L., Brennan, K., Arias, A. M., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-interactions+between+Delta+and+Notch+modulate+neurogenic+signalling+in+Drosophila.">Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila.</a> Development. 125 (22), 4531-4540 (1998).</li><li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+late+embryonic+stages+of+Drosophila+melanogaster.">Cell lines derived from late embryonic stages of Drosophila melanogaster.</a> J Embryol Exp Morphol. 27 (2), 353-365 (1972).</li><li>Fehon, R. G., 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=Molecular+interactions+between+the+protein+products+of+the+neurogenic+loci+Notch+and+Delta,+two+EGF-homologous+genes+in+Drosophila.">Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.</a> Cell. 61 (3), 523-534 (1990).</li><li>Fleming, R. J., 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=An+extracellular+region+of+Serrate+is+essential+for+ligand-induced+cis-inhibition+of+Notch+signaling.">An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling.</a> Development. 140 (9), 2039-2049 (2013).</li><li>Saj, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+ex+vivo+and+in+vivo+RNAi+screen+for+notch+regulators+in+Drosophila+reveals+an+extensive+notch+interaction+network.">A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.</a> Dev Cell. 18 (5), 862-876 (2010).</li><li>Fiuza, U. M., Klein, T., Martinez Arias, A., Hayward, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+ligand-mediated+inhibition+in+Notch+signaling+activity+in+Drosophila.">Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila.</a> Dev Dyn. 239 (3), 798-805 (2010).</li><li>Ahimou, F., Mok, L. P., Bardot, B., Wesley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adhesion+force+of+Notch+with+Delta+and+the+rate+of+Notch+signaling.">The adhesion force of Notch with Delta and the rate of Notch signaling.</a> J Cell Biol. 167 (6), 1217-1229 (2004).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Okajima, T., Xu, A., Irvine, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+notch-ligand+binding+by+protein+O-fucosyltransferase+1+and+fringe.">Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe.</a> J Biol Chem. 278 (43), 42340-42345 (2003).</li><li>Acar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rumi+is+a+CAP10+domain+glycosyltransferase+that+modifies+Notch+and+is+required+for+Notch+signaling.">Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling.</a> Cell. 132 (2), 247-258 (2008).</li><li>Bruckner, K., Perez, L., Clausen, H., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosyltransferase+activity+of+Fringe+modulates+Notch-Delta+interactions.">Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.</a> Nature. 406 (6794), 411-415 (2000).</li><li>Yamamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+EGF+repeat-8+of+Notch+discriminates+between+Serrate/Jagged+and+Delta+family+ligands.">A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands.</a> Science. 338 (6111), 1229-1232 (2012).</li></ol>

作者没有利益冲突。

标准缺口信号依赖于缺口受体与它的配体5之间的相互作用。虽然大多数研究的缺口通路主要考虑到缺口和配体的结合在相邻的细胞 (跨), 缺口和相同细胞配基确实相互作用, 这些 so-called 的cis-相互作用可以发挥抑制作用的缺口信号3,4。因此, 要破译修饰符对标准缺口信号的影响的机制, 通常要检查两种类型的缺口配体相互?...

标准缺口信号是一个短程细胞的细胞通信机制, 需要物理接触相邻细胞, 以促进相互作用的缺口受体和他们的配体1。缺口受体与配体 (目前在信号接收细胞表面) 的相互作用 (目前在信号发送细胞的表面) 启动缺口信号, 称为反激活2。另一方面, 缺口与其配体在同一细胞中的相互作用导致切口通路的抑制, 被称为cis抑制3。跨-和cis-交互之间的平衡需要确保最佳配体依赖性缺口信号4。果蝇有一个缺口受体和两个配体 (三角洲和锯齿), 而不是哺乳动物, 其中有四缺口受体和五配体 [锯齿状 1 (JAG1), JAG2, 三角洲样 1 (DLL1), DLL3 和 DLL4]5。在这种简单的情况下,果蝇模型提供了易于解剖/研究通路修饰剂对缺口配体相互作用和随后的缺口信号的影响。在某些情况下在动物发展期间(包括翼发展在果蝇), 两个cis-和跨-相互作用参与获得适当的缺口信号和细胞命运16.在这些上下文中, 区分缺口路径修饰符对cis-与跨-缺口及其配体的相互作用的影响是很重要的。
我们的小组以前报告说, 添加一种叫做木糖的碳水化合物残渣到果蝇缺口在某些上下文中负调节缺口信号, 包括机翼开发7。损失的欺诈 (xylosylates 缺口的酶) 导致 "机翼静脉丢失" 表型 7。最近, 利用基因剂量试验和克隆分析表明, 欺诈的损失提高了三角洲介导的缺口. 要区分 "欺诈" 突变体中增强的缺口信号是否是由于减少了 cis抑制或增加了跨激活, 在幼虫翅形象盘中的缺口配体的异位表达研究已执行使用dpp-GAL4驱动程序。这些实验提供的证据表明, 在不影响凹槽cis的情况下, 在配体8的作用下, 该方法可调节反式--由 Delta 激活。然而, 反馈规则和内源性配体的作用可能使异位表达研究的解释复杂化1,6,9。
为解决此问题, 使用了果蝇S2 单元格10 , 它提供了一个简单的体外系统, 用于缺口配体相互作用研究11,12。S2 细胞不表达内源性缺口受体和三角洲配体11 , 表达低锯齿状的13, 这不影响缺口配体聚合实验8。因此, S2 细胞可以稳定或瞬时转染的缺口和/或个别配体 (三角洲或锯齿), 以产生细胞, 完全表达的缺口受体或其中一个配体, 或他们的组合。S2 细胞配体表达 S2 细胞的混合表现为由受体配体介导的异型团聚体的形成11,12,14。聚合形成的定量化提供了一种测量缺口与其配体之间的跨绑定的方法15 (图 1)。同样, S2 细胞可以 co-transfected 与缺口和三角洲或有锯齿配体 (即 cis-配体)。Cis-在这些凹槽表达 S2 细胞的配体中, 用反式配基废除凹槽的绑定, 并导致聚合形成减少8,12,14。由顺式-配体引起的集料形成的相对减少, 提供了一种在凹槽和反式配体 (图 2) 之间绑定的cis配体的抑制效果的测量。因此, 利用细胞聚集分析研究了 xylosylation 对反式和cis-缺口与配体之间相互作用的影响。
在这里, 我们提出了一个详细的细胞聚集试验的协议, 目的是评估的结合,反式配体和它的抑制, 由cis-配基使用果蝇S2 细胞。例如, 我们提供的数据使我们能够确定缺口 xylosylation 对缺口和跨-Delta8之间的绑定的影响。这些简单的分析提供了一个半定量评估缺口配体相互作用的体外和帮助确定的分子机制的基础上的体内的效果的缺口途径修饰。

<table><tbody><tr><td>BioWhittaker Schneider&amp;rsquo;s Drosophila medium, Modified</td><td>Lonza</td><td>04-351Q</td><td></td></tr><tr><td>HyClone Penicillin-Streptomycin 100X solution</td><td>GE Healthcare lifescience&amp;nbsp;</td><td>SV30010</td><td></td></tr><tr><td>CELLSTAR 6 well plate</td><td>Greiner Bio-One</td><td>657 160</td><td></td></tr><tr><td>CELLSTAR 24 well plate</td><td>Greiner Bio-One</td><td>662160</td><td></td></tr><tr><td>VWR mini shaker</td><td>Marshell Sceintific</td><td>12520-956</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher Sceintific&amp;nbsp;</td><td>267110</td><td></td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>MEGAscrip T7 Transcription Kit</td><td>Ambion</td><td>AM1334</td><td></td></tr><tr><td>Quick-RNA MiniPrep (RNA purification Kit)</td><td>Zymo Research</td><td>R1054</td><td></td></tr><tr><td>VistaVision Inverted microscope</td><td>VWR</td><td></td><td></td></tr><tr><td>9MP USB2.0 Microscope Digital Camera + Advanced Software</td><td>AmScope</td><td>MU-900</td><td>Image acquisition using ToupView software</td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K210012</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GenDepot</td><td>F0600-050</td><td></td></tr><tr><td>Methotrexate</td><td>Sigma-Aldrich</td><td>A6770-10</td><td></td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>HY068-L6</td><td></td></tr><tr><td>Copper sulphate</td><td>Macron Fine Chemicals</td><td>4448-02</td><td></td></tr><tr><td>S2 cells</td><td>Invitrogen</td><td>R69007</td><td></td></tr><tr><td>S2-Serrate&lt;sup&gt;Tom &lt;/sup&gt;cells</td><td></td><td></td><td>Gift from R. Fleming (Fleming et al, Development, 2013)</td></tr><tr><td>S2-Delta cells</td><td>DGRC</td><td>152</td><td></td></tr><tr><td>S2-Notch cells</td><td>DGRC</td><td>154</td><td></td></tr><tr><td>pMT-Delta vector</td><td>DGRC</td><td>1021</td><td>Gift from S. Artavanis-Tsakonas</td></tr><tr><td>pMT-Serrate vector</td><td></td><td></td><td>Gift from Ken Irvine (Okajima et al, JBC, 2003)</td></tr><tr><td>pMT-Notch vector</td><td>DGRC</td><td>1022</td><td>Gift from S. Artavanis-Tsakonas</td></tr><tr><td>pAc5.1-EGFP</td><td></td><td></td><td>Gift from Hugo Bellen</td></tr><tr><td>TaqMan RNA-to-Ct 1-&lt;em&gt;Step&lt;/em&gt; Kit</td><td>Applied Biosystem</td><td>1611091</td><td></td></tr><tr><td>TaqMan Gene Expression Assay for CG9996 (Shams)</td><td>Applied Biosystem</td><td>Dm02144576_g1&amp;nbsp;</td><td>with FAM-MGB dye</td></tr><tr><td>TaqMan Gene Expression Assay for CG7939 (RpL32)</td><td>Applied Biosystem</td><td>Dm02151827_g1</td><td>with FAM-MGB dye</td></tr><tr><td>Applied Biosystems 7900HT Fast Real-Time PCR system</td><td>Applied Biosystem</td><td>4351405</td><td>96-well Block module</td></tr></tbody></table>

cell aggregation assays to evaluate the binding of the drosophila notch with trans-ligands and its inhibition by cis-ligands

缺口信号是一种进化保守的细胞-细胞通讯系统, 广泛用于动物发育和成人维护。缺口受体与相邻细胞配体的相互作用诱导信号通路激活 (反式激活), 而同一个细胞的配体相互作用抑制信号传导 (cis抑制)。在跨激活和cis抑制之间的适当平衡有助于在动物发育过程中的某些环境中建立最佳的缺口信号水平。由于凹槽及其配体在许多细胞类型中的重叠表达域和反馈机制的存在, 研究了给定的修饰修改对跨-与cis-缺口相互作用的影响它的配体在体内是困难的。在这里, 我们描述了一个协议, 使用果蝇S2 细胞在细胞聚集的分析, 以评估的影响, 敲下一个缺口通路修饰符的约束力, 每个配体在跨和cis。S2 细胞稳定或瞬时转染一个缺口表达载体与表达每个缺口配体 (S2-Delta 或 S2-Serrate) 的细胞混合。反式-受体与配体之间的结合导致异型细胞聚集物的形成, 并以每毫升和 #62 组成的集料数计算; 6 细胞。为了研究顺式-配体的抑制作用, S2 细胞 co-expressing 凹槽和各配基与 S2-Delta 或 S2-Serrate 细胞混合, 聚合数量按上述方法量化。由于顺式-配体的存在而导致的骨料数量相对减少, 提供了一种测量顺式-配基介导的抑制反式绑定的措施。这些简单的化验可以提供半定量的数据关于基因或药理操作对切口的结合的作用对它的配体, 并且能帮助破译基础的分子机制在体内作用这种对缺口信号的操控。

我们的在体内的观察表明, xylosyltransferase 基因的损失, 欺诈导致增益缺口信号由于增加三角洲介导的 跨-激活的缺口不影响cis抑制凹槽由配体8。为了测试这一概念,体外细胞聚合化验被执行。首先, S2 单元格中的欺诈表达式被使用 dsRNA 击倒. EGFP dsRNA 用作控件。用 real-time PCR 法检测了击倒 (KD) 的效率, ?...

Watch this Scientific Journal Video about Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands at JoVE.com

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

复杂的在体内系统, 使得很难区分的激活和抑制的缺口受体的跨-和cis配体, 分别。在这里, 我们提出了一个基于体外细胞聚合的方法, 用于定性和半定量评价的结合的果蝇缺口, 以跨配基与顺式配基。

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

cell-aggregation-assays-to-evaluate-binding-drosophila-notch-with

Research

JoVE Journal

Developmental Biology

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

7.5K 次观看.  Baylor College of Medicine. 复杂的在体内系统, 使得很难区分的激活和抑制的缺口受体的跨-和cis配体, 分别。在这里, 我们提出了一个基于体外细胞聚合的方法, 用于定性和半定量评价的结合的果蝇缺口, 以跨配基与顺式配基。

视频: Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

科研

生物工程

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

神经科学

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

发育生物学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

生物学

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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 ...

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

摘要

探索更多视频

此视频中的章节

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

对检测方法果蝇行为

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

检测

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用