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1.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34494097

RESUMO

Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MCOLN1), enabling the local activation of myosin II at the cell rear. Taken together, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling. This article has an associated First Person interview with the first author of the paper.


Assuntos
Actomiosina , Lisossomos , Citoesqueleto , Células Dendríticas , Endossomos , Humanos
2.
Plant Cell ; 30(10): 2573-2593, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30018157

RESUMO

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.


Assuntos
Arabidopsis/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brefeldina A/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
3.
Immunol Rev ; 272(1): 39-51, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27319341

RESUMO

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.


Assuntos
Apresentação de Antígeno , Linfócitos B/imunologia , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Movimento Celular , Endocitose , Antígenos de Histocompatibilidade/metabolismo , Humanos , Sinapses Imunológicas , Ativação Linfocitária , Peptídeos/metabolismo
4.
EMBO J ; 34(6): 798-810, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25637353

RESUMO

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.


Assuntos
Cálcio/metabolismo , Movimento Celular/fisiologia , Células Dendríticas/imunologia , Espaço Extracelular/imunologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miosina Tipo II/metabolismo , Animais , Polaridade Celular , Primers do DNA/genética , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Immunoblotting , Camundongos , Microscopia de Fluorescência , Microscopia de Vídeo , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real
5.
Dev Cell ; 59(12): 1571-1592.e9, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38626765

RESUMO

Neuronal endosomal and lysosomal abnormalities are among the early changes observed in Alzheimer's disease (AD) before plaques appear. However, it is unclear whether distinct endolysosomal defects are temporally organized and how altered γ-secretase function or amyloid precursor protein (APP) metabolism contribute to these changes. Inhibiting γ-secretase chronically, in mouse embryonic fibroblast and hippocampal neurons, led to a gradual endolysosomal collapse initiated by decreased lysosomal calcium and increased cholesterol, causing downstream defects in endosomal recycling and maturation. This endolysosomal demise is γ-secretase dependent, requires membrane-tethered APP cytoplasmic domains, and is rescued by APP depletion. APP C-terminal fragments (CTFs) localized to late endosome/lysosome-endoplasmic reticulum contacts; an excess of APP-CTFs herein reduced lysosomal Ca2+ refilling from the endoplasmic reticulum, promoting cholesterol accretion. Tonic regulation by APP-CTFs provides a mechanistic explanation for their cellular toxicity: failure to timely degrade APP-CTFs sustains downstream signaling, instigating lysosomal dyshomeostasis, as observed in prodromal AD. This is the opposite of substrates such as Notch, which require intramembrane proteolysis to initiate signaling.


Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Retículo Endoplasmático , Endossomos , Lisossomos , Neurônios , Lisossomos/metabolismo , Animais , Endossomos/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Camundongos , Retículo Endoplasmático/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Neurônios/metabolismo , Colesterol/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Cálcio/metabolismo , Humanos , Fibroblastos/metabolismo , Transdução de Sinais , Proteólise
6.
Nat Commun ; 14(1): 2847, 2023 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-37225734

RESUMO

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.


Assuntos
DNA Mitocondrial , Nucleotídeos , Mitocôndrias , Nucleotidiltransferases , Proteínas Amiloidogênicas , Cromogranina A , Fosfolipases
7.
Ageing Res Rev ; 71: 101444, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34391945

RESUMO

Microglia, the brain-resident immune cells, play an essential role in the upkeep of brain homeostasis. They actively adapt into specific activation states based on cues from the microenvironment. One of these encompasses the activated response microglia (ARMs) phenotype. It arises along a healthy aging process and in a range of neurodegenerative diseases, including Alzheimer's disease (AD). As the phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and secretion of neuroprotective agents, it leaves to reason that the phenotype is adapted in an attempt to restore homeostasis. This is important to the conundrum of inflammatory processes. Inflammation per se may not be deleterious; it is only when microglial reactions become chronic or the microglial subtype is made dysfunctional by (multiple) risk proteins with single-nucleotide polymorphisms that microglial involvement becomes deleterious instead of beneficial. Interestingly, the ARMs up- and downregulate many late-onset AD-associated risk factor genes, the products of which are particularly active in the endolysosomal system. Hence, in this review, we focus on how the endolysosomal system is placed at the crossroad of inflammation and microglial capacity to keep pace with degradation.


Assuntos
Doença de Alzheimer , Microglia , Doença de Alzheimer/genética , Humanos , Lisossomos , Fagocitose
8.
J Neurosci ; 28(34): 8470-6, 2008 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-18716205

RESUMO

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have a key role in membrane fusion. It is commonly assumed that pairing of SNARE proteins anchored in opposing membranes overcomes the repulsion energy between membranes, thereby catalyzing fusion. In this study, we have increased the distance between the coiled-coil SNARE motif and the transmembrane domain of the vesicular SNARE synaptobrevin-2 by insertion of a flexible linker to analyze how an increased intermembrane distance affects exocytosis. Synaptobrevin-2 lengthening did not change the frequency of exocytotic events measured at 1 mum free calcium but prevented the increase in the secretory activity triggered by higher calcium concentration. Exocytotic events monitored in adrenal chromaffin cells by means of carbon fiber amperometry were classified in two groups according to the rate and extent of fusion pore expansion. Lengthening the juxtamembrane region of synaptobrevin-2 severely reduced the occurrence of rapid single events, leaving slow ones unchanged. It also impaired the increase in the fast-fusion mode that normally follows elevation of intracellular Ca2+ levels. We conclude that mild stimuli trigger slow fusion events that do not rely on a short intermembrane distance. In contrast, a short intermembrane distance mediated by tight zippering of SNAREs is essential to a component of the secretory response elicited by robust stimuli and characterized by rapid dilation of the fusion pore.


Assuntos
Exocitose/fisiologia , Fusão de Membrana/fisiologia , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Medula Suprarrenal/citologia , Motivos de Aminoácidos , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Células Cromafins/fisiologia , Eletrofisiologia , Membranas Intracelulares/metabolismo , Mutagênese Insercional , Concentração Osmolar , Mutação Puntual , Probabilidade , Estrutura Terciária de Proteína , Fatores de Tempo , Transfecção , Proteína 2 Associada à Membrana da Vesícula/genética
9.
Mol Neurodegener ; 14(1): 20, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159836

RESUMO

Increasing evidence supports that cellular dysregulations in the degradative routes contribute to the initiation and progression of neurodegenerative diseases, including Alzheimer's disease. Autophagy and endolysosomal homeostasis need to be maintained throughout life as they are major cellular mechanisms involved in both the production of toxic amyloid peptides and the clearance of misfolded or aggregated proteins. As such, alterations in endolysosomal and autophagic flux, as a measure of degradation activity in these routes or compartments, may directly impact as well on disease-related mechanisms such as amyloid-ß clearance through the blood-brain-barrier and the interneuronal spreading of amyloid-ß and/or Tau seeds, affecting synaptic function, plasticity and metabolism. The emerging of several genetic risk factors for late-onset Alzheimer's disease that are functionally related to endocytic transport regulation, including cholesterol metabolism and clearance, supports the notion that in particular the autophagy/lysosomal flux might become more vulnerable during ageing thereby contributing to disease onset. In this review we discuss our current knowledge of the risk genes APOE4, BIN1, CD2AP, PICALM, PLD3 and TREM2 and their impact on endolysosomal (dys)regulations in the light of late-onset Alzheimer's disease pathology.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Autofagia/fisiologia , Humanos , Fatores de Risco
10.
Sci Immunol ; 2(16)2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-29079589

RESUMO

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.


Assuntos
Movimento Celular , Células Dendríticas/imunologia , Lisossomos/metabolismo , Transdução de Sinais , Animais , Apresentação de Antígeno , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/fisiologia , Regulação para Baixo , Lisossomos/imunologia , Camundongos , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Pinocitose , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo
12.
Nat Cell Biol ; 18(1): 43-53, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26641718

RESUMO

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.


Assuntos
Actinas/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Células Dendríticas/metabolismo , Animais , Células Cultivadas , Camundongos
13.
Nat Commun ; 6: 7526, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26109323

RESUMO

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos/metabolismo , Movimento Celular/fisiologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Miosina Tipo II/metabolismo , Ovalbumina/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Células da Medula Óssea , Catepsinas/genética , Catepsinas/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Masculino , Camundongos , Técnicas Analíticas Microfluídicas , Miosina Tipo II/genética
14.
Mol Biol Cell ; 25(20): 3195-209, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25143404

RESUMO

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.


Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Vesículas Secretórias/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Transporte Biológico , Humanos , Miosina Tipo II/metabolismo
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