Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay
Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay
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DOI：
10.1080/15548627.2015.1063871
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发表时间：
2015-08-01
期刊：
影响因子：
13.3
通讯作者：
Jaattela, Marja
中科院分区：
文献类型：
--
作者：
Aits, Sonja;Kricker, Jennifer;Jaattela, Marja
溶酶体膜透化（LMP）有助于组织退化，退行性疾病和癌症治疗。然而，由于缺乏敏感的方法，调查工作受到阻碍。在这里，我们表征并验证了泄漏溶酶体中半乳凝素点状物的检测是一种高度灵敏且易于管理的LMP检测方法。LGALS 1/半乳糖凝集素-1和LGALS 3/半乳糖凝集素-3最适合于此目的，因为它们广泛表达，快速易位到泄漏的溶酶体和高亲和力抗体的可用性。半乳糖凝集素染色在溶酶体细胞死亡期间早期标记单个泄漏的溶酶体，并且在确定LMP是细胞死亡的主要原因还是次要原因时是有用的。这种敏感的方法还揭示了细胞可以在有限的LMP中存活，并证实了在半乳糖凝集素斑点部位快速形成自噬结构。重要的是，半乳糖凝集素染色检测石蜡包埋组织中的单个渗漏溶酶体，使我们能够证明用阳离子两亲性药物治疗的小鼠肿瘤异种移植物中的LMP，并鉴定在退化小鼠乳腺中启动LMP的溶酶体亚群。异位荧光半乳糖凝集素的使用使得半乳糖凝集素斑点测定法适用于活细胞和动物中LMP的自动筛选和可视化。因此，溶酶体半乳糖凝集素斑点试验为研究LMP在细胞死亡中的作用及其在其他细胞过程如自噬、衰老、老化和炎症中的作用开辟了新的可能性。
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.