Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.

Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes. For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1.

How cells recognize and remove the perforated lysosome.

Macroautophagy (hereafter autophagy) is a highly conserved intracellular degradation system to maintain cellular homeostasis by degrading cellular components such as misfolded proteins, nonfunctional organelles, pathogens, and cytosol. Conversely, selective autophagy targets and degrades specific cargo, such as organelles, bacteria, etc. We previously reported that damaged lysosomes are autophagy targets, via a process called lysophagy. However, how cells target damaged lysosomes through autophagy is not known. We performed proteomics analysis followed by siRNA screening to identify genes involved in targeting damaged lysosomes and identified a new E3 ligase complex, involving CUL4A (cullin 4A), as a regulatory complex in lysophagy. We also found that this complex mediates K48-linked poly-ubiquitination on lysosome protein LAMP2 during lysosomal damage; particularly, the lumenal side of LAMP2 is important to recruit the complex to damaged lysosomes. This protein modification is thus critical to initiate the clearance of damaged lysosomes.

Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.