State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, China.
Department of Chemical Biology, College of Chemistry and Chemical Engineering, the Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory for Physical Chemistry of Solid Surfaces, the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Xiamen University, Xiamen, China.
Autophagy. 2021 Nov;17(11):3475-3490. doi: 10.1080/15548627.2021.1875597. Epub 2021 Jan 26.
Dysfunctional organelles and defective turnover of organelles are engaged in multiple human diseases, but are elusive to image with conventional organelle probes. To overcome this, we developed intra-mitochondrial CLICK to assess mitophagy (IMCLAM), using a pair of conventional ΔΨm probes, where each probe alone fails to track dysfunctional mitochondria. The formed optical triad is stably trapped in mitochondria without resorting to ΔΨm. Utilizing an acidity-responsive ΔΨm probe, IMCLAM enabled fluorescence-on detection of mitophagy by sensing pH acidification upon delivery of mitochondria into lysosomes. Moreover, we applied IMCLAM to assay mitophagy induced by pharmacological compounds in living cells and wild-type zebrafish embryos. Thus, IMCLAM offers a simplified tool to study mitochondria and mitophagy and provide a basis for screening mitophagy-inducing compounds. CCCP, carbonyl cyanide m-chlorophenylhydrazone; IMCLAM, intra-mitochondrial CLICK to assess mitophagy; ROX, X-rhodamine; SPAAC, stain-promoted azide-alkyne Click Chemistry; TPP, triphenylphosphonium.
功能失调的细胞器和细胞器的缺陷性更新参与了多种人类疾病，但用传统的细胞器探针很难对其进行成像。为了克服这一问题，我们开发了用于评估线粒体自噬（IMCLAM）的线粒体内 CLICK，使用了一对常规的ΔΨm 探针，其中每个探针本身都无法追踪功能失调的线粒体。形成的光学三联体在不依赖于ΔΨm 的情况下稳定地被困在线粒体中。利用对酸度有响应的ΔΨm 探针，IMCLAM 通过感测线粒体递送到溶酶体时 pH 值的酸化，实现了荧光开启检测线粒体自噬。此外，我们还将 IMCLAM 应用于在活细胞和野生型斑马鱼胚胎中测定药物化合物诱导的线粒体自噬。因此，IMCLAM 提供了一种简化的工具来研究线粒体和线粒体自噬，并为筛选诱导线粒体自噬的化合物提供了基础。CCCP，羰基氰化物 m-氯代苯腙；IMCLAM，线粒体内 CLICK 评估线粒体自噬；ROX，X-罗丹明；SPAAC，染色促进叠氮-炔点击化学；TPP，三苯基膦。
