我们想感谢 Bouchier-海斯实验室的所有成员, 他们为这一技术的发展做出了贡献。这项工作部分是由德州儿童医院儿科试验奖给 LBH 提供的。我们感谢 Joya 钱德拉 (马里兰州休斯顿, 得克萨斯州) 允许包括与她的团队合作发布的数据。所描述的试剂的开发得到了贝勒医学院的细胞分选核心和 NIH (NIAID P30AI036211, P30CA125123 和 NCRR S10RR024574) 的资助, 以及乔尔 m. Sederstrom 的帮助。

1. 细胞和培养皿的制备
注意: 在组织培养层流罩中执行步骤1-3。戴上手套。
<ol><li>如果使用玻璃底菜, 涂玻璃纤维连接器。如果使用塑料盘子, 请跳到步骤1.2。<ol>
<li>用0.1 毫克/毫升的纤维连接蛋白溶液: 稀释1毫升1毫克/毫升纤维连接蛋白溶液在9毫升 1 X PBS。</li>
		<li>用 0.5-1 毫升的纤连蛋白覆盖每个井的玻璃底部, 室温下孵育1-5 分钟。</li>
		<li>使用吸管, 收集纤维连接蛋白溶液和存储在4摄氏度。 注: 纤维连接蛋白溶液可多次重复使用。</li>
		<li>用1-2 毫升 1 X pbs 清洗玻璃, 然后从 pbs 中抽出。</li>
	</ol>
</li>
	<li>板 1 x 105单元每井的6井板在2毫升适当的细胞培养媒介 (参见表 1为用于不同大小的井的细胞数字)。如果使用稳定表达 caspase BiFC 组件的单元格线 (参见讨论), 则板 2 x 105单元格, 然后继续处理步骤 (步骤 3)。</li>
	<li>允许细胞在37摄氏度的组织培养孵化器中一夜之间坚持盘子。</li>
</ol>2. 转染细胞以引入 caspase BiFC 成分
<ol><li>染细胞与适当的转染试剂。 注意: 本协议概述了脂质体2000的说明, 该指令已被优化为最小毒性。该协议已成功地用于 Hela 细胞、MCF7 细胞和 LN18 细胞。如果使用其他转染试剂遵循制造商的指示, 并根据需要进行优化。<ol>
<li>在无菌管中添加12µL 的转染试剂到750µL 的减少血清培养基中。</li>
		<li>室温孵育5分钟。</li>
		<li>添加 10 ng 的报告质粒 (一种荧光蛋白编码的质粒,例如红色荧光蛋白 [RFP], 它将标记被转染的细胞) 到不育的1.5 毫升管为每个井被转染。</li>
		<li>将每个 caspase BiFC 质粒 (例如, 20 吴 caspase-2 prodomain [C2 pro] VC 和 20 ng C2 亲氮) 添加到每个管 (表 2) 中20。</li>
		<li>通过添加减少的血清培养基, 使每根管子的总容积达到100µL。</li>
		<li>使用 p200 吸管, 添加100µL 转染试剂溶液从步骤2.1.2 到质粒混合物滴状方式。</li>
		<li>室温下孵育质粒转染试剂混合物20分钟。</li>
		<li>用吸管或吸力从细胞中吸出培养基, 然后用 p1000 吸管将800µL 的血清游离培养基轻轻地放在井侧。</li>
		<li>使用 p200 吸管, 添加200µL 的 DNA 脂复合体滴向每个井。</li>
		<li>在37摄氏度的组织培养孵化器中孵化出3小时的细胞。</li>
		<li>注意不要破坏单层, 取出含有 DNA 脂复合物的血清游离培养基, 吸入或用吸管抽吸。</li>
		<li>吸管2毫升预热 (37 °c) 完全生长介质轻轻地向下的每一个井的一侧。</li>
	</ol>
</li>
	<li>在组织培养孵化器中孵育37摄氏度的细胞, 以达到最佳蛋白质表达的24-48 小时。</li>
</ol>3. 诱导 caspase 活化
<ol><li>治疗的药物或刺激, 诱导 caspase 激活约24小时后转染。<ol>
<li>添加所需的药物浓度的预热 (37 °c) 成像介质 (完全生长介质补充与 Hepes [20 毫米, pH 值 7.2-7.5] 和 2-巯基乙醇 [55 µM]), 并轻轻混合(表 3)。</li>
		<li>用吸管或吸力从细胞中吸出培养基, 然后用吸管将溶液中的2毫升吸管从3.1.1 的一侧轻轻地移到井边。</li>
		<li>将没有药物的成像介质添加到一个井中作为未经处理的控制。</li>
	</ol>
</li>
	<li>在37摄氏度的组织培养孵化器中孵化细胞, 或进行4.3 步的时间推移实验。</li>
</ol>4.Caspase BiFC 数据采集
<ol><li>
Caspase BiFC 单时间点采集
	<ol>
<li>按照制造商的说明打开显微镜和荧光灯光源。</li>
		<li>在 RFP 过滤器下找到细胞, 聚焦于报告基因荧光。</li>
		<li>使用手动计数计数器, 计算可视字段中所有的 RFP 阳性单元格。记录数字。</li>
		<li>在保持相同的视觉场的同时, 切换到 GFP 过滤器 (或 YFP 过滤器 (如果可用), 并使用手计数计数器, 计算红细胞的数量也是绿色 (金星阳性)。在两个过滤器之间来回切换, 以确保只有红细胞被评估为金星阳性。记录数字 (图 3)。</li>
		<li>从板块的三个单独区域中计算出至少100个 RFP 阳性细胞。</li>
		<li>在每个区域, 计算金星阳性转染细胞的百分比 (即红细胞也是绿色的)。平均产生的百分比以获得标准偏差。</li>
	</ol>
</li>
	<li>
caspase BiFC 的三维成像
	<ol>
<li>根据制造商的指示打开显微镜并启动采集软件。</li>
		<li>选择60x 或63x 油目标, 并在目标上放置一滴油</li>
		<li>使用正确的板架将培养皿放在显微镜台上。</li>
		<li>使用荧光光源和显微镜眼片或共焦激光器和计算机屏幕, 导航到感兴趣的领域。</li>
		<li>关注记者荧光使用 RFP 激光或过滤器。</li>
		<li>如果荧光已经用到这一点, 将光源切换到共焦激光器, 并可视化相机获取的单元格的实时图像, 并在计算机屏幕上显示采集软件。</li>
		<li>使用操纵杆控制和焦点轮, 微调单元格的焦点和位置, 使单元格位于视图查找器的中心。选择彼此分离且不重叠的单元格。</li>
		<li>输入的百分比激光功率为50% 和曝光时间在100毫秒的金星和 RFP 作为一个出发点, 以确定最佳设置, 用于实验。</li>
		<li>打开实时捕获并检查生成的图像以及两个通道的随附显示直方图。</li>
		<li>如果信号很低, 图像很难制作出来, 增加激光功率和/或曝光时间的增量, 直到图像中的信号看起来很好。</li>
		<li>确保信号不达到饱和。检查显示直方图, 确保每个流体都能看到明显的峰值。如果峰值包含整个直方图显示, 则输入较低的激光功率和曝光设置 (图 2)。</li>
		<li>在相同的条件下可视化控制 (未经处理或未转染) 细胞, 以确保信号是特定的。 注意: 单色 transfectants 也可以用来控制交叉, 但如果信号在空间上是不同的 (例如线粒体与胞浆), 这是不必要的。</li>
		<li>在显微镜软件中选择或打开 Z 堆栈模块。</li>
		<li>使用焦点旋钮, 近似与单元格中心相对应的焦点位置。</li>
		<li>将焦点调整为一个方向, 直到单元格不再可见时, 选择此项作为顶部位置。</li>
		<li>将焦点调整到相反方向, 直到单元格不再可见, 然后选择此作为底部位置。</li>
		<li>选择最佳步长 (通常约为20µm), 并开始获取, 以指示相机自动在每个通道中的20个µm 步骤之间的顶部和底部位置的单个图像。</li>
		<li>使用3D 渲染软件重建3D 映像 (图 4, 电影 1)。</li>
	</ol>
</li>
	<li>
caspase BiFC 的时间推移成像
	<ol>
<li>实验前至少1小时, 打开显微镜, 将温度设置为37摄氏度。</li>
		<li>选择40x、60x 或63x 油目标, 并在目标上放置一滴油。</li>
		<li>使用正确的板架将培养皿放在显微镜台上。</li>
		<li>如果 co2源可用, 请将 co2传递设备 (通常是连接到提供 co2的管道的有机玻璃盖子) 放在板架上。将 co2级别设置为 5%, 并从软件内部打开 co2控制器。如果 CO2源不可用, 则包括 20 mM Hepes 以缓冲介质。</li>
		<li>导航到转染细胞的领域, 并可视化相机获取的单元格的实时图像, 并使用 RFP 激光器在计算机屏幕上显示采集软件。</li>
		<li>按照步骤 4.2. 8-4. 2.12, 输入对 RFP 激光器的激光功率和曝光时间百分比的设置。保持这些值尽可能低, 同时仍然能够检测到荧光信号。</li>
		<li>使用正则控件示例 (请参见表 3中的示例), 按照步骤 4.2. 8-4. 2.12 输入 YFP 激光光功率和曝光时间百分比的设置。保持这些值尽可能低, 同时仍能探测到金星信号。</li>
		<li>对于每个板块的井, 选择多个不同的位置, 其中包含一个或多个表达记者的单元格。</li>
		<li>输入时间间隔的每个帧和要采取的帧总数之间的时间段。确保有时间获得所有选定的职位之前, 将采取的下一个框架。</li>
		<li>重新访问每个位置, 并根据需要更正和更新焦点。</li>
		<li>为了纠正可能因温度或外部振动变化而引起的焦漂移, 请打开焦点漂移校正系统 (如果可用)。</li>
		<li>开始时间推移并保存数据。</li>
		<li>使用可用的成像软件分析数据 (图 5, 电影 2)。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Salvesen, G. S., Riedl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+mechanisms.">Caspase mechanisms.</a> Adv Exp Med Biol. 615, 13-23 (2008).</li><li>Boatright, K. M., Salvesen, G. S. <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+caspase+activation.">Mechanisms of caspase activation.</a> Curr Opin Cell Biol. 15 (6), 725-731 (2003).</li><li>Luthi, A. U., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CASBAH:+a+searchable+database+of+caspase+substrates.">The CASBAH: a searchable database of caspase substrates.</a> Cell Death Differ. 14 (4), 641-650 (2007).</li><li>Baliga, B. C., Read, S. H., Kumar, 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+biochemical+mechanism+of+caspase-2+activation.">The biochemical mechanism of caspase-2 activation.</a> Cell Death Differ. 11 (11), 1234-1241 (2004).</li><li>Boatright, K. 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=A+unified+model+for+apical+caspase+activation.">A unified model for apical caspase activation.</a> Mol Cell. 11 (2), 529-541 (2003).</li><li>Aravind, L., Dixit, V. M., Koonin, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+domains+of+death:+evolution+of+the+apoptosis+machinery.">The domains of death: evolution of the apoptosis machinery.</a> Trends Biochem Sci. 24 (2), 47-53 (1999).</li><li>Salvesen, G. S., Dixit, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation:+the+induced-proximity+model.">Caspase activation: the induced-proximity model.</a> Proc Natl Acad Sci U S A. 96 (20), 10964-10967 (1999).</li><li>Oberst, 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=Inducible+dimerization+and+inducible+cleavage+reveal+a+requirement+for+both+processes+in+caspase-8+activation.">Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation.</a> J Biol Chem. 285 (22), 16632-16642 (2010).</li><li>Riedl, S. J., Salvesen, G. 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+apoptosome:+signalling+platform+of+cell+death.">The apoptosome: signalling platform of cell death.</a> Nat Rev Mol Cell Biol. 8 (5), 405-413 (2007).</li><li>Kischkel, F. C., 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=Cytotoxicity-dependent+APO-1+(Fas/CD95)-associated+proteins+form+a+death-inducing+signaling+complex+(DISC)+with+the+receptor.">Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.</a> EMBO J. 14 (22), 5579-5588 (1995).</li><li>Martinon, F., Burns, K., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+molecular+platform+triggering+activation+of+inflammatory+caspases+and+processing+of+proIL-beta.">The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.</a> Mol Cell. 10 (2), 417-426 (2002).</li><li>Tinel, A., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIDDosome,+a+protein+complex+implicated+in+activation+of+caspase-2+in+response+to+genotoxic+stress.">The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress.</a> Science. 304 (5672), 843-846 (2004).</li><li>Shyu, Y. J., Liu, H., Deng, X., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+fluorescent+protein+fragments+for+bimolecular+fluorescence+complementation+analysis+under+physiological+conditions.">Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions.</a> Biotechniques. 40 (1), 61-66 (2006).</li><li>Bouchier-Hayes, 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=Characterization+of+cytoplasmic+caspase-2+activation+by+induced+proximity.">Characterization of cytoplasmic caspase-2 activation by induced proximity.</a> Mol Cell. 35 (6), 830-840 (2009).</li><li>Manzl, C., 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=Caspase-2+activation+in+the+absence+of+PIDDosome+formation.">Caspase-2 activation in the absence of PIDDosome formation.</a> J Cell Biol. 185 (2), 291-303 (2009).</li><li>Manzl, C., 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=PIDDosome-independent+tumor+suppression+by+Caspase-2.">PIDDosome-independent tumor suppression by Caspase-2.</a> Cell Death Differ. 19 (10), 1722-1732 (2012).</li><li>Ando, K., 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=NPM1+directs+PIDDosome-dependent+caspase-2+activation+in+the+nucleolus.">NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.</a> J Cell Biol. , (2017).</li><li>Sanders, M. 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=Single-cell+imaging+of+inflammatory+caspase+dimerization+reveals+differential+recruitment+to+inflammasomes.">Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.</a> Cell Death Dis. 6, e1813 (2015).</li><li>Aaronson, D. S., Horvath, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+road+map+for+those+who+don't+know+JAK-STAT.">A road map for those who don't know JAK-STAT.</a> Science. 296 (5573), 1653-1655 (2002).</li><li>Manton, C. 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=Induction+of+cell+death+by+the+novel+proteasome+inhibitor+marizomib+in+glioblastoma+in+vitro+and+in+vivo.">Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo.</a> Sci Rep. 6, 18953 (2016).</li><li>Fan, J. 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=Split+mCherry+as+a+new+red+bimolecular+fluorescence+complementation+system+for+visualizing+protein-protein+interactions+in+living+cells.">Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.</a> Biochem Biophys Res Commun. 367 (1), 47-53 (2008).</li><li>Chu, 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=A+novel+far-red+bimolecular+fluorescence+complementation+system+that+allows+for+efficient+visualization+of+protein+interactions+under+physiological+conditions.">A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions.</a> Biosens Bioelectron. 25 (1), 234-239 (2009).</li><li>Karbowski, M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+mitochondrial+morphology+in+healthy+cells+and+during+apoptosis.">Dynamics of mitochondrial morphology in healthy cells and during apoptosis.</a> Cell Death Differ. 10 (8), 870-880 (2003).</li><li>Proell, M., Gerlic, M., Mace, P. D., Reed, J. C., Riedl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CARD+plays+a+critical+role+in+ASC+foci+formation+and+inflammasome+signalling.">The CARD plays a critical role in ASC foci formation and inflammasome signalling.</a> Biochem J. 449 (3), 613-621 (2013).</li><li>Szymczak, A. L., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Chang, D. W., Xing, Z., Capacio, V. L., Peter, M. E., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdimer+processing+mechanism+of+procaspase-8+activation.">Interdimer processing mechanism of procaspase-8 activation.</a> EMBO J. 22 (16), 4132-4142 (2003).</li><li>Stennicke, H. 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=Caspase-9+can+be+activated+without+proteolytic+processing.">Caspase-9 can be activated without proteolytic processing.</a> J Biol Chem. 274 (13), 8359-8362 (1999).</li><li>Slee, E. 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=Ordering+the+cytochrome+c-initiated+caspase+cascade:+hierarchical+activation+of+caspases-2,+-3,+-6,+-7,+-8,+and+-10+in+a+caspase-9-dependent+manner.">Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.</a> J Cell Biol. 144 (2), 281-292 (1999).</li><li>McStay, G. P., Salvesen, G. S., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+cleavage+motif+selectivity+of+caspases:+implications+for+analysis+of+apoptotic+pathways.">Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.</a> Cell Death Differ. 15 (2), 322-331 (2008).</li></ol>

作者声明他们没有竞争的财政利益。

该协议讨论了使用分裂荧光蛋白测量 caspase 诱导接近度。因为它非常明亮, 高度 photostable, 而且复用速度快13, 所以选择了分离金星。因此, 对 caspase 诱导接近度的金星复性分析可以提供接近 caspase 蛋白相互作用动力学的实时估计。金星被分成两个稍微重叠的片段, 金星的 n 终点 (金星 n 或钒氮), 包括氨基酸1-173 和 C 末端片段 (金星 c 或 VC), 包括残留物155-239。其他分裂荧光蛋白存在, ...

caspase 蛋白酶家族以其在细胞凋亡和先天免疫中的重要作用而闻名1。由于它们的重要性, 确定何时、在何处以及如何有效地激活特定的半胱氨酸蛋白酶, 可以为 caspase 激活通路的机制提供重要的洞察力。此处描述的基于映像的协议启用了 caspase 激活级联中最早步骤的可视化。这种技术利用动态蛋白质: 推动 caspase 活化的蛋白质相互作用。
半胱氨酸蛋白酶可分为两组: 启动器半胱氨酸蛋白酶 (caspase-1,-2,-4,-5,-8,-9, 10, 和-12) 和刽子手半胱氨酸蛋白酶 (caspase-3,-6, 和-7)。刽子手半胱氨酸蛋白酶是存在于细胞作为预制的脂肪酸并且由分裂激活在大和小亚基2 之间。当激活时, 它们会切割许多结构和调节蛋白, 导致细胞凋亡3。启动器半胱氨酸蛋白酶是第一个在通路中激活的半胱氨酸蛋白酶, 通常触发刽子手半胱氨酸蛋白酶的活化。与刽子手半胱氨酸蛋白酶相反, 启动器半胱氨酸蛋白酶由二聚化4、5激活。这种二聚化是通过招募不活跃的单体到特定的大型分子量复合物称为活化平台来促进的。活化平台的组装受一系列特定蛋白质的控制: 蛋白质相互作用。这些都是由在启动器 caspase 的 proform 中存在的保守蛋白交互图案介导的, 包括死亡域 (DD)、死亡效应域 () 和 caspase 招聘域 (卡) 6 (图 1A)。激活平台通常包括受体蛋白和适配器蛋白。受体通常被激活后, 结合的配体, 诱导构象变化, 使许多分子齐聚。然后, 受体要么直接招募 caspase 或适配器分子, 然后将 caspase 带到复合体。因此, 许多 caspase 分子进入接近的接近度允许二聚化。这称为诱导接近模型7。一旦二聚, caspase 经历 autoprocessing, 这有助于稳定活性酶 4, 8. 例如, Apaf1 apoptosome 的组装是由细胞色素 c 在线粒体外膜通透 (MOMP) 的过程中释放出来后触发的。Apaf1 反过来通过一个由两种蛋白质9中存在的卡介导的交互来招募 caspase-9。相似的蛋白质相互作用导致 CD95 死亡诱导信号复合体 (椎间盘) 的组装导致 caspase-8 活化;PIDDosome, 可以激活 caspase-2;和各种 inflammasome 复合体, 启动 caspase-1 激活10,11,12。因此, 启动器半胱氨酸蛋白酶通过一个共同的机制被招募到特定的激活平台上, 导致了诱导接近和二聚化, 没有它的激活就不会发生。
Caspase 分子荧光互补 (BiFC) 是一种基于成像的检测方法, 用于测量启动器半胱氨酸蛋白酶激活的第一步, 允许在激活平台后直接可视化 Caspase 诱导的接近度。大会.该方法利用了分裂荧光蛋白金星的性质。金星是一个更明亮和更 photostable 的黄色荧光蛋白 (YFP) 的版本, 可分为两个非荧光和略微重叠的片段: 金星 n 终点 (金星 n 或钒氮) 和金星的 c 终点 (金星 c 或 VC)。这些片段保留在接近接近13时再折叠餐巾和变为荧光的能力。每个金星碎片被融合到 caspase 的 prodomain, 这是绑定到激活平台的 caspase 的最小部分。这确保了半胱氨酸蛋白酶不保留酶活性, 因此可以同时分析与内源事件相关的下游事件。当 caspase prodomains 被招募到活化平台并经过诱导接近时, 金星碎片再折叠餐巾。(图 1B)。由此产生的金星荧光可用于准确和具体地监测 caspase 激活平台在单细胞中的亚细胞定位、动力学和组装效率。成像数据可以通过共聚焦显微镜或标准荧光显微镜获得, 并可适应多种不同的显微方法, 包括: 时间推移成像实时跟踪 caspase 激活;高分辨率成像对亚细胞定位的精确测定和末端点量化的活化效率。
首先开发了此技术来调查 caspase-214的激活。虽然 caspase-2 的活化平台被认为是 PIDDosome, 包括受体 PIDD (p53-induced 蛋白与死亡域) 和适配器 RAIDD (RIP 相关 ICH-1/CAD-3 同源蛋白与死亡域),PIDDosome 独立的 caspase-2 激活已被报道。这表明 caspase-2 的其他激活平台存在15,16。尽管不知道 caspase-2 激活平台的全部组件, caspase BiFC 技术还是允许在分子级14、17上成功地审问 caspase-2 信号通路。我们还成功地修改了该协议的炎症半胱氨酸蛋白酶 (caspase-1,-4,-5 和-12 ) 18, 原则上, 同样的方法应该足以同样分析每一个其余的发起人半胱氨酸蛋白酶。该协议也可以类似地被修改, 以调查其他途径是二聚化是一个主要的激活信号。例如, 通过二聚化激酶 (JAK )19 磷酸化后, 统计蛋白被激活。因此, BiFC 系统可以用于可视化的统计活化以及许多其他途径调控的动态蛋白质相互作用。下面的协议提供分步说明, 介绍如何将记者引入细胞, 以及图像获取和分析方法。

<table>
	<tbody>
		<tr>
			<td>6-well Uncoated No. 1.5 20 mm glass bottom dishes</td>
			<td>Mattek</td>
			<td>P06G-1.5-20-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin Purified Protein</td>
			<td>Millipore</td>
			<td>FC010-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>D8537-6x500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI MEM I</td>
			<td>Invitrogen</td>
			<td>31985088</td>
			<td></td>
		</tr>
		<tr>
			<td>C2-Pro VC plasmid</td>
			<td>Addgene</td>
			<td>49261</td>
			<td></td>
		</tr>
		<tr>
			<td>C2-Pro VN plasmid</td>
			<td>Addgene</td>
			<td>49262</td>
			<td></td>
		</tr>
		<tr>
			<td>Inflammatory caspase BiFC plasmids</td>
			<td></td>
			<td></td>
			<td>available by request from LBH</td>
		</tr>
		<tr>
			<td>HeLa cells stably expressing the C2-Pro BiFC components</td>
			<td></td>
			<td></td>
			<td>available by request from LBH</td>
		</tr>
		<tr>
			<td>DsRed mito plasmid</td>
			<td>Clontech</td>
			<td>632421</td>
			<td>similar plasmids that can be used as fluorescent reporters can be found on Addgene</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Mercaptoethanol 1000X</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>q-VD-OPH</td>
			<td>Apex Bio</td>
			<td>A1901</td>
			<td></td>
		</tr>
	</tbody>
</table>

lighting up the pathways to caspase activation using bimolecular fluorescence complementation

蛋白酶 caspase 家族在细胞凋亡和先天免疫中起着至关重要的作用。其中, 一个称为发起方半胱氨酸蛋白酶的子群是第一个在这些路径中激活的分组。该组包括 caspase-2,-8, 和-9, 以及炎症半胱氨酸蛋白酶, caspase-1,-4, 和-5。发起人半胱氨酸蛋白酶都是由二聚化激活后, 招募到特定的复合复合体称为活化平台。Caspase 分子荧光互补 (BiFC) 是一种以成像为基础的方法, 将分离的荧光蛋白融合到引发剂半胱氨酸蛋白酶用于可视化引发剂半胱氨酸蛋白酶到它们的活化平台, 由此产生的诱导接近。此荧光提供了启动器 caspase 激活所需的最早步骤之一的读数。利用多种不同的显微手术方法, 该技术可以提供定量的数据, caspase 活化的效率, 人口水平以及 caspase 活化的动力学和大小和数量的 caspase 活化在每个细胞基础上的配合物。

在图 3中显示了 DNA 损伤引起的 caspase-2 BiFC 的例子。喜树碱, 一种拓扑异构酶抑制剂, 被用来诱导 DNA 损伤和 caspase-2 活化。红色荧光蛋白 mCherry 作为一个记者, 以表明细胞表达的 BiFC 探针和帮助可视化的总数量的细胞。金星荧光显示在绿色和大 puncta 代表 caspase-2 诱导接近度。这些细胞可以计数, 以确定金星阳性细胞的百分比。结论也可以从这些图像中?...

Watch this Scientific Journal Video about Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation at JoVE.com

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

本协议描述 caspase 分子荧光互补 (BiFC);一种基于图像的方法, 可用于可视化引发器半胱氨酸蛋白酶的诱导接近度, 这是它们激活的第一步。

利用分子荧光互补的方法照亮 Caspase 活化的途径

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

lighting-up-pathways-to-caspase-activation-using-bimolecular

Research

JoVE Journal

Biochemistry

9.0K 次观看.  Baylor College of Medicine. 本协议描述 caspase 分子荧光互补 (BiFC);一种基于图像的方法, 可用于可视化引发器半胱氨酸蛋白酶的诱导接近度, 这是它们激活的第一步。

视频: Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

研究鞘氨醇1-磷酸受体结构和信号通路的管道

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...