Necroptosis MLKL 介导的血浆膜破裂特征
Summary
我们报告的方法, MLKL 介导的等离子膜破裂在 necroptosis 包括常规和共聚焦活细胞显微成像, 扫描电镜, 核磁共振基脂结合。
Abstract
Necroptosis 是由受体相互作用蛋白激酶 3 (RIPK3) 激活触发的程序化细胞死亡通路, phosphorylates 并激活混合谱系激酶样域 pseudokinase, MLKL, 破裂或 permeabilize 等离子膜.Necroptosis 是一种炎症通路, 涉及多种病理, 包括自身免疫、传染性和心血管疾病、中风、变性和癌症。在这里, 我们描述的协议, 可以用来表征 MLKL 作为刽子手的等离子膜破裂在 necroptosis。我们用常规和共聚焦荧光显微学的活细胞成像和固定细胞中的电子显微术来可视化细胞 necroptosis 的过程, 这一起揭示了 MLKL 从细胞质向血浆的再分布膜前诱导大孔的等离子体膜。我们采用体外核磁共振 (NMR) 分析方法, 用脂质来鉴定 MLKL 介导 necroptosis 的调节剂。基于这种方法, 我们确定了定量脂结合偏好和磷脂肌醇磷酸盐 (点子) 作为 MLKL 的关键粘合剂, 这是需要的血浆膜靶向和通透在 necroptosis。
Introduction
确定 necroptosis 的遗传成分有助于使用动物模型测试 necroptosis 在生理学和疾病1、2、3、4、5中的含义。RIPK3 或 MLKL 在小鼠中的击倒对发育和成人稳态的影响最小, 表明 necroptosis 对生命3,6不重要。此外, 某些物种不包含 RIPK3 或 MLKL 基因, 支持 necroptosis 在动物7,8的非必要的作用。另一方面, 在实验室诱发的各种病态的击倒动物模型揭示了 necroptosis 在炎症、先天免疫和病毒感染方面的重要作用,9,10,11,12。
Necroptosis 可以通过多种方式激活, 通过不同的先天免疫传感器发出信号, 所有这些都导致激活 RIPK31,13,14。活动 RIPK3 依次 phosphorylates 并激活 MLKL3、4、5、6、7、8、9、10 ,11,12,13,14,15,16,17,18。最受研究的, 也许是最复杂的方式, 导致激活的 RIPK3 涉及死亡受体结扎, 分叉的基础上的信号复合物的下游组成, 以诱导凋亡或 necroptosis1。Necroptosis 发生时, 通过 RIPK1 信号是青睐和结果参与 RIPK319,20。这一结果很容易被青睐的药理抑制或基因删除 caspase 8, 一个假定的内源性抑制剂 necroptosis, 保持 necroptosis 在海湾。RIPK1 绑定并激活 RIPK3。激活 necroptosis 的另一种方法是通过类似于 TLR3/TLR4 信号的像样的受体, 它通过域的适配器诱导的干扰素β (TRIF)21参与并激活 RIPK3。另一种死于 necroptosis 的方法是激活 DNA 传感器戴, 它直接参与并激活 RIPK322。
MLKL 是由一个 n-端螺旋束 (NB) 域和一个 C 终端 pseudokinase 域 (psKD) 组成的胞浆蛋白, 由一个调节支撑区3连接。在正常细胞中, MLKL 被发现在细胞质中, 它被认为是在一个不活跃的复杂与 RIPK314。激活 necroptosis 触发器 RIPK3 磷酸化的 MLKL 在 psKD 的活化回路, 并有可能在 NB 和支撑3,15,23的其他地点。磷酸化诱导 MLKL 的构象变化, 导致从 RIPK314的离解。不清楚的构象变化释放 psKD24的支撑。支撑, 其中包含2螺旋, 介导 MLKL 的齐聚三聚体通过 C 端螺旋25。支撑的 n-端螺旋抑制 NB 域, 这对于膜通透24,26是必不可少的。在分离的情况下, NB 域足以诱发等离子膜通透和 necroptosis16、24、27。在 MLKL (MLKL MEFs) 小鼠胚胎成纤维细胞中, necroptotic 活性为铌的重组。NB 是一种脂质结合领域, 优先从事磷脂磷酸肌醇45磷酸 (PIP2)。我们提出了 MLKL 活化的逐步机制, 其中大括号齐聚通过与 PIP2极性头组24的弱相互作用, 促进了 MLKL 到等离子膜的招募。在膜上, NB 对2的附加高亲和结合点进行了规范的曝光, 在非活动 MLKL 中由大括号屏蔽。总的来说, NB 与 PIP2的多重相互作用破坏了等离子膜的破裂, 尽管这些事件的分子机制尚未被阐明。
在这里, 我们说明了一些具体的方法来描述 MLKL 作为 necroptosis24的刽子手的功能。特别是, 我们专注于最微小的领域 MLKL, NB 和支撑 (NBB), 这是由支撑抑制, 可以通过强制二聚化激活, 以诱导等离子膜破裂和 necroptosis。我们描述了我们的诱导表达系统结合强迫药物诱导 FKBP 介导的二聚化的活细胞成像和电子显微手术细胞 necroptosis。此外, 我们还说明了我们的体外核磁共振分析的相互作用的 NBB 与 phosphatidylinositols (点子)。
Protocol
Representative Results
Discussion
我们提供的技术协议, 我们结合到牵连 MLKL 作为推定刽子手的等离子膜破裂24。除了破译调节 MLKL 介导的 necroptosis 的规管网络外, 这些技术还可以独立用于其他合适的生物系统。实际上, 这些技术是中等到低吞吐量的发现工具。
我们经常使用活细胞成像 NBB140-2 xfv-金星介导 necroptosis, 并诱导其他 MLKL 构造, 机械化解剖 MLKL 的调节 necroptosis 通过诱变和使?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| Cloning and cell line generation | |||
| pRetroX-TRE3G | Clontech | 631188 | |
| Tet-On transactivator plasmid | Llambi et al., 2016 | ||
| Mouse Embryonic Fibroblasts (MEFs) mlkl-/- | Dillon et al., 2014 | ||
| Blasticidin S Hydrochloride | Thermo Fisher Scientific | BP2647100 CAS#3513-03-9 | |
| Cell death quantification and live-cell microscopy | |||
| Doxycycline | Clontech | 631311 CAS# 24390-14-5 | |
| B/B Homodimerizer AP20187 | Takara | 635059 CAS# 195514-80-8 | |
| SYTOX Green | Thermo Fisher Scientific | S7020 | |
| Syto16 | Thermo Fisher Scientific | S7578 | |
| NMR | |||
| 15N Ammonium Chloride | Cambridge Isotope Laboratories | NLM-467-10 CAS# 12125-02-9 | |
| Deuterated DTT | Cambridge Isotope Laboratories | DLM-2622-1 | |
| Deuterium Oxide | Sigma Aldrich | 617385-1 CAS# 7789-20-0 | |
| n-Dodecyl-β-D-Maltopyranoside | Anatrace | D310 CAS# 69227-93-6 | |
| L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt) | Avanti Polar Lipids | 840046X CAS# 383907-42-4 | |
| 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI) | Avanti Polar Lipids | 850143 CAS# 849412-67-5 | |
| 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt) (18:1) | Avanti Polar Lipids | 850149 CAS# 799268-53-4 | |
| Specialized Equipment | |||
| IncuCyte FLR or ZOOM | Essen BioScience, Inc. | Live-cell microscopy imaging | |
| Helios NanoLab 660 DualBeam | Thermo Fisher Scientific | Electron microscope | |
| Software | |||
| IncuCyte 2011A Rev2 v20111.3.4288 (FLR) | Essen BioScience, Inc. | http://www.essenbioscience.com | Imaging analysis |
| FEI MAPS | Thermo Fisher Scientific | https://www.fei.com/software/maps/ | EM analysis |
| TopSpin v3.2 | Bruker BioSpin | http://www.bruker.com | NMR data collection |
| CARA v1.9.1.7 | http://cara.nmr.ch/ | NMR data analysis | |
| Slidebook | 3i (Intelligent Imaging Innovations) | https://www.intelligent-imaging.com/slidebook | Confocal microscopy |
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