Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes

Abu-Remaileh, Monther; Wyant, Gregory A.; Kim, Choah; Laqtom, Nouf N.; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M.

doi:10.1126/science.aan6298

Cited by 466 publications

(473 citation statements)

References 46 publications

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“…It is thought that association and dissociation of V 1 -ATPase complex with the V 0 complex is a key regulatory step for many cellular pathways [74] including exocytosis of synaptic vesicles [75]. The V 0 -V 1 ATPase complex has been shown to recruit key signaling machinery such as the mTOR kinase that sense endolysosomal function and generate an appropriate response to regulate protein synthesis and cholesterol transport (reviewed in [76,77]). Kozik et al, found an important role for V-ATPase in clathrin-coated vesicle formation [78].…”

Section: Discussionmentioning

confidence: 99%

A cell-based assay for CD63-containing extracellular vesicles

Cashikar

Hanson

2019

PLoS ONE

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Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.

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Section: Discussionmentioning

confidence: 99%

A cell-based assay for CD63-containing extracellular vesicles

Cashikar

Hanson

2019

PLoS ONE

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“…Although previously viewed as “intracellular garbage disposal”, an emerging body of work indicates that lysosomes are a highly active site for integration of the metabolome (1). A key lysosomal function, namely autophagy, is not just a process of garbage collection and removal in the cell, but a key cellular mechanism for energetic homeostasis as well as maintenance of cell survival in the cardiovascular system (reviewed in (134) and (68)).…”

Section: Lysosomes As Regulators Of Cardiac Homeostasis and The Fastimentioning

confidence: 99%

Lysosomes Mediate Benefits of Intermittent Fasting in Cardiometabolic Disease: The Janitor Is the Undercover Boss

Mani

Javaheri

Diwan

2018

Comprehensive Physiology

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Adaptive responses that counter starvation have evolved over millennia to permit organismal survival, including changes at the level of individual organelles, cells, tissues, and organ systems. In the past century, a shift has occurred away from disease caused by insufficient nutrient supply toward overnutrition, leading to obesity and diabetes, atherosclerosis, and cardiometabolic disease. The burden of these diseases has spurred interest in fasting strategies that harness physiological responses to starvation, thus limiting tissue injury during metabolic stress. Insights gained from animal and human studies suggest that intermittent fasting and chronic caloric restriction extend lifespan, decrease risk factors for cardiometabolic and inflammatory disease, limit tissue injury during myocardial stress, and activate a cardioprotective metabolic program. Acute fasting activates autophagy, an intricately orchestrated lysosomal degradative process that sequesters cellular constituents for degradation, and is critical for cardiac homeostasis during fasting. Lysosomes are dynamic cellular organelles that function as incinerators to permit autophagy, as well as degradation of extracellular material internalized by endocytosis, macropinocytosis, and phagocytosis. The last decade has witnessed an explosion of knowledge that has shaped our understanding of lysosomes as central regulators of cellular metabolism and the fasting response. Intriguingly, lysosomes also store nutrients for release during starvation; and function as a nutrient sensing organelle to couple activation of mammalian target of rapamycin to nutrient availability. This article reviews the evidence for how the lysosome, in the guise of a janitor, may be the "undercover boss" directing cellular processes for beneficial effects of intermittent fasting and restoring homeostasis during feast and famine. © 2018 American Physiological Society. Compr Physiol 8:1639-1667, 2018.

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“…The logic of this placement can be understood through an evolutionary context 39 ; in yeast, the TOR pathway is localized to the vacuole, the lysosomal orthologue 7 , which functions as a major storage repository for nutrients. Recent technological advances 40,41 have determined that the mammalian lysosome is likewise selectively enriched in certain nutrients, including certain amino acids. Thus, the localization of mTORC1 to the lysosome allows mTORC1 to immediately gauge the nutrient status of the cell and rapidly shift its activity as required to maintain homeostasis.…”

Section: Metabolic Signaling At the Lysosomal Surfacementioning

confidence: 99%

Lysosome: The metabolic signaling hub

Lamming

Bar-Peled

2018

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For the past five decades the lysosome has been characterized as an unglamorous cellular recycling center. This notion has undergone a radical shift in the last ten years, with new research revealing that this organelle serves as a major hub for metabolic signaling pathways. The discovery that master growth regulators, including the protein kinase mTOR (mechanistic Target Of Rapamycin) make their home at the lysosomal surface has generated intense interest in the lysosome’s key role in nutrient sensing and cellular homeostasis. The transcriptional networks required for lysosomal maintenance and function are just being unraveled and their connection to lysosome-based signaling pathways revealed. The catabolic and anabolic pathways that converge on the lysosome connect this organelle with multiple facets of cellular function; when these pathways are deregulated they underlie multiple human diseases, and promote cellular and organismal aging. Thus, understanding how lysosome-based signaling pathways function will not only illuminate the fascinating biology of this organelle but will also be critical in unlocking its therapeutic potentials.

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Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes

Cited by 466 publications

References 46 publications

A cell-based assay for CD63-containing extracellular vesicles

A cell-based assay for CD63-containing extracellular vesicles

Lysosomes Mediate Benefits of Intermittent Fasting in Cardiometabolic Disease: The Janitor Is the Undercover Boss

Lysosome: The metabolic signaling hub

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