我们感谢玉米 Borok 和晃平 Miyazono 为编写手稿提出建议。H.I.S. 和 M.H. 得到了上原纪念基金会研究金的支持, H.I.S. 得到了留学 Hayaishi 纪念奖学金的支持。这项工作得到了武田科学基金会 (犍为县) 的资助。

1. EndMT 的诱导
<ol><li>在标准培养条件下保持 MS-1 细胞, 避免融合。在材料表中描述了 MS-1 细胞的来源。对于 MS-1 细胞, 使用最低的基本中α (内存α) 与10% 胎小牛血清 (FCS), 50 毫升/ml 青霉素, 50 微克/毫升链霉素。</li>
	<li>用1x 磷酸盐缓冲盐水 (PBS) 在10厘米的盘子上洗 MS-1 细胞, 并在盘子中加入1.0 毫升的胰蛋白酶。孵育5分钟, 在37摄氏度。</li>
	<li>使用9毫升培养基分离细胞。收集细胞悬浮在15毫升管。</li>
	<li>在室温下离心 300–400 x g的细胞悬浮液5分钟。</li>
	<li>小心取出上清液, 在预热培养基中悬浮细胞颗粒。</li>
	<li>使用台盼蓝溶液和标准 hemocytometer 或自动单元计数器计数可行细胞。</li>
	<li>板 MS-1 细胞到未涂布的标准培养板上, 每 cm2 0.5 x 103细胞, 用于长期培养。例如, 板 5.0 x 103细胞每井在6个井文化板材。使用相同的文化媒体, 包括同一浓度的 FCS。在 MS-1 细胞中, TGF β诱导 EndMT 在长期培养 (至少 48–72 h) 与使用 FCS。</li>
	<li>刺激内皮细胞与 TGF-β2 (最终浓度为 1 ng/毫升) 后24小时。</li>
	<li>在一个湿润的孵化器中培养细胞 (5% CO2, 37 °c)。在关注形态学变化时, 每天监视细胞。</li>
	<li>在长期培养的 TGF 中, 用含有 TGF-β2的新鲜的预热培养基取代培养基, 并在48小时后进行治疗。</li>
	<li>进行下游分析。典型地, 在24小时治疗后观察肌动蛋白重组, 并观察 TGF β48–72治疗后下调内皮标记物和上调间充质标志物。</li>
</ol>2. 免疫细胞化学分析
<ol><li>在标准培养条件下保持 MS-1 细胞。</li>
	<li>板 MS-1 细胞到4井细胞培养室幻灯片在 1.0 x 103细胞每井 (1.7 厘米2)。幻灯片预先涂上明胶。每井都使用0.5–1.0 培养基。</li>
	<li>刺激内皮细胞与 TGF-β2 (最终浓度为 1 ng/毫升) 后24小时。当分析岩石在 EndMT 中的参与时, 添加一个岩石抑制剂 Y-27632 (10 微米) 1 小时之前, TGF β治疗。当评估 TGF β信号抑制的效果时, TGF β受体激酶抑制剂可以被使用。</li>
	<li>在一个湿润的孵化器中培养细胞 (5% CO2, 37 °c)。肌动蛋白重组是观察 MS-1 细胞后24小时的治疗与 TGF β。其他变化在48–72治疗后变得明显。对于 72 h 培养, 用 TGF β治疗后用含有 TGF β的新鲜培养基取代培养基。</li>
	<li>
f-肌动蛋白染色
	<ol>
<li>经过24小时的 TGF 治疗, 去除培养基, 固定细胞与4% 多聚甲醛 1x PBS 在室温下20分钟, 并冲洗细胞与 1x PBS。</li>
		<li>在室温下孵育0.2% 海卫 X-100 5 分钟。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>罗丹明-四甲基罗丹明 B 异硫氰酸酯与鹅蕈稀释150倍, 室温下阻塞缓冲1小时。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>室温下用菁核酸结合染料将细胞核染色5分钟。</li>
		<li>使用幻灯片安装介质冲洗幻灯片并在幻灯片上安装盖玻片面。</li>
		<li>用共焦显微镜观察。使用 543 nm 激光和560–615纳米发射过滤器检测罗丹明的荧光。使用 633 nm 激光和发射过滤器长于 650 nm 的核染色。经 TGF β治疗后, 可观察到厚应力纤维的形成。</li>
	</ol>
</li>
	<li>
VE 钙粘蛋白与α SMA 染色
	<ol>
<li>经过72小时的 TGF 治疗, 去除培养基, 固定细胞与冷50% 甲醇和50% 丙酮 (0.5–1.0 毫升每井) 为5分钟。</li>
		<li>冲洗细胞三倍 1x PBS (0.5–1.0 毫升每井)。</li>
		<li>根据制造商推荐的浓度, 在4摄氏度的黑暗中, 以主要抗体孵化, 在夜间阻塞缓冲区。使用 VE 钙黏蛋白单克隆抗体 (BV13, 1:100 稀释) 和 Cy3-conjugated α SMA 单克隆抗体 (1A4, 1:200 稀释)20。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>用绿色染料共轭二级抗体孵育细胞, 在1小时的室温下, 根据制造商推荐的浓度对 VE 粘附蛋白抗体进行阻塞缓冲。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>室温下用菁核酸结合染料将细胞核染色5分钟。</li>
		<li>使用幻灯片安装介质冲洗幻灯片并在幻灯片上安装盖玻片面。</li>
		<li>用共焦显微镜观察。使用 543 nm 激光和 560–615 nm 的发射过滤器检测α SMA (Cy3)。使用 488 nm 激光和505–550纳米的发射过滤器检测 VE 钙黏蛋白。通常情况下, TGF 治疗后, VE 钙黏蛋白信号的减少和α SMA 信号的增加将被观察到。</li>
	</ol>
</li>
</ol>3. 三维胶原凝胶收缩试验
<ol><li>在胶原凝胶收缩试验前, 用/不 TGF β MS-1 细胞培养72小时。</li>
	<li>在冰上准备 i 型胶原凝胶。混合冷胶原溶液, 10x 浓缩的记忆培养基, 胶原稀释缓冲液, 含 0.05 N 氢氧化钠, 2.2% NaHCO3, 200 毫米 HEPES pH 7.4 在 8:1: 1 比例。</li>
	<li>混合200毫升控制或 TGF β处理内皮细胞悬浮 (1.0 x 106 cells/200 ul) 和 800 ul 的胶原凝胶溶液。对于 TGF β处理的样品, 添加 TGF-β2 (1 ng/毫升) 的细胞悬浮。</li>
	<li>加入1.0 毫升的混合物, 每井12井培养板, 并允许固化在孵化器37°c 为30–60分钟。</li>
	<li>糊化后, 覆盖1.0 毫升的记忆α含有 10% FCS, 50 U/毫升青霉素, 50 微克/毫升链霉素, 以漂浮凝胶。对于 TGF β处理的样品, 添加 TGF-β2 (1 ng/毫升) 的媒体。 注: 对于 TGF β治疗样品, 添加 TGF β的凝胶和培养基。</li>
	<li>在37摄氏度的48小时内孵化漂浮凝胶。</li>
	<li>使用扫描仪扫描印版底部的凝胶图像。或者, 用数码相机在凝胶上方的固定距离上记录凝胶的图像。</li>
	<li>使用 ImageJ 对基于像素数的凝胶表面积进行量化。选择使用手绘选择或相关选择工具的凝胶, 或选择凝胶使用 "图像 |调整 |颜色阈值 "工具, 然后确定使用的凝胶面积" 分析 |度量 "命令。</li>
</ol>4. 锁定核酸基 miRNA 抑制剂对 miRNA 活性的抑制作用
<ol><li>在标准培养条件下保持 MS-1 细胞。</li>
	<li>染锁定核酸 (低噪声放大器) miRNA 抑制剂, 合成 miRNA 复式或 siRNAs (20 或 50 nM) 根据制造商的指示使用 lipofection 试剂。我们在以前的报告26、27中描述了低噪声 miRNA 抑制剂、合成 miRNA 复式和 siRNAs 的详细资料和来源。在 MS-1 细胞中, 标准的转染程序的基础上制造商的指导工作很好, 引入低噪声 miRNA 抑制剂, 合成 miRNA 复式或 siRNAs。</li>
	<li>后 16–48 h, 刺激内皮细胞与 TGF-β2 (最终浓度 1 ng/毫升)。</li>
	<li>进行各种下游分析, 包括 rna 的表达分析 (定量 rt-pcr 和 rna 序列分析), 免疫细胞化学分析, 和记者化验。我们在过去的报告20、26、27中描述了下游分析。</li>
</ol>

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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=Endothelial-mesenchymal+transition+of+brain+endothelial+cells:+possible+role+during+metastatic+extravasation.">Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.</a> PLoS One. 10 (3), e0119655 (2015).</li><li>Arciniegas, E., Sutton, A. B., Allen, T. D., Schor, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta+1+promotes+the+differentiation+of+endothelial+cells+into+smooth+muscle-like+cells+in+vitro.">Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.</a> Journal of Cell Science. 103 (Pt 2), 521-529 (1992).</li><li>Deissler, H., Deissler, H., Lang, G. K., Lang, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+induces+transdifferentiation+of+iBREC+to+alphaSMA-expressing+cells.">TGFbeta induces transdifferentiation of iBREC to alphaSMA-expressing cells.</a> International Journal of Molecular Medicine. 18 (4), 577-582 (2006).</li><li>Paranya, 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=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> American Journal of Pathology. 159 (4), 1335-1343 (2001).</li><li>Maleszewska, 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=IL-1beta+and+TGFbeta2+synergistically+induce+endothelial+to+mesenchymal+transition+in+an+NFkappaB-dependent+manner.">IL-1beta and TGFbeta2 synergistically induce endothelial to mesenchymal transition in an NFkappaB-dependent manner.</a> Immunobiology. 218 (4), 443-454 (2013).</li><li>Ubil, E., 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=Mesenchymal-endothelial+transition+contributes+to+cardiac+neovascularization.">Mesenchymal-endothelial transition contributes to cardiac neovascularization.</a> Nature. 514 (7524), 585-590 (2014).</li><li>Xu, 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=Epigenetic+balance+of+aberrant+Rasal1+promoter+methylation+and+hydroxymethylation+regulates+cardiac+fibrosis.">Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis.</a> Cardiovasc Research. 105 (3), 279-291 (2015).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Endothelial+Cells+with+Distinct+Patterns+of+TGFbeta-Driven+Endothelial-to-Mesenchymal+Transition.">Tumor Endothelial Cells with Distinct Patterns of TGFbeta-Driven Endothelial-to-Mesenchymal Transition.</a> Cancer Research. 75 (7), 1244-1254 (2015).</li></ol>

作者没有什么可透露的。

据报道, 活化 Ras 和 TGF β治疗24小时诱导 EndMT 在 MS-1 细胞, 而 TGF β单独未能诱导 EndMT 在这短短的时间21。我们一贯地观察到, TGF β在 MS-1 细胞中的长时间治疗 (48–72 h) 后, 极大地诱发了 EndMT20。EndMT 在 TGF β (2–6天) 在各种内皮细胞如人脐静脉内皮细胞 (HUVEC)、人皮肤微血管内皮细胞 (HCMEC)、人冠状动脉内皮细胞等的长期治疗后反复观察 (HCEC), 小鼠肺内皮细胞 (《电...

内皮-间质转移 (EndMT) 是一种分化的内皮细胞经历多种分子变化的过程, 导致成纤维细胞样间质细胞1。EndMT 最初被描述作为内皮细胞变革在心脏的发展期间2,3。在早期心脏发育中, 心脏管由内心内膜和外心肌组成。这两层被一层细胞外基质所分离, 称为心脏果冻。胚胎心内膜细胞, 获得内皮细胞标志物, 转运到间充质细胞, 侵入底层的心脏果冻, 促进心脏垫的形成, 为房室瓣膜和隔膜提供基础和半月形阀门。此外, EndMT 已被认为是毛细血管和血管平滑肌细胞在其他胚胎血管系统的来源, 包括冠状动脉血管, 腹主动脉, 肺动脉4,5,6。此外, EndMT 与生理血管生成7有牵连。
积累的证据表明, EndMT 也参与多种心血管疾病和其他疾病1,8。EndMT 相关的条件包括血管钙化, 动脉粥样硬化, 肺动脉高压, 海绵状畸形, 器官纤维化, 静脉移植重塑, 移植肾功能障碍和癌症8, 9,10,11,12,13,14,15,16,17, 18. 最近的一份报告指出, 几种分子 EndMT 标记物可以作为诊断和预测肾脏移植肾功能障碍的一种工具,17。EndMT 相关的细胞信号通路的调制已被证明可以改善多种疾病的条件, 包括心脏纤维化和静脉移植重塑的动物模型8,15。因此, 了解 EndMT 所依据的机制对于制定针对 EndMT 的诊断和治疗策略是很重要的。
EndMT 的特点是细胞连接的丧失, 迁徙潜能的增加, 内皮特异基因的下调, 如 VE 钙粘蛋白, 上调间充质基因, 包括α平滑肌肌动蛋白 (α SMA)。此外, EndMT 和上皮间质转移 (急诊室), 一个类似的过程, 将上皮细胞转化为间充质细胞, 与各种细胞外基质成分的改变产生相关, 这可能有助于发展组织纤维化8,19。
最近, EndMT 的几项体外研究已经阐明了 EndMT15、20的分子机制的细节。EndMT 是由各种信号通路, 包括转化生长因子 (TGF) β, Wnt, 和缺口1诱导。其中, TGF β在急救和 EndMT 诱导中起着举足轻重的作用。在 EndMT, 长期暴露于 TGF β的结果 EndMT 在各种内皮细胞, 而短期暴露似乎不足21。我们这里描述了一个简单的 EndMT 诱导协议, 其中英里斯文 1 (MS-1) 小鼠胰腺微血管内皮细胞在长时间接触 TGF β20后,体外EndMT。在该模型中, 可以进行多种下游分析, 以研究 EndMT 的标志性特征, 包括形态学变化、内皮标记下调、间充质标志物上调和炎症基因骨架重排, 胶原凝胶收缩。
MicroRNAs (miRNAs) 是 22 nt 小规 rna, 直接转录后水平压制各种 mRNA 目标22,23。通过种子序列介导的目标识别, miRNAs 抑制数以百计的靶基因, 调节细胞分化、增殖和运动等多种细胞功能。这也是对急诊医师和 EndMT 的监管, 并且有几个 miRNAs 被报告为急诊医师和 EndMT24,25的监管者。本文提出的 EndMT 模型可以很容易地结合 miRNA 调制程序来测试 miRNAs 在 EndMT 中的作用。本文综述了 TGF β诱导 EndMT 在 MS-1 细胞中的实验方法, 并对其他内皮细胞中 TGF β的 EndMT 诱导条件进行了比较。

<table border="0" cellpadding="0" cellspacing="0" style="width:1435px;" width="1434">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:361px;">MS-1 cells</td>
			<td style="width:289px;">American Type Culture Collection</td>
			<td style="width:593px;">CRL-2279</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MEM-alpha</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">32571036</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TGF-beta2</td>
			<td>R&#38;D</td>
			<td style="width:593px;">302-B2-002</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4 well Lab-Tek II Chamber Slide</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">154526</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Y-27632&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:593px;">Y0503</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Blocking One</td>
			<td style="width:289px;">nacalai tesque</td>
			<td style="width:593px;">03953-95</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">phalloidin-tetramethylrhodamine B isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TOTO-3 iodide</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">T3604</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">VE cadherin monoclonal antibody (BV13)</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td>14-1441-82</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">alpha-SMA Cy3 monoclonal antibody (1A4)</td>
			<td>Sigma-Aldrich</td>
			<td>C6198</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">A-11001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cover slip</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td>174934</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Collagen solution</td>
			<td style="width:289px;">Nitta gelatin Inc.</td>
			<td>Cellmatrix I-P</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:361px;">Collagen dilution buffer</td>
			<td style="width:289px;">Nitta gelatin Inc.</td>
			<td>Cellmatrix I-P</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LNA miRNA inhibitor</td>
			<td>EXIQON&#160;</td>
			<td colspan="2">miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">synthetic miRNA duplex</td>
			<td>Qiagen&#160;</td>
			<td>miScript miRNA Mimic</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lipofectamine RNAiMAX</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">13778030</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lipofectamine 2000</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">11668027</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling

内皮细胞表型可塑性是心血管系统发展、心血管疾病以及与器官纤维化相关的各种条件的基础。在这些条件下, 分化的内皮细胞获得间充质样表型。这一过程称为内皮-间质转移 (EndMT), 其特点是下调的内皮标记, 上调的间充质标记, 和形态学的变化。EndMT 是由几种信号通路引起的, 包括转化生长因子 (TGF) β、Wnt 和凹槽, 并由类似于上皮-间质转移 (急诊) 的分子机制所调控, 对肠、组织纤维化和肿瘤转移。了解 EndMT 的机制对于发展针对 EndMT 的诊断和治疗方法非常重要。EndMT在体外的鲁棒诱导是一种常见的基因表达特征, 识别 druggable 分子机制和 EndMT 调节剂的筛查。在这里, 我们描述了一种诱导 EndMT 的体外方法。MS-1 小鼠胰腺微血管内皮细胞在长时间暴露于 TGF β后 EndMT, 显示上调的间充质标志物和形态学变化以及诱导多种炎症趋化因子和细胞因子。microRNA (miRNA) 调制的分析方法也包括在内。这些方法为调查 EndMT 的机制和 miRNAs 对 EndMT 的贡献提供了一个平台。

TGF β是 EndMT 在各种内皮细胞中的有力诱导剂。在对 MS-1 细胞进行24小时的 TGF 治疗后, 对 F 肌动蛋白的染色显示重组肌动蛋白应激纤维 (图 1A)20。用岩石抑制剂 Y-27632 预处理抑制肌动蛋白重组20的诱导。MS-1 内皮细胞从经典的鹅卵石状形态学转变为 TGF β治疗时的间充质纺锤形形态 (图 1B)。TGF β?...

Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-Î² Signaling at JoVE.com

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

本文介绍了一种体外诱导内皮-间质转移 (EndMT) 的方法, 该协议对于研究 EndMT 中涉及的细胞信号通路是有用的。在该实验模型中, EndMT 是由 TGF β在 MS-1 内皮细胞中的治疗引起的。

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

molecular-analysis-endothelial-mesenchymal-transition-induced

Research

JoVE Journal

Biology

11.2K 次观看.  Massachusetts Institute of Technology. 本文介绍了一种体外诱导内皮-间质转移 (EndMT) 的方法, 该协议对于研究 EndMT 中涉及的细胞信号通路是有用的。在该实验模型中, EndMT 是由 TGF β在 MS-1 内皮细胞中的治疗引起的。

视频: Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

科研

生物学

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

蓝莓虫害诱导挥发物和植物内信令

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层:牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

追踪药物诱导Colocalizational变化分析受体后内部贩运

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...