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.

Lysosomal Amino Acid Transporter: Cystinosis is characterized by intralysosomal accumulation of free cystine, which results in age-dependent problems in the kidney, muscle, retina, and central nervous system. The disease-causing gene encodes a lysosomal cystine transporter. The most effective therapeutic agent for cystinosis, cysteamine, depletes lysosomal free cystine by converting it to cysteine and the mixed disulfide cysteine-cysteamine, which can then be exported from lysosomes as a lysine analog through a putative lysine/cationic amino acid transporter. Using an unbiased genetic screen for Caenorhabditis elegans mutants with increased accumulation of apoptotic cells or autophagosomes, Liu et al. (p. 351 ) now reveal the molecular identity of a lysosomal lysine/arginine transporter that plays a role in maintaining lysosome function and amino acid homeostasis and that can explain how cysteamine alleviates a lysosomal storage disease.

The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA‐ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc‐25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV‐3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.

Inducible gene expression systems are favored over stable expression systems in a wide variety of basic and applied research areas, including functional genomics, gene therapy, tissue engineering, biopharmaceutical protein production and drug discovery. This is because they are mostly reversible and thus more flexible to use. Furthermore, compared to constitutive expression, they generally exhibit a higher efficiency and have fewer side effects, such as cell death and delayed growth or development. Empowered by decades of development of inducible gene expression systems, researchers can now efficiently activate or suppress any gene, temporarily and quantitively at will, depending on experimental requirements and designs. Here, we review a number of most commonly used mammalian inducible expression systems and provide basic standards and criteria for the selection of the most suitable one.

Recognition of apoptotic cells by phagocytes is not fully understood. In C. elegans, the transthyretin-like protein, TTR-52, is secreted by the endoderm and clusters around apoptotic cells, inducing their engulfment through the CED-1/6/7 pathway. During apoptosis, dying cells are swiftly removed by phagocytes. It is not fully understood how apoptotic cells are recognized by phagocytes. Here we report the identification and characterization of the Caenorhabditis elegans ttr-52 gene, which encodes a transthyretin-like protein and is required for efficient cell corpse engulfment. The TTR-52 protein is expressed in, and secreted from, C. elegans endoderm and clusters around apoptotic cells. Genetic analysis indicates that TTR-52 acts in the cell corpse engulfment pathway mediated by CED-1, CED-6 and CED-7 and affects clustering of the phagocyte receptor CED-1 around apoptotic cells. TTR-52 recognizes surface-exposed phosphatidylserine (PtdSer) in vivo and binds to both PtdSer and the extracellular domain of CED-1 in vitro. TTR-52 is therefore the first bridging molecule identified in C. elegans that mediates recognition of apoptotic cells by crosslinking the PtdSer 'eat me' signal with the phagocyte receptor CED-1.

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.