Chu Y-P, Hung Y-H, Chang H-Y, Yang W Y
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
Methods Enzymol. 2017;588:231-244. doi: 10.1016/bs.mie.2016.09.083. Epub 2016 Nov 29.
Lysophagy belongs to one of the many pathways cells activate in response to lysosomal damage. Damaged lysosomes attract glycan-binding galectins, become ubiquitinated, and are later on targeted for engulfment and degradation through lysophagy. Many triggers that are known to cause lysosomal membrane permeabilization have all been shown to induce lysophagy and can therefore be used to construct platforms for further molecular-level characterization of this process. In this chapter, we describe experimental parameters for triggering lysophagy through combined use of lysosome-specific dyes and light illumination. Within single cells, this optogenetic scheme allows easy manipulation on the amount of lysosomes to be impaired, the degree of damage desired, as well as when and where this should happen. On the other hand it can also be used to target all lysosomes within the entire cell population of a culture, allowing screening or bulk biochemical analyses to be carried out. The methodology will find use not only in monitoring lysophagy but also in probing lysosome damage responses in general.
溶酶体自噬是细胞在应对溶酶体损伤时激活的众多途径之一。受损的溶酶体吸引聚糖结合半乳糖凝集素，发生泛素化，随后通过溶酶体自噬被靶向吞噬和降解。许多已知可导致溶酶体膜通透性增加的触发因素均已被证明可诱导溶酶体自噬，因此可用于构建平台，对这一过程进行进一步的分子水平表征。在本章中，我们描述了通过联合使用溶酶体特异性染料和光照来触发溶酶体自噬的实验参数。在单细胞内，这种光遗传学方案允许轻松操控受损溶酶体的数量、所需的损伤程度以及损伤发生的时间和位置。另一方面，它也可用于靶向培养物整个细胞群体中的所有溶酶体，从而进行筛选或大量生化分析。该方法不仅将用于监测溶酶体自噬，还将普遍用于探究溶酶体损伤反应。
