Singh Sanjna, Bensalem Julien, Hein Leanne K, Casey Aaron, Mäkinen Ville-Petteri, Sargeant Timothy J
South Australian Health and Medical Research Institute, Adelaide, SA, Australia.
University of Adelaide, Adelaide, SA, Australia.
Cell Death Discov. 2024 Apr 17;10(1):179. doi: 10.1038/s41420-024-01952-1.
The efficient removal of apoptotic cells via efferocytosis is critical for maintaining optimal tissue function. This involves the binding and engulfment of apoptotic cells by phagocytes and the subsequent maturation of the phagosome, culminating in lysosomal fusion and cargo destruction. However, current approaches to measure efferocytosis rely on labelling apoptotic targets with fluorescent dyes, which do not sufficiently distinguish between changes to the engulfment and acidification of apoptotic material. To address this limitation, we have developed a genetically coded ratiometric probe epHero which when expressed in the cytoplasm of target cells, bypasses the need for additional labelling steps. We demonstrate that epHero is a pH-sensitive reporter for efferocytosis and can be used to simultaneously track changes to apoptotic cell uptake and acidification, both in vitro and in mice. As proof-of-principle, we modify extracellular nutrition to show how epHero can distinguish between changes to cargo engulfment and acidification. Thus, tracking efferocytosis with epHero is a simple, cost-effective improvement on conventional techniques.
通过胞葬作用有效清除凋亡细胞对于维持最佳组织功能至关重要。这涉及吞噬细胞对凋亡细胞的结合和吞噬以及随后吞噬体的成熟，最终导致溶酶体融合和货物破坏。然而，目前测量胞葬作用的方法依赖于用荧光染料标记凋亡靶点，这不足以区分凋亡物质吞噬和酸化的变化。为了解决这一局限性，我们开发了一种基因编码的比率探针epHero，当在靶细胞的细胞质中表达时，无需额外的标记步骤。我们证明epHero是一种用于胞葬作用的pH敏感报告基因，可用于在体外和小鼠体内同时跟踪凋亡细胞摄取和酸化的变化。作为原理验证，我们改变细胞外营养以展示epHero如何区分货物吞噬和酸化的变化。因此，用epHero跟踪胞葬作用是对传统技术的一种简单、经济高效的改进。
