Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
High-throughput Screening Center, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
J Mol Biol. 2024 Jul 1;436(13):168628. doi: 10.1016/j.jmb.2024.168628. Epub 2024 May 24.
Necroptosis is a programmed lytic cell death involving active cytokine production and plasma membrane rupture through distinct signaling cascades. However, it remains challenging to delineate this inflammatory cell death pathway at specific signaling nodes with spatiotemporal accuracy. To address this challenge, we developed an optogenetic system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, to enable ligand-free, optical induction of RIPK3 oligomerization. La-RIPK3 activation dissects RIPK3-centric lytic cell death through the induction of RIPK3-containing necrosome, which mediates cytokine production and plasma membrane rupture. Bulk RNA-Seq analysis reveals that RIPK3 oligomerization results in partially overlapped gene expression compared to pharmacological induction of necroptosis. Additionally, La-RIPK3 activates separated groups of genes regulated by RIPK3 kinase-dependent and -independent processes. Using patterned light stimulation delivered by a spatial light modulator, we demonstrate precise spatiotemporal control of necroptosis in La-RIPK3-transduced HT-29 cells. Optogenetic control of proinflammatory lytic cell death could lead to the development of innovative experimental strategies to finetune the immune landscape for disease intervention.
细胞程序性坏死是一种涉及到细胞因子产生和质膜破裂的细胞程序性死亡方式，其通过不同的信号通路来实现。然而，要在特定的信号节点上以时空精度来描绘这种炎症性细胞死亡途径仍然具有挑战性。为了解决这个问题，我们开发了一种光遗传学系统，称为光激活受体相互作用蛋白激酶 3 或 La-RIPK3，用于实现无配体、光学诱导的 RIPK3 寡聚化。La-RIPK3 的激活通过诱导含有 RIPK3 的坏死小体来剖析以 RIPK3 为中心的细胞裂解死亡，该坏死小体介导细胞因子的产生和质膜的破裂。批量 RNA-Seq 分析表明，与坏死诱导的药理学方法相比，RIPK3 寡聚化导致了部分重叠的基因表达。此外，La-RIPK3 激活了由 RIPK3 激酶依赖性和非依赖性过程调节的分离基因群。我们使用空间光调制器提供的图案化光刺激，在 La-RIPK3 转导的 HT-29 细胞中演示了精确的细胞程序性坏死的时空控制。对促炎细胞程序性坏死的光遗传学控制可能会导致开发出创新的实验策略，以微调免疫景观以进行疾病干预。
