RESUMEN
Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.
RESUMEN
Cells maintain optimal levels of lysosome degradative activity to protect against pathogens, clear waste, and generate nutrients. Here, we show that LRRK2, a protein that is tightly linked to Parkinson's disease, negatively regulates lysosome degradative activity in macrophages and microglia via a transcriptional mechanism. Depletion of LRRK2 and inhibition of LRRK2 kinase activity enhanced lysosomal proteolytic activity and increased the expression of multiple lysosomal hydrolases. Conversely, the kinase hyperactive LRRK2 G2019S Parkinson's disease mutant suppressed lysosomal degradative activity and gene expression. We identified MiT-TFE transcription factors (TFE3, TFEB, and MITF) as mediators of LRRK2-dependent control of lysosomal gene expression. LRRK2 negatively regulated the abundance and nuclear localization of these transcription factors and their depletion prevented LRRK2-dependent changes in lysosome protein levels. These observations define a role for LRRK2 in controlling lysosome degradative activity and support a model wherein LRRK2 hyperactivity may increase Parkinson's disease risk by suppressing lysosome degradative activity.
Asunto(s)
Enfermedad de Parkinson , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Microglía/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismoRESUMEN
The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.
Asunto(s)
Proteínas , Saposinas , Humanos , Transporte Biológico , Lisosomas/metabolismo , Progranulinas/metabolismo , Transporte de Proteínas/fisiología , Proteínas/metabolismo , Saposinas/genética , Saposinas/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFß3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.
Asunto(s)
Calcio , Leche , Femenino , Animales , Leche/metabolismo , Calcio/metabolismo , Muerte Celular , Lactancia , Lisosomas/metabolismo , Glándulas Mamarias Animales/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues.
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Membrana Celular/fisiología , Dinaminas/fisiología , Animales , Membrana Celular/metabolismo , Dinaminas/química , Dinaminas/genética , Dinaminas/metabolismo , Endocitosis/genética , Endocitosis/fisiología , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/fisiología , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Mamíferos/genética , Mamíferos/metabolismo , Fluidez de la Membrana/genética , Ratones , Modelos Biológicos , Modelos Moleculares , Conformación ProteicaRESUMEN
PQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex composed of C9orf72, SMCR8, and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we present evidence that the WDR41-PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 ß-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. The data support a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41-binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes.
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Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos/metabolismo , Lisosomas/metabolismo , Secuencias de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/química , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Sitios de Unión , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos/metabolismo , Mutagénesis , Conformación Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Phospholipase D3 (PLD3) is a protein of unclear function that structurally resembles other members of the phospholipase D superfamily. A coding variant in this gene confers increased risk for the development of Alzheimer's disease (AD), although the magnitude of this effect has been controversial. Because of the potential significance of this obscure protein, we undertook a study to observe its distribution in normal human brain and AD-affected brain, determine whether PLD3 is relevant to memory and cognition in sporadic AD, and to evaluate its molecular function. In human neuropathological samples, PLD3 was primarily found within neurons and colocalized with lysosome markers (LAMP2, progranulin, and cathepsins D and B). This colocalization was also present in AD brain with prominent enrichment on lysosomal accumulations within dystrophic neurites surrounding ß-amyloid plaques. This pattern of protein distribution was conserved in mouse brain in wild type and the 5xFAD mouse model of cerebral ß-amyloidosis. We discovered PLD3 has phospholipase D activity in lysosomes. A coding variant in PLD3 reported to confer AD risk significantly reduced enzymatic activity compared to wild-type PLD3. PLD3 mRNA levels in the human pre-frontal cortex inversely correlated with ß-amyloid pathology severity and rate of cognitive decline in 531 participants enrolled in the Religious Orders Study and Rush Memory and Aging Project. PLD3 levels across genetically diverse BXD mouse strains and strains crossed with 5xFAD mice correlated strongly with learning and memory performance in a fear conditioning task. In summary, this study identified a new functional mammalian phospholipase D isoform which is lysosomal and closely associated with both ß-amyloid pathology and cognition.
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Enfermedad de Alzheimer/genética , Disfunción Cognitiva/genética , Predisposición Genética a la Enfermedad , Fosfolipasa D/genética , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Animales , Autopsia , Disfunción Cognitiva/enzimología , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Células HeLa , Humanos , Lisosomas/enzimología , Lisosomas/patología , Ratones , Neuronas/enzimología , Neuronas/patología , Placa Amiloide/enzimología , Placa Amiloide/genética , Placa Amiloide/patologíaRESUMEN
The MiT-TFE family of basic helix-loop-helix leucine-zipper transcription factors includes four members: TFEB, TFE3, TFEC, and MITF Originally described as oncogenes, these factors play a major role as regulators of lysosome biogenesis, cellular energy homeostasis, and autophagy. An important mechanism by which these transcription factors are regulated involves their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. Such dynamic changes in subcellular localization occur in response to nutrient fluctuations and various forms of cell stress and are mediated by changes in the phosphorylation of multiple conserved amino acids. Major kinases responsible for MiT-TFE protein phosphorylation include mTOR, ERK, GSK3, and AKT In addition, calcineurin de-phosphorylates MiT-TFE proteins in response to lysosomal calcium release. Thus, through changes in the phosphorylation state of MiT-TFE proteins, lysosome function is coordinated with the cellular metabolic state and cellular demands. This review summarizes the evidence supporting MiT-TFE regulation by phosphorylation at multiple key sites. Elucidation of such regulatory mechanisms is of fundamental importance to understand how these transcription factors contribute to both health and disease.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Núcleo Celular/genética , Citoplasma/genética , Metabolismo Energético/genética , Autofagia/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation.
Asunto(s)
Demencia Frontotemporal/etiología , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Demencia Frontotemporal/genética , Técnicas de Sustitución del Gen , Granulinas , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/farmacología , Progranulinas , ARN MensajeroRESUMEN
The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith-Magenis chromosome region 8 (SMCR8) and WD repeat-containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72-containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.
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Lisosomas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Proteínas/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rabRESUMEN
We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.
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Linfoma de Células B/tratamiento farmacológico , Morfolinas/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Triazinas/uso terapéutico , Antineoplásicos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Evaluación Preclínica de Medicamentos/métodos , Endosomas/efectos de los fármacos , Endosomas/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazonas , Lisosomas/efectos de los fármacos , Lisosomas/genética , Fosfatidilinositol 3-Quinasas , PirimidinasRESUMEN
Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.
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Dinamina II/fisiología , Endocitosis , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales/química , Autofagia , Membrana Celular/metabolismo , Proliferación Celular , Regulación de la Expresión Génica , Inmunoterapia , Ratones , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Linfocitos T/citología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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 ß-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, ß-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 ß-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.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Axones/enzimología , Lisosomas/enzimología , Placa Amiloide/enzimología , Animales , Modelos Animales de Enfermedad , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , RatonesRESUMEN
Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of ß1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of ß1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.
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Vasos Sanguíneos/embriología , Dinamina II/fisiología , Endocitosis/genética , Integrinas/metabolismo , Neovascularización Fisiológica/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Animales Recién Nacidos , Vasos Sanguíneos/crecimiento & desarrollo , Células Cultivadas , Dinamina II/genética , Embrión de Mamíferos , Femenino , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors.
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Dinamina III/metabolismo , Dinamina II/metabolismo , Dinamina I/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Dinamina I/genética , Dinamina II/genética , Dinamina III/genética , Endocitosis/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Hidrazonas/farmacología , Ratones , Naftoles/farmacologíaRESUMEN
The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.
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Membrana Celular/metabolismo , Dinaminas/metabolismo , Receptores ErbB/metabolismo , Animales , Clatrina/metabolismo , Endocitosis , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Fibroblastos/citología , GTP Fosfohidrolasas/metabolismo , Ligandos , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Fosforilación , Transducción de SeñalRESUMEN
Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.
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Dinaminas/metabolismo , Mutación/genética , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Corteza Cerebral/patología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/ultraestructura , Regulación hacia ArribaRESUMEN
Dynamin 1 (Dyn1) and Dyn2 are neuronal and ubiquitously expressed isoforms, respectively, of the multidomain GTPase required for clathrin-mediated endocytosis (CME). Although they are 79% identical, Dyn1 and Dyn2 are not fully functionally redundant. Through direct measurements of basal and assembly-stimulated GTPase activities, membrane binding, self-assembly, and membrane fission on planar and curved templates, we have shown that Dyn1 is an efficient curvature generator, whereas Dyn2 is primarily a curvature sensor. Using Dyn1/Dyn2 chimeras, we identified the lipid-binding pleckstrin homology domain as being responsible for the differential in vitro properties of these two isoforms. Remarkably, their in vitro activities were reversed by a single amino acid change in the membrane-binding variable loop 3. Reconstitution of KO mouse embryo fibroblasts showed that both the pleckstrin homology and the Pro/Arg-rich domains determine the differential abilities of these two isoforms to support CME. These domains are specific to classical dynamins and are involved in regulating their activity. Our findings reveal opportunities for fundamental differences in the regulation of Dyn1, which mediates rapid endocytosis at the synapse, vs. Dyn2, which regulates early and late events in CME in nonneuronal cells.
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Dinaminas/fisiología , Isoformas de Proteínas/fisiología , Animales , Membrana Celular , Dinaminas/química , Endocitosis , Ratones , Ratones Noqueados , Isoformas de Proteínas/químicaRESUMEN
Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease (PD). Here we show that VPS13C, a bridge-like lipid transport protein and a PD gene, is a sensor of lysosome stress/damage. Upon lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires release of a brake, most likely an intramolecular interaction within VPS13C, which hinders access of its VAB domain to lysosome-bound Rab7. While another PD protein, LRRK2, is also recruited to stressed/damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the putative role of VPS13 proteins in bulk lipid transport, these findings suggest lipid delivery to lysosomes by VPS13C is part of an early response to lysosome damage.
RESUMEN
Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. At lysosomes, this TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.