CRISPR knockout screen implicates three genes in lysosome function.

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.

Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages.

As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously, we reported a phenomenon, termed "inducible renitence," in which lipopolysaccharide (LPS) activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads. To gain mechanistic insight into the process, we analyzed the kinetics of renitence and morphological features of LPS-activated versus resting macrophages following silica bead-mediated injury. We discovered novel vacuolar structures that form in LPS-activated but not resting macrophages following silica bead phagocytosis. Because of their correlation with renitence and damage-resistant nature, we termed these structures "renitence vacuoles" (RVs). RVs formed coincident with silica bead uptake in a process associated with membrane ruffling and macropinocytosis. However, unlike normal macropinosomes (MPs), which shrink within 20 min of formation, RVs persisted around bead-containing phagosomes. RVs fused with lysosomes, whereas associated phagosomes typically did not. These findings are consistent with a model in which RVs, as persistent MPs, prevent fusion between damaged phagosomes and intact lysosomes and thereby preserve endolysosomal integrity.

Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.

The cellular movements that construct a macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of macropinosome closure.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Inducible renitence limits Listeria monocytogenes escape from vacuoles in macrophages.

Membranes of endolysosomal compartments in macrophages are often damaged by physical or chemical effects of particles ingested through phagocytosis or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macrophages that increases resistance of phagosomes, late endosomes, and lysosomes to membrane damage. Pretreatment of murine macrophages with LPS, peptidoglycan, TNF-α, or IFN-γ conferred protection against subsequent damage to intracellular membranes caused by photooxidative chemistries or by phagocytosis of ground silica or silica microspheres. Phagolysosome damage was partially dependent on reactive oxygen species but was independent of the phagocyte oxidase. IFN-γ-stimulated macrophages from mice lacking the phagocyte oxidase inhibited escape from vacuoles by the intracellular pathogen Listeria monocytogenes, which suggested a role for this inducible renitence (resistance to pressure) in macrophage resistance to infection by pathogens that damage intracellular membranes. Renitence and inhibition of L. monocytogenes escape were partially attributable to heat shock protein-70. Thus, renitence is a novel, inducible activity of macrophages that maintains or restores the integrity of endolysosomal membranes.