Name: lighting up the pathways to caspase activation using bimolecular fluorescence complementation
Uploaded: 2018-03-05T13:00:00
Description: Watch this Scientific Journal Video about Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation at JoVE.com

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?...

<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=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>

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 ...

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

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

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

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

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

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

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

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

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

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃƒÅ½Ã‚Â±-Synuclein Monomer at a Time

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

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

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

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 ...