Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins are uniformly present on each LEL, or if there are cell-type-dependent LEL subpopulations with unique protein compositions is unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging to examine the distribution of seven key LEL proteins (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, and LAMTOR4). While LAMP1, LAMP2, and Cathepsin D were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type-specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts.

中文翻译：

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多重 DNA-PAINT 成像显示晚期内体/溶酶体的异质性。


晚期内体/溶酶体 （REL） 对许多生理过程至关重要，它们的功能障碍与许多疾病有关。蛋白质组学分析已经鉴定了数百种 LEL 蛋白;然而，这些蛋白质是否均匀存在于每个 LEL 上，或者是否存在具有独特蛋白质组成的细胞类型依赖性 LEL 亚群尚不清楚。我们采用定量、多路复用 DNA-PAINT 超分辨率成像来检查七种关键 LEL 蛋白 （LAMP1 、 LAMP2 、 CD63 、组织蛋白酶 D 、 TMEM192 、 NPC1 和 LAMTOR4 ）的分布。虽然 LAMP1 、 LAMP2 和组织蛋白酶 D 在 LEL 中含量丰富，标志着一个共同的群体，但大多数分析的蛋白质与特定的 LEL 亚群相关。我们的多重成像方法根据独特的膜蛋白组成确定了多达八个不同的 LEL 亚群。此外，我们对这些亚群与线粒体之间的空间关系的分析揭示了 NPC1 阳性 LELs 与线粒体密切相关的细胞类型特异性趋势。我们的方法将广泛适用于在许多生物学环境中确定具有单个细胞器分辨率的细胞器异质性。