bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021–05–23
27 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes.
  2. Lysosomes to the rescue: Anterograde axonal lysosome transport and neuronal proteostasis.
  3. Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB.
  4. mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.
  5. Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.
  6. Naturally occurring and tumor-associated variants of RNF167 promotes lysosomal exocytosis and plasma membrane resealing.
  7. The small GTPase Rab32 resides on lysosomes to regulate mTORC1 signaling.
  8. An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling.
  9. Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction.
  10. Lysosome Function in Cardiovascular Diseases.
  11. Mitochondria Control mTORC1 Activity Linked Compartmentalization of eIF4E to Regulate Extracellular Export of microRNAs.
  12. A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.
  13. The Rheb-TORC1 signaling axis functions as a developmental checkpoint.
  14. Retromer retrieves the Wilson Disease protein ATP7B from endolysosomes in a copper-dependent mode.
  15. Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.
  16. Early evidence of delayed oligodendrocyte maturation in the mouse model of mucolipidosis type IV.
  17. Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.
  18. Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells.
  19. Recent Updates on the Involvement of PI3K/AKT/mTOR Molecular Cascade in the Pathogenesis of Hyperproliferative Skin Disorders.
  20. S. pombe Pef1/CDK5 regulates sexual differentiation through control of the TORC1 pathway and autophagy.
  21. AHCYL1 senses SAH to inhibit autophagy through interaction with PIK3C3 in an MTORC1-independent manner.
  22. Amyloid Beta oligomers prevents Lysosomal targeting of miRNP to stop its recycling and target repression in glial cells.
  23. Genetic background modifies phenotypic severity and longevity in a mouse model of Niemann-Pick disease type C1.
  24. Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma.
  25. Cholesterol transport between cellular membranes: A balancing act between interconnected lipid fluxes.
  26. More writing: mTORC1 promotes m6A mRNA methylation.
  27. Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway.
  1. EMBO J. 2021 May 21. e105990
    NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes.
    Candice Kutchukian, Oscar Vivas, Maria Casas, Julia G Jones, Scott A Tiscione, Sergi Simó, Daniel S Ory, Rose E Dixon, Eamonn J Dickson.
      Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIβ-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.
    Keywords:  Niemann-Pick Type C; mTORC; membrane contact sites; neurodegeneration; phosphoinositides
    DOI:  https://doi.org/10.15252/embj.2020105990
  2. Dev Cell. 2021 May 17. pii: S1534-5807(21)00362-2. [Epub ahead of print]56(10): 1361-1362
    Lysosomes to the rescue: Anterograde axonal lysosome transport and neuronal proteostasis.
    Chan Choo Yap, Bettina Winckler.
      Niemann-Pick is a lysosomal storage disease caused by loss of the lysosomal cholesterol exporter NPC1 and leads to axon degeneration. Roney et al. report that immature autophagosomes accumulate in axons because cholesterol-laden lysosomes in the soma are not transported to the axon for autophagosome fusion and maturation because they aberrantly sequester non-functioning kinesin-1.
    DOI:  https://doi.org/10.1016/j.devcel.2021.04.024
  3. J Cell Sci. 2021 May 18. pii: jcs.246694. [Epub ahead of print]
    Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB.
    Cristina Escrevente, Liliana Bento-Lopes, José S Ramalho, Duarte C Barral.
      Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.
    Keywords:  Exocytosis; GRAB; Lysosome; Rab11; Rab3a; Sec15
    DOI:  https://doi.org/10.1242/jcs.246694
  4. Cell Mol Biol Lett. 2021 May 18. 26(1): 18
    mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.
    Alicja Koscielny, Ewa Liszewska, Katarzyna Machnicka, Michalina Wezyk, Katarzyna Kotulska, Jacek Jaworski.
       BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway.
    METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts.
    RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced.
    CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.
    Keywords:  Endoplasmic reticulum; Golgi apparatus; MTOR; Retention using selective hooks; Tuberous sclerosis complex; VSVg
    DOI:  https://doi.org/10.1186/s11658-021-00262-z
  5. J Biol Chem. 2021 May 14. pii: S0021-9258(21)00573-1. [Epub ahead of print] 100780
    Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.
    Kaito Mimura, Jun-Ichi Sakamaki, Hideaki Morishita, Masahito Kawazu, Hiroyuki Mano, Noboru Mizushima.
      Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
    Keywords:  CRISPR/Cas; mammalian target of rapamycin (mTOR); nucleoside/nucleotide biosynthesis; nucleoside/nucleotide metabolism; nucleotide; phosphoribosylformylglycinamidine synthase (PFAS); tuberous sclerosis complex (TSC)
    DOI:  https://doi.org/10.1016/j.jbc.2021.100780
  6. J Cell Sci. 2020 Jan 01. pii: jcs.239335. [Epub ahead of print]
    Naturally occurring and tumor-associated variants of RNF167 promotes lysosomal exocytosis and plasma membrane resealing.
    Sreeja V Nair, Nikhil Dev Narendradev, Rithwik P Nambiar, Rakesh Kumar, Srinivasa M Srinivasula.
      Lysosomal exocytosis and resealing of damaged plasma membrane are essential for cellular homeostasis and tumor invasion. However, very little is known of the molecular machinery that regulates these physiological processes. Moreover, no mutations in any of the known regulators of lysosomal exocytosis in primary tumors of patients have been characterized. Here we demonstrate that RNF167-a, a lysosomal associated ubiquitin ligase, negatively regulates lysosomal exocytosis by inducing perinuclear clustering of lysosomes. Importantly, we also characterized a set of novel natural mutations in RNF167-a, which are commonly found in diverse tumor types. We found that RNF167-a-K97N mutant, unlike the wild-type, localizes in the cytoplasm and does not promote perinuclear lysosomal clustering and that cells expressing RNF167-a-K97N exhibit dispersed lysosomes, increased exocytosis, and enhanced plasma membrane repair. Interestingly, these functional features of RNF167-a-K97N were shared with a naturally occurring short version of RNF167, i.e. isoform b. In brief, the results presented here reveal a novel role of RNF167-a as well as its natural variants, RNF167-a-K97N and RNF167-b as an upstream regulator of lysosomal exocytosis and plasma membrane resealing.
    Keywords:  Isoform b; Lysosomal exocytosis; Mutant; Plasma membrane repair; RNF167; Tumor; Ubiquitin ligase
    DOI:  https://doi.org/10.1242/jcs.239335
  7. J Cell Sci. 2020 Jan 01. pii: jcs.236661. [Epub ahead of print]
    The small GTPase Rab32 resides on lysosomes to regulate mTORC1 signaling.
    Kristina Drizyte-Miller, Jing Chen, Hong Cao, Micah B Schott, Mark A McNiven.
      Epithelial cells such as liver-resident hepatocytes rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rabs can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports mTORC1 signaling under basal and amino acid stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling.
    Keywords:  Lysosome; MTORC1; S6K; Small Rab GTPase; TFEB
    DOI:  https://doi.org/10.1242/jcs.236661
  8. J Cell Sci. 2020 Jan 01. pii: jcs.248336. [Epub ahead of print]
    An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling.
    Tadayoshi Murakawa, Amy A Kiger, Yuriko Sakamaki, Mitsunori Fukuda, Naonobu Fujita.
      Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.
    Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17
    DOI:  https://doi.org/10.1242/jcs.248336
  9. Front Cell Dev Biol. 2021 ;9 652651
    Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction.
    Leonardo Ermini, Abby Farrell, Sruthi Alahari, Jonathan Ausman, Chanho Park, Julien Sallais, Megan Melland-Smith, Tyler Porter, Michael Edson, Ori Nevo, Michael Litvack, Martin Post, Isabella Caniggia.
      Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.
    Keywords:  SMPD1; exosomes; lysosomes; placenta; preeclampsia
    DOI:  https://doi.org/10.3389/fcell.2021.652651
  10. Cell Physiol Biochem. 2021 May 22. 55(3): 277-300
    Lysosome Function in Cardiovascular Diseases.
    Owais M Bhat, Pin-Lan Li.
      The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.
    Keywords:  Lysosome; Smooth Muscle Cells; Exosomes; Sphingolipids; Vascular Calcification
    DOI:  https://doi.org/10.33594/000000373
  11. J Cell Sci. 2020 Jan 01. pii: jcs.250241. [Epub ahead of print]
    Mitochondria Control mTORC1 Activity Linked Compartmentalization of eIF4E to Regulate Extracellular Export of microRNAs.
    Susanta Chatterjee, Yogaditya Chakrabarty, Saikat Banerjee, Souvik Ghosh, Suvendra N Bhattacharyya.
      Defective intracellular trafficking and export of microRNAs have been observed in growth retarded mammalian cells having impaired mitochondrial potential and dynamics. Uncoupling Protein 2 mediated depolarization of mitochondrial membrane also results in progressive sequestration of microRNAs with polysomes and lowered their release via extracellular vesicles. Interestingly, impaired miRNA-trafficking process in growth retarded human cells could be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum, observed in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to endoplasmic reticulum attached polysomes. It causes retarded translation process accompanied by enhanced retention of miRNAs and target mRNAs with endoplasmic reticulum attached polysomes to restrict extracellular export of miRNAs. Reduced compartment specific activity of mTORC1 complex, the master regulator of protein synthesis, in mitochondria defective or ER- detethered cells, causes reduced phosphorylation of eIF4E-BP1 to prevent eIF-4E targeting to ER attached polysome and microRNA export. These data suggest how mitochondrial membrane potential and dynamics, by affecting mTORC1 activity and compartmentalization, determine sub-cellular localization and export of microRNAs.
    Keywords:  EIF4E and mTORC1; Exosomes; Extracellular vesicles; MiRNA; Mitochondria; P-body; Polysome; Processing bodies
    DOI:  https://doi.org/10.1242/jcs.250241
  12. J Cell Sci. 2020 Jan 01. pii: jcs.247080. [Epub ahead of print]
    A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.
    Lena Dehnen, Maren Janz, Jitender Kumar Verma, Olympia Ekaterini Psathaki, Lars Langemeyer, Florian Fröhlich, Jürgen J Heinisch, Heiko Meyer, Christian Ungermann, Achim Paululat.
      Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanosine exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli. Bulli localises to Rab7 positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, and (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role of Bulli in Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.
    Keywords:  Endosome; GEF; Lysosome; Mon1-Ccz1 complex; Nephrocytes; Pericardial cells
    DOI:  https://doi.org/10.1242/jcs.247080
  13. Development. 2020 Jan 01. pii: dev.181727. [Epub ahead of print]
    The Rheb-TORC1 signaling axis functions as a developmental checkpoint.
    Tam Duong, Neal R Rasmussen, Elliot Ballato, F Sefakor Mote, David J Reiner.
      In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development, and autophagy. Our CRISPR-generated, fluorescently tagged endogenous C. elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. Disruption of LET-363/TOR and DAF-15/Raptor are required for development past the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs at each stage the decision of the animal to progress through development.
    Keywords:  Ral; RalGAP; TSC; Tuberous sclerosis complex; mTOR; mTORC1
    DOI:  https://doi.org/10.1242/dev.181727
  14. J Cell Sci. 2020 Jan 01. pii: jcs.246819. [Epub ahead of print]
    Retromer retrieves the Wilson Disease protein ATP7B from endolysosomes in a copper-dependent mode.
    Santanu Das, Saptarshi Maji, Rutu Raj, Indira Bhattacharya, Tanusree Saha, Nabanita Naskar, Arnab Gupta.
      Wilson disease protein, ATP7B maintains copper homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and fro endolysosomes is not well understood. We investigated the fate of ATP7B, post-copper export. At high copper ATP7B traffics primarily to acidic, active hydrolase (Cathepsin-B) positive endolysosomes and upon subsequent copper chelation, returns to trans-Golgi network. At high copper, ATP7B co-localizes with endolysosomal markers and with core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to TGN that was rescued by overexpressing wtVPS35. Overexpressing mutants of retromer complex associated proteins, Rab7 and COMMD1 yielded similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in-vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B and it is dependent upon intracellular copper.
    Keywords:  ATP7B; Copper metabolism; Endolysosome; Retromer; VPS35; Wilson disease
    DOI:  https://doi.org/10.1242/jcs.246819
  15. Dis Model Mech. 2020 Jan 01. pii: dmm.046425. [Epub ahead of print]
    Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.
    Lena M Westermann, Lutz Fleischhauer, Jonas Vogel, Zsuzsa Jenei-Lanzl, Nataniel Floriano Ludwig, Lynn Schau, Fabio Morellini, Anke Baranowsky, Timur A Yorgan, Giorgia Di Lorenzo, Michaela Schweizer, Bruna de Souza Pinheiro, Nicole Ruas Guarany, Fernanda Sperb-Ludwig, Fernanda Visioli, Thiago Oliveira Silva, Jamie Soul, Gretl Hendrickx, J Simon Wiegert, Ida V D Schwartz, Hauke Clausen-Schaumann, Frank Zaucke, Thorsten Schinke, Sandra Pohl, Tatyana Danyukova.
      Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptgko mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgkochondrocytes and their impaired differentiation, as well as with an altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptabki mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a Rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint.
    Keywords:  Cartilage; Extracellular matrix; Joints; Lysosomal enzymes; MLIII gamma; Tendon
    DOI:  https://doi.org/10.1242/dmm.046425
  16. Dis Model Mech. 2020 Jan 01. pii: dmm.044230. [Epub ahead of print]
    Early evidence of delayed oligodendrocyte maturation in the mouse model of mucolipidosis type IV.
    Molly Mepyans, Livia Andrzejczuk, Jahree Sosa, Sierra Smith, Shawn Herron, Samantha DeRosa, Susan Slaugenhaupt, Albert Misko, Yulia Grishchuk, Kirill Kiselyov.
      Mucolipidosis type IV (MLIV) is a lysosomal disease caused by mutations in the MCOLN1 gene that encodes the endolysosomal transient receptor potential channel mucolipin-1, or TRPML1. MLIV results in developmental delay, motor and cognitive impairments, and vision loss. Brain abnormalities include thinning and malformation of the corpus callosum, white matter abnormalities, accumulation of undegraded intracellular "storage" material and cerebellar atrophy in older patients. Identification of the early events in the MLIV course is key to understanding the disease and deploying therapies. Mcoln1-/- mouse model reproduces all major aspects of the human disease. We have previously reported hypomyelination in the MLIV mouse brain. Here we investigated the onset of hypomyelination and compared oligodendrocyte maturation between the cortex/forebrain and cerebellum. We found significant delays in expression of mature oligodendrocyte markers Mag, Mbp, and Mobp in the Mcoln1-/-cortex manifesting as early as 10 days after birth and persisting later in life. Such delays were less pronounced in the cerebellum. Despite our previous finding of diminished accumulation of the ferritin-bound iron in the Mcoln1-/- brain, we report no significant changes in expression of the cytosolic iron reporters, suggesting that iron-handling deficits in MLIV occur in the lysosomes and do not involve broad iron deficiency. These data demonstrate very early deficits of oligodendrocyte maturation and critical regional differences in myelination between the forebrain and cerebellum in the mouse model of MLIV. Furthermore, they establish quantitative readouts of the MLIV impact on early brain development, useful to gauge efficacy in pre-clinical trials.
    Keywords:  Lysosome; Mucolipidosis IV; Mucolipin-1; Myelination; Oligodendrocyte; TRPML1
    DOI:  https://doi.org/10.1242/dmm.044230
  17. J Cell Sci. 2020 Jan 01. pii: jcs.245555. [Epub ahead of print]
    Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.
    Satoshi Uemura, Takahiro Mochizuki, Kengo Amemiya, Goyu Kurosaka, Miho Yazawa, Keiko Nakamoto, Yu Ishikawa, Shingo Izawa, Fumiyoshi Abe.
      Mechanical stresses including high hydrostatic pressure elicit diverse physiological effects on organisms. Gtr1/Gtr2 and Ego1/Ego3, central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased the glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ, and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1, and PIB2 with GLN3. These results suggested that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrated the regulatory circuit that maintained the intracellular amino acid homeostasis and suggested the critical roles the EGOC-TORC1 and Pib2-TORC1 complexes played in the growth of yeast under high hydrostatic pressure.
    Keywords:  EGO complex; Glutamine; Gtr1/Gtr2; High hydrostatic pressure; Pib2; Polysome profile; TORC1
    DOI:  https://doi.org/10.1242/jcs.245555
  18. Proc Natl Acad Sci U S A. 2021 May 25. pii: e2002486118. [Epub ahead of print]118(21):
    Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells.
    Song Chen, Caroline Chandra Tjin, Xiang Gao, Yi Xue, Haoyan Jiao, Ruilin Zhang, Mengnan Wu, Zunyu He, Jonathan Ellman, Ya Ha.
      Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.
    Keywords:  chemical biology; lipid kinase; p53; pip4k; synthetic lethality
    DOI:  https://doi.org/10.1073/pnas.2002486118
  19. Front Med (Lausanne). 2021 ;8 665647
    Recent Updates on the Involvement of PI3K/AKT/mTOR Molecular Cascade in the Pathogenesis of Hyperproliferative Skin Disorders.
    Laura Mercurio, Cristina Albanesi, Stefania Madonna.
      PhosphoInositide-3 Kinase (PI3K) represents a family of different classes of kinases which control multiple biological processes in mammalian cells, such as cell growth, proliferation, and survival. Class IA PI3Ks, the main regulators of proliferative signals, consists of a catalytic subunit (α, β, δ) that binds p85 regulatory subunit and mediates activation of AKT and mammalian Target Of Rapamycin (mTOR) pathways and regulation of downstream effectors. Dysregulation of PI3K/AKT/mTOR pathway in skin contributes to several pathological conditions characterized by uncontrolled proliferation, including skin cancers, psoriasis, and atopic dermatitis (AD). Among cutaneous cancers, basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) display PI3K/AKT/mTOR signaling hyperactivation, implicated in hyperproliferation, and tumorigenesis, as well as in resistance to apoptosis. Upregulation of mTOR signaling proteins has also been reported in psoriasis, in association with enhanced proliferation, defective keratinocyte differentiation, senescence-like growth arrest, and resistance to apoptosis, accounting for major parts of the overall disease phenotypes. On the contrary, PI3K/AKT/mTOR role in AD is less characterized, even though recent evidence demonstrates the relevant function for mTOR pathway in the regulation of epidermal barrier formation and stratification. In this review, we provide the most recent updates on the role and function of PI3K/AKT/mTOR molecular axis in the pathogenesis of different hyperproliferative skin disorders, and highlights on the current status of preclinical and clinical studies on PI3K-targeted therapies.
    Keywords:  AKT; PI3K; apoptosis; atopic dermatitis; hyperproliferation; non-melanoma skin cancer; psoriasis
    DOI:  https://doi.org/10.3389/fmed.2021.665647
  20. J Cell Sci. 2020 Jan 01. pii: jcs.247817. [Epub ahead of print]
    S. pombe Pef1/CDK5 regulates sexual differentiation through control of the TORC1 pathway and autophagy.
    Shinya Matsuda, Ushio Kikkawa, Haruka Uda, Akio Nakashima.
      In Schizosaccharomyces pombe, a general strategy for survival in response to environmental changes is sexual differentiation, which is triggered by TORC1 inactivation. However, mechanisms of TORC1 regulation in fission yeast remain poorly understood. In this study, we found that Pef1, which is an ortholog of mammalian CDK5, regulates the initiation of sexual differentiation through positive regulation of TORC1 activity. Conversely, deletion of pef1 leads to activation of autophagy and subsequent excessive TORC1 reactivation during the early phases of the nitrogen starvation response. This excessive TORC1 reactivation results in the silencing of the Ste11-Mei2 pathway and mating defects. Additionally, we found that pef1 genetically interacts with tsc1/2 in TORC1 regulation, and physically interacts with three types of cyclins, Clg1, Pas1, and Psl1. The double deletion of clg1 and pas1 promotes activation of autophagy and TORC1 during nitrogen starvation, similar to pef1Δ cells. Overall, our work suggests that Pef1-Clg1 and Pef1-Pas1 complexes regulate initiation of sexual differentiation through control of the TSC-TORC1 pathway and autophagy.
    Keywords:  Autophagy; CDK5; Cyclin; Pef1; Sexual differentiation; TORC1
    DOI:  https://doi.org/10.1242/jcs.247817
  21. Autophagy. 2021 May 16. 1-11
    AHCYL1 senses SAH to inhibit autophagy through interaction with PIK3C3 in an MTORC1-independent manner.
    Wei Huang, Na Li, Yi Zhang, Xu Wang, Miao Yin, Qun-Ying Lei.
      S-adenosyl-l-homocysteine (SAH), an amino acid derivative, is a key intermediate metabolite in methionine metabolism, which is normally considered as a harmful by-product and hydrolyzed quickly once formed. AHCY (adenosylhomocysteinase) converts SAH into homocysteine and adenosine. There are two other members in the AHCY family, AHCYL1 (adenosylhomocysteinase like 1) and AHCYL2 (adenosylhomocysteinase like 2). Here we define AHCYL1 function as a SAH sensor to inhibit macroautophagy/autophagy through PIK3C3. The C terminus of AHCYL1 interacts with SAH specifically and the interaction with SAH promotes the binding of the N terminus to the catalytic domain of PIK3C3, resulting in inhibition of PIK3C3. More importantly, this observation was further validated in vivo, indicating that SAH functions as a signaling molecule. Our study uncovers a new axis of SAH-AHCYL1-PIK3C3, which senses the intracellular level of SAH to inhibit autophagy in an MTORC1-independent manner.Abbreviations: ADOX: adenosine dialdehyde; AHCY: adenosylhomocysteinase; AHCYL1: adenosylhomocysteinase like 1; cLEU: cycloleucine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; SAH: S-adenosyl-l-homocysteine; SAM: S-adenosyl-l-methionine.
    Keywords:  Metabolite; metabolite sensing; methionine cycle; methyltransferase; one-carbon metabolism; signaling molecule
    DOI:  https://doi.org/10.1080/15548627.2021.1924038
  22. J Cell Sci. 2021 May 18. pii: jcs.258360. [Epub ahead of print]
    Amyloid Beta oligomers prevents Lysosomal targeting of miRNP to stop its recycling and target repression in glial cells.
    Dipayan De, Suvendra N Bhattacharyya.
      On exposure to Amyloid Beta Oligomers (Aβ1-42), glial cells start expressing proinflammatory cytokines although there has been increase in repressive miRNAs levels as well. Exploring the mechanism of this potential immunity of target cytokine mRNAs against repressive miRNAs in amyloid beta exposed glial cells, we have identified differential compartmentalization of repressive miRNAs in glial cells to explain this aberrant miRNA function. While the target mRNAs were found to be associated with polysomes attached to endoplasmic reticulum, the miRNPs found to be present predominantly with endosomes that failed to recycle to endoplasmic reticulum attached polysomes to repress mRNA targets in Aβ1-42 treated cells. Aβ1-42 oligomers, by masking the Rab7 proteins on endosomal surface, affects Rab7 interaction with Rab Interacting Lysosomal Protein (RILP) to restrict lysosomal targeting and recycling of miRNPs. RNA processing body or P-body localization of the miRNPs also get enhanced in amyloid beta treated cells as a consequence of enhanced endosomal retention of miRNPs. Interestingly, depletion of P-body components partly rescues the miRNA function in glial cells exposed to amyloid beta and restricts the excess cytokine expression there.
    Keywords:  Endosome Lysosome interaction; RNA processing bodies; mRNA compartmentalization; miRNA mediated translation repression; miRNP recycling
    DOI:  https://doi.org/10.1242/jcs.258360
  23. Dis Model Mech. 2020 Jan 01. pii: dmm.042614. [Epub ahead of print]
    Genetic background modifies phenotypic severity and longevity in a mouse model of Niemann-Pick disease type C1.
    Jorge L Rodriguez-Gil, Dawn E Watkins-Chow, Laura L Baxter, Gene Elliot, Ursula L Harper, Stephen M Wincovitch, Julia C Wedel, Arturo A Incao, Mylene Huebecker, Frederick J Boehm, William S Garver, Forbes D Porter, Karl W Broman, Frances M Platt, William J Pavan.
      Niemann-Pick disease type C1 (NPC1) is a rare, fatal neurodegenerative disorder characterized by lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration, and premature death. NPC1 is extremely heterogeneous in the timing of clinical presentation and is associated with a wide spectrum of causative NPC1 mutations. To study the genetic architecture of NPC1, including the clinical and genetic heterogeneity seen in this patient population, we have generated a new NPC1 mouse model, Npc1em1Pav. Npc1em1Pav/em1Pav mutants showed notably reduced NPC1 protein compared to controls and displayed the pathological and biochemical hallmarks of NPC1. Interestingly, Npc1em1Pav/em1Pav mutants on a C57BL/6J genetic background showed more severe visceral pathology, quantified by the presence of CD68+ foam cells, compared to Npc1em1Pav/em1Pav mutants on a BALB/cJ background. Furthermore, C57BL/6J mutants exhibited a significantly shorter lifespan (mean=70 days) than Npc1em1Pav/em1Pav mutants on a BALB/cJ genetic background (mean=84 days), suggesting strain-specific modifiers contribute to disease severity and survival. QTL analysis for lifespan of 202 backcross N2 mutants on a mixed C57BL/6J and BALB/cJ background detected significant linkage to markers on chromosomes 1 (LOD=5.57) and 7 (LOD=8.91). The discovery of these modifier regions demonstrates that mouse models are powerful tools for analyzing the genetics underlying rare human disease, which can be used to improve understanding of the variability in NPC1 phenotypes and advance options for patient diagnosis and therapy.
    Keywords:  Genetic modifiers; Mouse models; NPC1; Niemann-Pick disease type C1
    DOI:  https://doi.org/10.1242/dmm.042614
  24. Sci Rep. 2021 May 19. 11(1): 10609
    Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma.
    Marie-Anaïs Locquet, Gabriel Ichim, Joseph Bisaccia, Aurelie Dutour, Serge Lebecque, Marie Castets, Kathrin Weber.
      In cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a "default" death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.
    DOI:  https://doi.org/10.1038/s41598-021-89793-1
  25. Dev Cell. 2021 May 17. pii: S1534-5807(21)00363-4. [Epub ahead of print]56(10): 1430-1436
    Cholesterol transport between cellular membranes: A balancing act between interconnected lipid fluxes.
    Elina Ikonen, Xin Zhou.
      Cholesterol represents the most abundant single lipid in mammalian cells. How its asymmetric distribution between subcellular membranes is achieved and maintained attracts considerable interest. One of the challenges is that cholesterol rarely is transported alone, but rather is coupled with heterotypic transport and metabolism of other lipids, in particular phosphoinositides, phosphatidylserine, and sphingolipids. This perspective summarizes the major exo- and endocytic cholesterol transport routes and how lipid transfer proteins at membrane contacts and membrane transport intersect along these routes. It discusses the co-transport of cholesterol with other lipids in mammalian cells and reviews emerging evidence related to the physiological relevance of this process.
    Keywords:  lipid transfer protein; membrane contact sites; membrane domain; membrane transport; sterol transport
    DOI:  https://doi.org/10.1016/j.devcel.2021.04.025
  26. Mol Cell. 2021 May 20. pii: S1097-2765(21)00325-7. [Epub ahead of print]81(10): 2057-2058
    More writing: mTORC1 promotes m6A mRNA methylation.
    Sunil Shetty, Michael N Hall.
      Cho et al. (2021) and Villa et al. (2021) demonstrate that mTORC1 stimulates m6A mRNA methylation via WTAP expression and SAM synthesis. Increased mRNA methylation in turn promotes cell growth by enhancing mRNA degradation or translation.
    DOI:  https://doi.org/10.1016/j.molcel.2021.04.020
  27. J Cell Biol. 2021 Jul 05. pii: e202010179. [Epub ahead of print]220(7):
    Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway.
    Sydney E Cason, Peter J Carman, Claire Van Duyne, Juliet Goldsmith, Roberto Dominguez, Erika L F Holzbaur.
      Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health.
    DOI:  https://doi.org/10.1083/jcb.202010179
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