Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

溶酶体膜通透性改变（Lysosomal membrane permeabilization, LMP）参与组织退化、退行性疾病及癌症治疗进程。然而，由于缺乏高灵敏度检测手段，相关研究长期受到阻碍。本研究对“以渗漏溶酶体处的半乳糖凝集素斑点（galectin puncta）检测LMP”这一高灵敏度且易于操作的检测方法进行了表征与验证。
LGALS1/半乳糖凝集素1（galectin-1）与LGALS3/半乳糖凝集素3（galectin-3）因表达范围广泛、可快速转位至渗漏溶酶体，且已有高亲和力抗体可供使用，成为该检测的最优选择。
半乳糖凝集素染色可在溶酶体细胞死亡早期标记单个渗漏溶酶体，有助于明确LMP是细胞死亡的原发诱因还是继发诱因。该高灵敏度方法还揭示，细胞可耐受有限程度的LMP，并证实渗漏溶酶体处可快速形成自噬结构（autophagic structures）。
尤为关键的是，半乳糖凝集素染色同样可在石蜡包埋组织中标记单个渗漏溶酶体，借此我们证实了阳离子两亲性药物（cationic amphiphilic drugs）处理的小鼠肿瘤异种移植物（tumor xenografts）中存在LMP，并鉴定出小鼠退化乳腺中可启动LMP的溶酶体亚群。
采用异位表达荧光标记半乳糖凝集素（ectopic fluorescent galectins）的半乳糖凝集素斑点检测法，可实现活细胞与活体动物内LMP的自动化筛选与可视化观测。综上，溶酶体半乳糖凝集素斑点检测法为研究LMP在细胞死亡中的作用，及其在自噬、细胞衰老、机体老化与炎症等其他细胞过程中的功能提供了全新的研究途径。