RESUMEN
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.
Asunto(s)
Actomiosina , Lisosomas , Citoesqueleto , Células Dendríticas , Endosomas , HumanosRESUMEN
Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper.
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Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Endosomas/enzimología , Endosomas/genética , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi/enzimología , Aparato de Golgi/genética , Humanos , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.
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Movimiento Celular , Embrión no Mamífero/patología , Neoplasias/patología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Citoesqueleto de Actina , Animales , Embrión no Mamífero/metabolismo , Humanos , Microtúbulos , Invasividad Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Transporte de Proteínas , Transducción de Señal , Células Tumorales Cultivadas , Pez Cebra , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22â¯N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Retículo Endoplásmico/genética , Homeostasis/genética , Proteínas de Unión al GTP rab/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas Nogo/genética , Unión Proteica , ARN Interferente Pequeño/genética , Ácido Tauroquenodesoxicólico/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Unión a GTP rab7RESUMEN
The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.
RESUMEN
L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.
Asunto(s)
Catepsinas/metabolismo , Cisteína/metabolismo , Citosol/metabolismo , Dipéptidos/farmacología , Lisosomas/metabolismo , Apoptosis/efectos de los fármacos , Citosol/efectos de los fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lisosomas/ultraestructura , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ProtonesRESUMEN
Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Cisteína Endopeptidasas/genética , Regulación de la Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al GTP rab/deficiencia , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Rab7a, a small GTPase of the Rab family, is localized to late endosomes and controls late endocytic trafficking. The discovery of several Rab7a interacting proteins revealed that Rab7a function is closely connected to cytoskeletal elements. Indeed, Rab7a recruits on vesicles RILP and FYCO that are responsible for the movement of Rab7a-positive vesicles and/or organelles on microtubule tracks, but also directly interacts with Rac1, a fundamental regulator of actin cytoskeleton, and with peripherin and vimentin, two intermediate filament proteins. Considering all these interactions and, in particular, the fact that Rac1 and vimentin are key factors for cellular motility, we investigated a possible role of Rab7a in cell migration. We show here that Rab7a is needed for cell migration as Rab7a depletion causes slower migration of NCI H1299 cells affecting cell velocity and directness. Rab7a depletion negatively affects adhesion and spreading onto fibronectin substrates, altering ß1-integrin activation, localization and intracellular trafficking, and myosin X localization. In fact, Rab7a-depleted cells show 40% less filopodia and active integrin accumulates at the leading edge of migrating cells. Furthermore, Rab7a depletion decreases the amount of active Rac1 but not its abundance and reduces the number of cells with vimentin filaments facing the wound, indicating that Rab7a has a role in the orientation of vimentin filaments during migration. In conclusion, our results demonstrate a key role of Rab7a in the regulation of different aspects of cell migration.
Asunto(s)
Movimiento Celular/fisiología , Vimentina/fisiología , Proteínas de Unión al GTP rab/fisiología , Proteína de Unión al GTP rac1/fisiología , Línea Celular Tumoral , Humanos , Cicatrización de Heridas , Proteínas de Unión a GTP rab7RESUMEN
Rab9 is a small GTPase that localizes to the trans-Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose-6-phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation-independent (CI-MPR) away from the Golgi yet, has no effect on the retrograde transport of CI-MPR. We also show that CI-MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. CI-MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI-MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI-MPR to the endosomal pathway, entering the maturing endosome at the early-to-late transition.
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Endosomas/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Perros , Endocitosis , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Glicoproteínas de Membrana/metabolismo , Mutación Missense , Transporte de Proteínas , Proteínas de Unión al GTP rab/genética , Red trans-Golgi/metabolismoRESUMEN
The bidirectional transport between the Golgi complex and the endocytic pathway has to be finely regulated in order to ensure the proper delivery of newly synthetized lysosomal enzymes and the return of sorting receptors from degradative compartments. The high complexity of these routes has led to experimental difficulties in properly dissecting and separating the different pathways. As a consequence, several models have been proposed during the past decades. However, recent advances in our understanding of endosomal dynamics have helped to unify these different views. We provide here an overview of the current insights into the transport routes between Golgi and endosomes in mammalian cells. The focus of the Commentary is on the key molecules involved in the trafficking pathways between these intracellular compartments, such as Rab proteins and sorting receptors, and their regulation. A proper understanding of the bidirectional traffic between the Golgi complex and the endolysosomal system is of uttermost importance, as several studies have demonstrated that mutations in the factors involved in these transport pathways result in various pathologies, in particular lysosome-associated diseases and diverse neurological disorders, such as Alzheimer's and Parkinson's disease.
Asunto(s)
Endosomas/metabolismo , Aparato de Golgi/metabolismo , Animales , Humanos , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide-MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells "dummy T cells" and propose to use them for safety validation of new TCRs prior to therapy.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Familia-src Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , Muerte Celular , Células Cultivadas , Humanos , Fosforilación , Unión Proteica , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Linfocitos T/citología , Familia-src Quinasas/genéticaRESUMEN
Rab proteins are small GTPases that regulate transport between the different compartments of the endomembrane system in eukaryotic cells. Here, we show that Rab7b, a Rab that controls the transport between late endosomes and the trans Golgi network, interacts directly with myosin II. We illustrate the functional relevance of this interaction, demonstrating that myosin II mediates the transport of Rab7b endosomes, as Rab7b dynamics are strongly affected after myosin II depletion or inhibition. We also demonstrate that a member of the Rab family regulates actin remodeling and, consequently, influences cell adhesion, polarization and migration. We find the molecular mechanism by which Rab7b influences stress fiber formation - through controlling the activation status of the small GTPase RhoA and therefore influencing myosin light chain phosphorylation. Our findings reveal a newly identified role for Rab proteins outside of their canonical role in intracellular trafficking, identifying Rab7b as a coordinator of cytoskeletal organization.
Asunto(s)
Actomiosina/metabolismo , Movimiento Celular/fisiología , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Fosforilación , Transporte de Proteínas , Transfección , Proteínas de Unión a GTP rab7RESUMEN
Rab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Membranas Intracelulares/enzimología , Ubiquitinación , ATPasas de Translocación de Protón Vacuolares/metabolismo , Complejo Dinactina , Endosomas/enzimología , Expresión Génica , Células HeLa , Humanos , Lisosomas/enzimología , Proteínas Asociadas a Microtúbulos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Proteolisis , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The small GTPase Rab7b localizes to late endosomes-lysosomes and to the Golgi, regulating the transport between these two intracellular compartments. We have recently demonstrated that depletion of Rab7b causes missorting of the cation-independent mannose 6-phosphate receptor (CI-MPR), suggesting that Rab7b may control the trafficking of this receptor. Here we further investigated the function of this small GTPase with special attention to its role in the trafficking of sorting receptors and dynamics in living cells. Using endosome-to-Golgi retrieval assays we show that Rab7b is involved not only in CI-MPR transport but also in the MPRs independent pathway. Indeed, we find that it regulates and interacts with sortilin, a mannose 6-phosphate-independent sorting receptor. CI-MPR and sortilin are sorted from the trans-Golgi network (TGN) in tubular structures and the expression of Rab7b mutants or its silencing reduces CI-MPR and sortilin tubulation. In addition, the constitutively active mutant Rab7b Q67L impairs the formation of carriers from TGN. Collectively, our observations show for the first time that Rab7b is required for transport from endosomes to the TGN, not only of the CI-MPR, but also of sortilin, and that alterations in this transport result in impaired carrier formation from TGN.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Mutación Missense , Dominios y Motivos de Interacción de Proteínas/genética , Transporte de Proteínas , ARN Interferente Pequeño , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7 , Red trans-Golgi/metabolismoRESUMEN
Intermediate filaments are cytoskeletal elements important for cell architecture. Recently it has been discovered that intermediate filaments are highly dynamic and that they are fundamental for organelle positioning, transport and function thus being an important regulatory component of membrane traffic. We have identified, using the yeast two-hybrid system, vimentin, a class III intermediate filament protein, as a Rab7a interacting protein. Rab7a is a member of the Rab family of small GTPases and it controls vesicular membrane traffic to late endosomes and lysosomes. In addition, Rab7a is important for maturation of phagosomes and autophagic vacuoles. We confirmed the interaction in HeLa cells by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using bacterially expressed recombinant proteins. Immunofluorescence analysis on HeLa cells indicate that Rab7a-positive vesicles sometimes overlap with vimentin filaments. Overexpression of Rab7a causes an increase in vimentin phosphorylation at different sites and causes redistribution of vimentin in the soluble fraction. Consistently, Rab7a silencing causes an increase of vimentin present in the insoluble fraction (assembled). Also, expression of Charcot-Marie-Tooth 2B-causing Rab7a mutant proteins induces vimentin phosphorylation and increases the amount of vimentin in the soluble fraction. Thus, modulation of expression levels of Rab7a wt or expression of Rab7a mutant proteins changes the assembly of vimentin and its phosphorylation state indicating that Rab7a is important for the regulation of vimentin function.
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Proteínas Mutantes/metabolismo , Proteínas Recombinantes/metabolismo , Vimentina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Western Blotting , Endosomas , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Proteínas Mutantes/genética , Fosforilación , Proteínas Recombinantes/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Charcot-Marie-Tooth type 2B (CMT2B) is a peripheral ulcero-mutilating neuropathy caused by four missense mutations in the rab7a gene. CMT2B is clinically characterized by prominent sensory loss, distal muscle weakness leading to muscle atrophy, high frequency of foot ulcers and infections that often results in toe amputations. RAB7A is a ubiquitous small GTPase, which controls transport to late endocytic compartments. Although the biochemical and functional properties of disease-causing RAB7A mutant proteins have been investigated, it is not yet clear how the disease originates. To understand how mutations in a ubiquitous protein specifically affect peripheral neurons, we performed a two-hybrid screen using a dorsal root ganglia cDNA library with the purpose of identifying RAB7A interactors specific for these cells. We identified peripherin, an intermediate filament protein expressed primarily in peripheral neurons, as a putative RAB7A interacting protein. The interaction was confirmed by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using recombinant proteins. Silencing or overexpression of wild type RAB7A changed the soluble/insoluble rate of peripherin indicating that RAB7A is important for peripherin organization and function. In addition, disease-causing RAB7A mutant proteins bind more strongly to peripherin and their expression causes a significant increase in the amount of soluble peripherin. Since peripherin plays a role not only in neurite outgrowth during development but also in axonal regeneration after injury, these data suggest that the altered interaction between disease-causing RAB7A mutants and peripherin could play an important role in CMT2B neuropathy.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Filamentos Intermediarios/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Proteínas de Unión al GTP rab/fisiología , Animales , Western Blotting , Células Cultivadas , Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de Filamentos Intermediarios/genética , Laminopatías , Glicoproteínas de Membrana/genética , Ratones , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Células PC12 , Periferinas , Plásmidos/genética , Células del Asta Posterior/fisiología , ARN/genética , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Focal adhesions (FAs) are contact points of the cell with the extracellular matrix (ECM) and play a major role in several cellular functions including migration, proliferation, differentiation, and growth. During cell migration, FAs are continuously assembled and disassembled. It is well established that FA dynamics are regulated by the cytoskeleton, motor proteins, small GTPases, and specific kinases and phosphatases. However, more recently, the establishment of contacts between FAs and the endoplasmic reticulum (ER) has been shown to be another factor implicated in the regulation of FA dynamics. The transport of ER tubules along microtubules to contact FAs is indeed crucial to support FA growth. Alteration of such ER-FA contacts affects FA growth, dynamics, and thus cell migration. Here, we present a protocol for live-cell imaging and analysis of ER-FA contact points during cell migration. Our analysis pipeline includes two examples showing physiological conditions and disruption of ER-FA contacts upon nocodazole treatment. The described method can be adapted to different cell lines.
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Adhesiones Focales , Cinesinas , Adhesiones Focales/metabolismo , Movimiento Celular , Línea Celular , Cinesinas/metabolismo , Retículo Endoplásmico , Adhesión Celular/fisiologíaRESUMEN
Invariant chain (Ii, CD74) is a type II transmembrane glycoprotein that acts as a chaperone and facilitates the folding and transport of MHC II chains. By assisting the assembly and subcellular targeting of MHC II complexes, Ii has a wide impact on the functions of antigen-presenting cells such as antigen processing, endocytic maturation, signal transduction, cell migration, and macropinocytosis. Ii is a multifunctional molecule that can alter endocytic traffic and has several interacting molecules. To understand more about Ii's function and to identify further Ii interactors, a yeast two-hybrid screening was performed. Retinoic Acid-Induced 14 (Rai14) was detected as a putative interaction partner, and the interaction was confirmed by co-immunoprecipitation. Rai14 is a poorly characterized protein, which is believed to have a role in actin cytoskeleton and membrane remodeling. In line with this, we found that Rai14 localizes to membrane ruffles, where it forms macropinosomes. Depletion of Rai14 in antigen-presenting cells delays MHC II internalization, affecting macropinocytic activity. Intriguingly, we demonstrated that, similar to Ii, Rai14 is a positive regulator of macropinocytosis and a negative regulator of cell migration, two antagonistic processes in antigen-presenting cells. This antagonism is known to depend on the interaction between myosin II and Ii. Here, we show that Rai14 also binds to myosin II, suggesting that Ii, myosin II, and Rai14 work together to coordinate macropinocytosis and cell motility.
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Antígenos de Histocompatibilidad Clase II , Tretinoina , Pinocitosis/fisiología , Proteínas del Citoesqueleto , Miosina Tipo IIRESUMEN
Major histocompatibility complex (MHC) class I and II are crucial for the adaptive immune system because they are involved in peptide presentation to T cells. Until recently, it was believed that MHC genes and their associated immune components had been conserved since their evolutionary emergence in jawed fish. However, sequencing of the Atlantic cod (Gadus morhua) genome revealed a loss of MHC class II genes, and an extreme expansion of MHC class I genes. These findings lead to the hypothesis that a loss of the MHC class II pathway coincided with a more versatile use of MHC class I, but so far there is no direct experimental evidence in support of this. To gain a deeper understanding of the function of the expanded MHC class I, we selected five MHC class I gene variants representing five of the six clades identified in previous studies and investigated their intracellular localization in human and Atlantic cod larval cells. Intriguingly, we uncovered that all selected MHC class I variants localize to endolysosomal compartments in Atlantic cod cells. Additionally, by introducing point mutations or deletions in the cytosolic tail, we found that hypothetical sorting signals in the MHC class I cytosolic tail do not influence MHC class I trafficking. Moreover, we demonstrated that in Atlantic cod, tapasin and MHC class I colocalize on endolysosomes suggesting that peptide-loading assistance and stabilization of MHC class I occurs outside the endoplasmic reticulum. Altogether, our results demonstrate that MHC class I from Atlantic cod is sorted to the endolysosomal system, which may indicate that it interacts with exogenous peptides for potential cross presentation.
RESUMEN
Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to Rab7. It has been reported that Rab7b is lysosome associated, that it is involved in monocytic differentiation and that it promotes lysosomal degradation of TLR4 and TLR9. Here we investigated further the localization and function of this GTPase. We found that wild-type Rab7b is lysosome associated whereas an activated, GTP-bound form of Rab7b localizes to the Golgi apparatus. In contrast to Rab7, Rab7b is not involved in EGF and EGFR degradation. Depletion of Rab7b or expression of Rab7b T22N, a Rab7b dominant-negative mutant, impairs cathepsin-D maturation and causes increased secretion of hexosaminidase. Moreover, expression of Rab7b T22N or depletion of Rab7b alters TGN46 distribution, cation-independent mannose-6-phosphate receptor (CI-MPR) trafficking, and causes an increase in the levels of the late endosomal markers CI-MPR and cathepsin D. Vesicular stomatitis virus G protein (VSV-G) trafficking, by contrast, is normal in Rab7b-depleted or Rab7b-T22N-expressing cells. In addition, depletion of Rab7b prevents cholera toxin B-subunit from reaching the Golgi. Altogether, these data indicate that Rab7b is required for normal lysosome function, and, in particular, that it is an essential factor for retrograde transport from endosomes to the trans-Golgi network (TGN).