Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca2+ release from nearly all. Chelating cytoplasmic Ca2+ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca2+ in lysosomal membrane resilience. GPN-elicited Ca2+ release causes the Ca2+-sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF122 splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca2+, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.

ABSTRACT Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the cathepsin C-metabolized osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that widely used concentrations of GPN rupture only a small fraction of lysosomes, but surprisingly trigger Ca 2+ release from nearly all. Chelating cytoplasmic Ca 2+ using BAPTA makes lysosomes more likely to rupture under GPN-induced stress, suggesting that Ca 2+ plays a role in protecting or rapidly repairing lysosomal membranes. Mechanistically, we establish that GPN causes the Ca 2+ -sensitive protein Apoptosis Linked Gene-2 (ALG-2) and interacting ESCRT proteins to redistribute onto lysosomes, improving their resistance to membrane stress created by GPN as well as the lysosomotropic drug chlorpromazine. Furthermore, we show that activating the cation channel TRPML1, with or without blocking the endoplasmic reticulum Ca 2+ pump, creates local Ca 2+ signals that protect lysosomes from rupture by recruiting ALG-2 and ESCRTs without any membrane damage. These findings reveal that Ca 2+ , through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress. SIGNIFICANCE As the degradative hub of the cell, lysosomes are full of toxic content that can spill into the cytoplasm. There has been much recent interest in how cells sense and repair lysosomal membrane damage using ESCRTs and cholesterol to rapidly fix “nanoscale damage”. Here, we extend understanding of how ESCRTs contribute by uncovering a preventative role of the ESCRT machinery. We show that ESCRTs, when recruited by the Ca 2+ -sensor ALG-2, play a critical role in stabilizing the lysosomal membrane against osmotically-induced rupture. This finding suggests that cells have mechanisms not just for repairing but also for actively protecting lysosomes from stress-induced membrane damage.

While the distant organ environment is known to support metastasis of primary tumors, its metabolic roles in this process remain underdetermined. Here, in breast cancer models, we found lung-resident mesenchymal cells (MCs) accumulating neutral lipids at the pre-metastatic stage. This was partially mediated by interleukin-1β (IL-1β)-induced hypoxia-inducible lipid droplet-associated (HILPDA) that subsequently represses adipose triglyceride lipase (ATGL) activity in lung MCs. MC-specific ablation of the ATGL or HILPDA genes in mice reinforced and reduced lung metastasis of breast cancer respectively, suggesting a metastasis-promoting effect of lipid-laden MCs. Mechanistically, lipid-laden MCs transported their lipids to tumor cells and natural killer (NK) cells via exosome-like vesicles, leading to heightened tumor cell survival and proliferation and NK cell dysfunction. Blockage of IL-1β, which was effective singly, improved the efficacy of adoptive NK cell immunotherapy in mitigating lung metastasis. Collectively, lung MCs metabolically regulate tumor cells and anti-tumor immunity to facilitate breast cancer lung metastasis.
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Induction of distinct cell death pathways is critical to deal with tumor heterogeneity and therapeutic resistance. In our previous study, we reported a promising saponin analog (AG-08) for cancer therapy inducing non-canonical necrotic cell death. Here, we describe that AG-08 forms unique supramolecular structures responsible for its biological activity. After internalization via non-canonical endocytosis pathway, these structures affect several cell signaling pathways including unfolded protein response, immune response, oxidative stress and heat stress. Moreover, we prepared 18 analogs to reveal the role of residues on the formation of supramolecular structures and biological activities. The results have demonstrated that unique structural features are required for particulate structures and unprecedented cell death mechanism. Although small molecule based supramolecular assemblies have widely been accepted as nuisance for drug discovery studies, our results indicate that they may provide a new research field for anti-cancer drug development studies.