Nemegeer Josephine, Lemeire Kelly, Vandenabeele Peter, Maelfait Jonathan
VIB-UGent Center for Inflammation Research; Department of Biomedical Molecular Biology, Ghent University.
VIB-UGent Center for Inflammation Research; Department of Biomedical Molecular Biology, Ghent University;
J Vis Exp. 2022 Oct 20(188). doi: 10.3791/64332.
The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.
激酶相互作用丝氨酸/苏氨酸蛋白激酶3（RIPK3）及其底物混合谱系激酶结构域样蛋白（MLKL）是坏死性凋亡的关键调节因子，坏死性凋亡是一种具有重要抗病毒功能的炎症性细胞死亡形式。RIPK3的自磷酸化诱导坏死性凋亡的孔形成执行蛋白MLKL的磷酸化和激活。磷酸化的MLKL在细胞膜上的运输和寡聚化导致细胞裂解，这是坏死性凋亡细胞死亡的特征。核酸传感器ZBP1在感染RNA和DNA病毒后通过与左手Z型双链RNA（Z-RNA）结合而被激活。ZBP1激活通过诱导受感染宿主细胞的程序性细胞死亡（包括坏死性凋亡）来限制病毒感染。免疫荧光显微镜允许在单个细胞基础上可视化ZBP1介导的坏死性凋亡下游的不同信号步骤。然而，使用目前市售的针对人RIPK3和MLKL的磷酸化特异性抗体的标准荧光显微镜的灵敏度，使得这些标志物的可重复成像成为不可能。在这里，我们描述了一种针对感染单纯疱疹病毒1（HSV-1）的人HT-29细胞中丝氨酸（S）磷酸化的RIPK3（S227）和MLKL（S358）的优化染色程序。在免疫荧光染色方案中加入酪胺信号放大（TSA）步骤，可以特异性检测S227磷酸化的RIPK3。此外，TSA大大提高了S358磷酸化的MLKL的检测灵敏度。总之，这种方法能够在ZBP1诱导的坏死性凋亡诱导过程中可视化这两个关键信号事件。
