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Shawn M Ferguson - One of the best experts on this subject based on the ideXlab platform.
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minimal membrane interactions conferred by rheb c terminal farnesylation are essential for mtorc1 activation
bioRxiv, 2019Co-Authors: Brittany Angarola, Shawn M FergusonAbstract:Stable localization of the Rheb GTPase to Lysosomes is thought to be required for activation of mTORC1 signaling. However, the lysosome targeting mechanisms for Rheb remain unclear. We therefore investigated the relationship between Rheb subcellular localization and mTORC1 activation. Surprisingly, we found that although Rheb was prominently enriched at the endoplasmic reticulum (ER), Rheb was undetectable at Lysosomes. Functional assays in knockout human cells revealed that farnesylation of the C-terminal CaaX motif on Rheb was essential for both Rheb enrichment on ER membranes and mTORC1 activation. However, constitutively targeting Rheb to ER membranes did not support mTORC1 activation. Further systematic analysis of the Rheb hypervariable region revealed that weak, non-selective, farnesylation-dependent, membrane interactions confer Rheb function without the need for a specific lysosome targeting motif. Collectively, these results argue against stable interactions of Rheb with Lysosomes and instead that transient membrane interactions optimally allow Rheb to activate mTORC1 signaling.
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Axonal transport and maturation of Lysosomes.
Current Opinion in Neurobiology, 2018Co-Authors: Shawn M FergusonAbstract:Lysosomes perform degradative functions that are important for all cells. However, neurons are particularly dependent on optimal lysosome function due to their extremes of longevity, size and polarity. Axons in particular exemplify the major spatial challenges faced by neurons in the maintenance of lysosome biogenesis and function. What impact does this have on the regulation and functions of Lysosomes in axons? This review focuses on the mechanisms whereby axonal lysosome biogenesis, transport and function are adapted to meet neuronal demand. Important features include the dynamic relationship between endosomes, autophagosomes and Lysosomes as well as the transport mechanisms that support the movement of lysosome precursors in axons. A picture is emerging wherein intermediates in the lysosome maturation processes that would only exist transiently within the crowded confines of a neuronal cell body are spatially and temporally separated over the extreme distances encountered in axons. Axons may thus offer significant opportunities for the analysis of the mechanisms that control lysosome biogenesis. Insights from the genetics and pathology of human neurodegenerative diseases furthermore emphasize the importance of efficient axonal transport of Lysosomes and their precursors.
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Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology.
The Journal of cell biology, 2017Co-Authors: Swetha Gowrishankar, Shawn M FergusonAbstract:Lysosomes robustly accumulate within axonal swellings at Alzheimer’s disease (AD) amyloid plaques. However, the underlying mechanisms and disease relevance of such lysosome accumulations are not well understood. Motivated by these problems, we identified JNK-interacting protein 3 (JIP3) as an important regulator of axonal lysosome transport and maturation. JIP3 knockout mouse neuron primary cultures accumulate Lysosomes within focal axonal swellings that resemble the dystrophic axons at amyloid plaques. These swellings contain high levels of amyloid precursor protein processing enzymes (BACE1 and presenilin 2) and are accompanied by elevated Aβ peptide levels. The in vivo importance of the JIP3-dependent regulation of axonal Lysosomes was revealed by the worsening of the amyloid plaque pathology arising from JIP3 haploinsufficiency in a mouse model of AD. These results establish the critical role of JIP3-dependent axonal lysosome transport in regulating amyloidogenic amyloid precursor protein processing and support a model wherein Aβ production is amplified by plaque-induced axonal lysosome transport defects.
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massive accumulation of luminal protease deficient axonal Lysosomes at alzheimer s disease amyloid plaques
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Swetha Gowrishankar, Peng Yuan, Matthew Schrag, Summer Paradise, Jaime Grutzendler, Pietro De Camilli, Shawn M FergusonAbstract:Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that Lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal Lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal Lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.
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recruitment of folliculin to Lysosomes supports the amino acid dependent activation of rag gtpases
Journal of Cell Biology, 2013Co-Authors: Constance S Petit, Agnes Roczniakferguson, Shawn M FergusonAbstract:Birt-Hogg-Dube syndrome, a human disease characterized by fibrofolliculomas (hair follicle tumors) as well as a strong predisposition toward the development of pneumothorax, pulmonary cysts, and renal carcinoma, arises from loss-of-function mutations in the folliculin (FLCN) gene. In this study, we show that FLCN regulates lysosome function by promoting the mTORC1-dependent phosphorylation and cytoplasmic sequestration of transcription factor EB (TFEB). Our results indicate that FLCN is specifically required for the amino acid–stimulated recruitment of mTORC1 to Lysosomes by Rag GTPases. We further demonstrated that FLCN itself was selectively recruited to the surface of Lysosomes after amino acid depletion and directly bound to RagA via its GTPase domain. FLCN-interacting protein 1 (FNIP1) promotes both the lysosome recruitment and Rag interactions of FLCN. These new findings define the lysosome as a site of action for FLCN and indicate a critical role for FLCN in the amino acid–dependent activation of mTOR via its direct interaction with the RagA/B GTPases.
Juan S Bonifacino - One of the best experts on this subject based on the ideXlab platform.
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a ragulator borc interaction controls lysosome positioning in response to amino acid availability
Journal of Cell Biology, 2017Co-Authors: Jing Pu, Tal Kerenkaplan, Juan S BonifacinoAbstract:Lysosomes play key roles in the cellular response to amino acid availability. Depletion of amino acids from the medium turns off a signaling pathway involving the Ragulator complex and the Rag guanosine triphosphatases (GTPases), causing release of the inactive mammalian target of rapamycin complex 1 (mTORC1) serine/threonine kinase from the lysosomal membrane. Decreased phosphorylation of mTORC1 substrates inhibits protein synthesis while activating autophagy. Amino acid depletion also causes clustering of Lysosomes in the juxtanuclear area of the cell, but the mechanisms responsible for this phenomenon are poorly understood. Herein we show that Ragulator directly interacts with BLOC-1–related complex (BORC), a multi-subunit complex previously found to promote lysosome dispersal through coupling to the small GTPase Arl8 and the kinesins KIF1B and KIF5B. Interaction with Ragulator exerts a negative regulatory effect on BORC that is independent of mTORC1 activity. Amino acid depletion strengthens this interaction, explaining the redistribution of Lysosomes to the juxtanuclear area. These findings thus demonstrate that amino acid availability controls lysosome positioning through Ragulator-dependent, but mTORC1-independent, modulation of BORC.
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borc kinesin 1 ensemble drives polarized transport of Lysosomes into the axon
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Ginny G. Farías, Carlos M Guardia, Raffaella De Pace, Dylan J Britt, Juan S BonifacinoAbstract:The ability of Lysosomes to move within the cytoplasm is important for many cellular functions. This ability is particularly critical in neurons, which comprise vast, highly differentiated domains such as the axon and dendrites. The mechanisms that control lysosome movement in these domains, however, remain poorly understood. Here we show that an ensemble of BORC, Arl8, SKIP, and kinesin-1, previously shown to mediate centrifugal transport of Lysosomes in nonneuronal cells, specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. This transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. Our findings illustrate how a general mechanism for lysosome dispersal in nonneuronal cells is adapted to drive polarized transport in neurons, and emphasize the importance of this mechanism for critical axonal processes.
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mechanisms and functions of lysosome positioning
Journal of Cell Science, 2016Co-Authors: Jing Pu, Carlos Minchola Guardia, Tal Kerenkaplan, Juan S BonifacinoAbstract:ABSTRACT Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of Lysosomes in most of these processes is now known to depend on the ability of Lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of Lysosomes, and the importance of lysosome dynamics for cell physiology and pathology.
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borc functions upstream of kinesins 1 and 3 to coordinate regional movement of Lysosomes along different microtubule tracks
Cell Reports, 2016Co-Authors: Carlos M Guardia, Ginny G. Farías, Rui Jia, Juan S BonifacinoAbstract:The multiple functions of Lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of Lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multi-subunit complex named BORC that recruits the small GTPase Arl8 to Lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here, we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves Lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell.
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borc a multisubunit complex that regulates lysosome positioning
Developmental Cell, 2015Co-Authors: Jing Pu, Christina Schindler, Michal Jarnik, Peter S Backlund, Juan S BonifacinoAbstract:Summary The positioning of Lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multisubunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the kinesin-dependent movement of Lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of Lysosomes.
Li Wang - One of the best experts on this subject based on the ideXlab platform.
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Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte
Journal of Neuroscience, 2011Co-Authors: Liu Tao, Lei Sun, Yingfei Xiong, Shujiang Shang, Ning Guo, Sasa Teng, Yeshi Wang, Bin Liu, Changhe Wang, Li WangAbstract:Astrocytes release a variety of signaling molecules including glutamate, d-serine, and ATP in a regulated manner. Although the functions of these molecules, from regulating synaptic transmission to controlling specific behavior, are well documented, the identity of their cellular compartment(s) is still unclear. Here we set out to study vesicular exocytosis and glutamate release in mouse hippocampal astrocytes. We found that small vesicles and Lysosomes coexisted in the same freshly isolated or cultured astrocytes. Both small vesicles and lysosome fused with the plasma membrane in the same astrocytes in a Ca2+-regulated manner, although small vesicles were exocytosed more efficiently than Lysosomes. Blockade of the vesicle glutamate transporter or cleavage of synaptobrevin 2 and cellubrevin (both are vesicle-associated membrane proteins) with a clostridial toxin greatly inhibited glutamate release from astrocytes, while lysosome exocytosis remained intact. Thus, both small vesicles and Lysosomes contribute to Ca2+-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.
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Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte
journal of neuroscience, 2011Co-Authors: Liu Tao, Sun Lei, Xiong Yingfei, Shang Shujiang, Guo Ning, Teng Sasa, Wang Yeshi, Liu Bin, Wang Changhe, Li WangAbstract:Astrocytes release a variety of signaling molecules including glutamate, D-serine, and ATP in a regulated manner. Although the functions of these molecules, from regulating synaptic transmission to controlling specific behavior, are well documented, the identity of their cellular compartment(s) is still unclear. Here we set out to study vesicular exocytosis and glutamate release in mouse hippocampal astrocytes. We found that small vesicles and Lysosomes coexisted in the same freshly isolated or cultured astrocytes. Both small vesicles and lysosome fused with the plasma membrane in the same astrocytes in a Ca2+-regulated manner, although small vesicles were exocytosed more efficiently than Lysosomes. Blockade of the vesicle glutamate transporter or cleavage of synaptobrevin 2 and cellubrevin (both are vesicle-associated membrane proteins) with a clostridial toxin greatly inhibited glutamate release from astrocytes, while lysosome exocytosis remained intact. Thus, both small vesicles and Lysosomes contribute to Ca2+-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000292921100018&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701NeurosciencesSCI(E)PubMed47ARTICLE2910593-106013
Yamuna Krishnan - One of the best experts on this subject based on the ideXlab platform.
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A DNA nanomachine chemically resolves Lysosomes in live cells
Nature Nanotechnology, 2019Co-Authors: Kaho Leung, Kasturi Chakraborty, Anand Saminathan, Yamuna KrishnanAbstract:Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional Lysosomes underlie Alzheimer’s disease, Parkinson’s disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of Lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann–Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving Lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy. A DNA-based nanosensor that simultaneously measures pH and chloride concentrations can chemically resolve different subpopulations of Lysosomes in live cells derived from healthy individuals and patients with Niemann–Pick disease.
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High lumenal chloride in the lysosome is critical for lysosome function
eLife, 2017Co-Authors: Kasturi Chakraborty, Kaho Leung, Yamuna KrishnanAbstract:In cells, worn out proteins and other unnecessary materials are sent to small compartments called Lysosomes to be broken down and recycled. Lysosomes contain many different proteins including some that break down waste material into recyclable fragments and others that transport the fragments out of the lysosome. If any of these proteins do not work, waste products build up and cause disease. There are around 70 such lysosomal storage diseases, each arising from a different lysosomal protein not working correctly. A recently developed “nanodevice” called Clensor can measure the levels of chloride ions inside cells. Clensor is constructed from DNA, and its fluorescence changes when it detects chloride ions. Although chloride ions have many biological roles, chloride ion levels had not been measured inside a living organism. Now, Chakraborty et al. – including some of the researchers who developed Clensor – have used this nanodevice to examine chloride ion levels in the Lysosomes of the roundworm Caenorhabditis elegans. This revealed that the Lysosomes contain high levels of chloride ions. Furthermore, reducing the amount of chloride in the Lysosomes made them worse at breaking down waste. Do Lysosomes affected by lysosome storage diseases also contain low levels of chloride ions? To find out, Chakraborty et al. used Clensor to study C. elegans worms and mouse and human cells whose Lysosomes accumulate waste products. In all these cases, the levels of chloride in the diseased Lysosomes were much lower than normal. This had a number of effects on how the Lysosomes worked, such as reducing the activity of key lysosomal proteins. Chakraborty et al. also found that Clensor can be used to distinguish between different lysosomal storage diseases. This means that in the future, Clensor (or similar methods that directly measure chloride ion levels in Lysosomes) may be useful not just for research purposes. They may also be valuable for diagnosing lysosomal storage diseases early in infancy that, if left undiagnosed, are fatal.
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High lumenal chloride in the lysosome is critical for lysosome function
bioRxiv, 2017Co-Authors: Yamuna Krishnan, Kasturi Chakraborty, Kaho LeungAbstract:Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional Lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~10 4 -fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca 2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function.
Rosa Puertollano - One of the best experts on this subject based on the ideXlab platform.
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lysosome enlargement during inhibition of the lipid kinase pikfyve proceeds through lysosome coalescence
Journal of Cell Science, 2018Co-Authors: Christopher H Choy, Roya M. Dayam, Rosa Puertollano, Golam Saffi, Matthew A Gray, Callen T Wallace, Zhenyi A Ou, Guy M Lenk, Simon C Watkins, Roberto J. BotelhoAbstract:Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux, and a massive enlargement of Lysosomes. Collectively, this leads to various physiological defects including embryonic lethality, neurodegeneration and overt inflammation. While being the most dramatic phenotype, the reasons for lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged Lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.
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tfeb regulates lysosomal positioning by modulating tmem55b expression and jip4 recruitment to Lysosomes
Nature Communications, 2017Co-Authors: Rose Willett, Jose A Martina, James P Zewe, Rachel C Wills, Gerald R.v. Hammond, Rosa PuertollanoAbstract:Lysosomal distribution is linked to the role of Lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of Lysosomes toward the microtubules minus-end. TMEM55B overexpression causes Lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome–lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions. Lysosomal distribution is linked to the role of Lysosomes in many cellular functions. Here the authors show that the lysosomal protein TMEM55B is regulated by TFEB and recruits JIP4 to the lysosomal surface inducing dynein-dependent transport of Lysosomes toward the cell center in response to stress conditions.
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tfeb regulates lysosomal positioning by modulating tmem55b expression and jip4 recruitment to Lysosomes
Nature Communications, 2017Co-Authors: Rose Willett, Jose A Martina, James P Zewe, Rachel C Wills, Gerald R.v. Hammond, Rosa PuertollanoAbstract:Lysosomal distribution is linked to the role of Lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of Lysosomes toward the microtubules minus-end. TMEM55B overexpression causes Lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.
