Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration.

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves hitchhiking of RNA granules on lysosome-related vesicles. In the present study, we leveraged the ability to prevent transport of these vesicles into the axon by knockout of the lysosome-kinesin adaptor BLOC-one-related complex (BORC) to identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport. We found that BORC knockout causes depletion of a large group of axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins. This depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)-derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection of BORC deficiency with common neurodegenerative disorders. These results demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration.

The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Inhibition of endolysosome fusion increases exosome secretion.

Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications.

Polycomb Ezh1 maintains murine muscle stem cell quiescence through non-canonical regulation of Notch signaling.

Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.

VpdC is a ubiquitin-activated phospholipase effector that regulates Legionella vacuole expansion during infection.

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.

The autophagy protein ATG9A enables lipid mobilization from lipid droplets.

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.

Neto-α Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction.

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking.

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.

A myosin-7B-dependent endocytosis pathway mediates cellular entry of α-synuclein fibrils and polycation-bearing cargos.

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.

IRBIT Directs Differentiation of Intestinal Stem Cell Progeny to Maintain Tissue Homeostasis.

The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.

Reversible association with motor proteins (RAMP): A streptavidin-based method to manipulate organelle positioning.

We report the development and characterization of a method, named reversible association with motor proteins (RAMP), for manipulation of organelle positioning within the cytoplasm. RAMP consists of coexpressing in cultured cells (i) an organellar protein fused to the streptavidin-binding peptide (SBP) and (ii) motor, neck, and coiled-coil domains from a plus-end-directed or minus-end-directed kinesin fused to streptavidin. The SBP-streptavidin interaction drives accumulation of organelles at the plus or minus end of microtubules, respectively. Importantly, competition of the streptavidin-SBP interaction by the addition of biotin to the culture medium rapidly dissociates the motor construct from the organelle, allowing restoration of normal patterns of organelle transport and distribution. A distinctive feature of this method is that organelles initially accumulate at either end of the microtubule network in the initial state and are subsequently released from this accumulation, allowing analyses of the movement of a synchronized population of organelles by endogenous motors.

Noncanonical autophagy at ER exit sites regulates procollagen turnover.

Type I collagen is the main component of bone matrix and other connective tissues. Rerouting of its procollagen precursor to a degradative pathway is crucial for osteoblast survival in pathologies involving excessive intracellular buildup of procollagen that is improperly folded and/or trafficked. What cellular mechanisms underlie this rerouting remains unclear. To study these mechanisms, we employed live-cell imaging and correlative light and electron microscopy (CLEM) to examine procollagen trafficking both in wild-type mouse osteoblasts and osteoblasts expressing a bone pathology-causing mutant procollagen. We found that although most procollagen molecules successfully trafficked through the secretory pathway in these cells, a subpopulation did not. The latter molecules appeared in numerous dispersed puncta colocalizing with COPII subunits, autophagy markers and ubiquitin machinery, with more puncta seen in mutant procollagen-expressing cells. Blocking endoplasmic reticulum exit site (ERES) formation suppressed the number of these puncta, suggesting they formed after procollagen entry into ERESs. The punctate structures containing procollagen, COPII, and autophagic markers did not move toward the Golgi but instead were relatively immobile. They appeared to be quickly engulfed by nearby lysosomes through a bafilomycin-insensitive pathway. CLEM and fluorescence recovery after photobleaching experiments suggested engulfment occurred through a noncanonical form of autophagy resembling microautophagy of ERESs. Overall, our findings reveal that a subset of procollagen molecules is directed toward lysosomal degradation through an autophagic pathway originating at ERESs, providing a mechanism to remove excess procollagen from cells.

Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction.

Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. Here we report that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, we show that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits αPS2/ßPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. Our study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome.

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-ß4-µ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as "AP-4 deficiency syndrome". In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the µ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.

Acute loss of iron-sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells.

Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.

VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.

UNLABELLED: Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. CONCLUSION: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).

The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function.

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.