RESUMEN
Endocytosis allows cells to transport particles and molecules across the plasma membrane. In addition, it is involved in the termination of signalling through receptor downmodulation and degradation. This traditional outlook has been substantially modified in recent years by discoveries that endocytosis and subsequent trafficking routes have a profound impact on the positive regulation and propagation of signals, being key for the spatiotemporal regulation of signal transmission in cells. Accordingly, endocytosis and membrane trafficking regulate virtually every aspect of cell physiology and are frequently subverted in pathological conditions. Two key aspects of endocytic control over signalling are coming into focus: context-dependency and long-range effects. First, endocytic-regulated outputs are not stereotyped but heavily dependent on the cell-specific regulation of endocytic networks. Second, endocytic regulation has an impact not only on individual cells but also on the behaviour of cellular collectives. Herein, we will discuss recent advancements in these areas, highlighting how endocytic trafficking impacts complex cell properties, including cell polarity and collective cell migration, and the relevance of these mechanisms to disease, in particular cancer.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Animales , Transporte Biológico , Movimiento Celular , Polaridad Celular , Humanos , Morfogénesis , Transducción de SeñalRESUMEN
Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.
Asunto(s)
Neoplasias de la Mama , Células Neoplásicas Circulantes , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Separación Celular , Femenino , Humanos , Dispositivos Laboratorio en un Chip , Ratones , Células Neoplásicas Circulantes/patología , PronósticoRESUMEN
Although endocytosis was first described as the process mediating macromolecule or nutrient uptake through the plasma membrane, it is now recognised as a critical component of the cellular infrastructure involved in numerous processes, ranging from receptor signalling, proliferation and migration to polarity and stem cell regulation. To realise these varying roles, endocytosis needs to be finely regulated. Accordingly, multiple endocytic mechanisms exist that require specialised molecular machineries and an array of endocytic adaptor proteins with cell-specific functions. This review provides some examples of specialised functions of endocytic adaptors and other components of the endocytic machinery in different cell physiological processes, and how the alteration of these functions is linked to cancer. In particular, we focus on: (i) cargo selection and endocytic mechanisms linked to different adaptors; (ii) specialised functions in clathrin-mediated versus non-clathrin endocytosis; (iii) differential regulation of endocytic mechanisms by post-translational modification of endocytic proteins; (iv) cell context-dependent expression and function of endocytic proteins. As cases in point, we describe two endocytic protein families, dynamins and epsins. Finally, we discuss how dysregulation of the physiological role of these specialised endocytic proteins is exploited by cancer cells to increase cell proliferation, migration and invasion, leading to anti-apoptotic or pro-metastatic behaviours.
Asunto(s)
Endocitosis/fisiología , Neoplasias , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Apoptosis/fisiología , Membrana Celular/metabolismo , Movimiento Celular , Proliferación Celular , Clatrina/metabolismo , Exocitosis/fisiología , Humanos , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/fisiopatología , Transporte de Proteínas , Receptores de Superficie Celular/metabolismoRESUMEN
During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Línea Celular Tumoral , Proliferación Celular , Receptores ErbB/metabolismo , Humanos , Cinética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Our understanding of endocytosis has evolved remarkably in little more than a decade. This is the result not only of advances in our knowledge of its molecular and biological workings, but also of a true paradigm shift in our understanding of what really constitutes endocytosis and of its role in homeostasis. Although endocytosis was initially discovered and studied as a relatively simple process to transport molecules across the plasma membrane, it was subsequently found to be inextricably linked with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved in space and time. In essence, endocytosis provides the communications and supply routes (the logistics) of the cell. Although this may seem revolutionary, it is still likely to be only a small part of the entire story. A wealth of new evidence is uncovering the surprisingly pervasive nature of endocytosis in essentially all aspects of cellular regulation. In addition, many newly discovered functions of endocytic proteins are not immediately interpretable within the classical view of endocytosis. A possible framework, to rationalize all this new knowledge, requires us to "upgrade" our vision of endocytosis. By combining the analysis of biochemical, biological, and evolutionary evidence, we propose herein that endocytosis constitutes one of the major enabling conditions that in the history of life permitted the development of a higher level of organization, leading to the actuation of the eukaryotic cell plan.
Asunto(s)
Endocitosis/fisiología , Células Eucariotas/fisiología , Transducción de Señal/fisiología , Animales , Evolución Biológica , Comunicación Celular/fisiología , Células Eucariotas/citología , Hemostasis/fisiología , HumanosRESUMEN
Signaling from the epidermal growth factor receptor (EGFR) elicits multiple biological responses, including cell proliferation, migration, and survival. Receptor endocytosis and trafficking are critical physiological processes that control the strength, duration, diversification, and spatial restriction of EGFR signaling through multiple mechanisms, which we review in this chapter. These mechanisms include: (i) regulation of receptor density and activation at the cell surface; (ii) concentration of receptors into distinct nascent endocytic structures; (iii) commitment of the receptor to different endocytic routes; (iv) endosomal sorting and postendocytic trafficking of the receptor through distinct pathways, and (v) recycling to restricted regions of the cell surface. We also highlight how communication between organelles controls EGFR activity along the endocytic route. Finally, we illustrate how abnormal trafficking of EGFR oncogenic mutants, as well as alterations of the endocytic machinery, contributes to aberrant EGFR signaling in cancer.
Asunto(s)
Endocitosis/genética , Endosomas/genética , Neoplasias/genética , Membrana Celular/genética , Movimiento Celular/genética , Endosomas/metabolismo , Receptores ErbB/genética , Humanos , Neoplasias/patología , Transducción de Señal/genéticaRESUMEN
How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.
Asunto(s)
Endocitosis/fisiología , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Ubiquitinación/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/genética , Proteína Adaptadora GRB2/genética , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-cbl/genéticaRESUMEN
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
One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.
Asunto(s)
Adenosina Trifosfato , Endocitosis , Retículo Endoplásmico , Receptores ErbB , Mitocondrias , Transducción de Señal , Mitocondrias/metabolismo , Receptores ErbB/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismoRESUMEN
The blood-brain barrier (BBB) acquires unique properties to regulate neuronal function during development. The formation of the BBB, which occurs in tandem with angiogenesis, is directed by the Wnt/ß-catenin signaling pathway. Yet the exact molecular interplay remains elusive. Our study reveals the G protein-coupled receptor GPR126 as a critical target of canonical Wnt signaling, essential for the development of the BBB's distinctive vascular characteristics and its functional integrity. Endothelial cell-specific deletion of the Gpr126 gene in mice induced aberrant vascular morphogenesis, resulting in disrupted BBB organization. Simultaneously, heightened transcytosis in vitro compromised barrier integrity, resulting in enhanced vascular permeability. Mechanistically, GPR126 enhanced endothelial cell migration, pivotal for angiogenesis, acting through an interaction between LRP1 and ß1 integrin, thereby balancing the levels of ß1 integrin activation and recycling. Overall, we identified GPR126 as a specifier of an organotypic vascular structure, which sustained angiogenesis and guaranteed the acquisition of the BBB properties during development.
Asunto(s)
Barrera Hematoencefálica , Integrina beta1 , Receptores Acoplados a Proteínas G , Animales , Ratones , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Movimiento Celular , Células Endoteliales/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones Noqueados , Neovascularización Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Vía de Señalización Wnt , Masculino , FemeninoAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Endosomas/genética , Células HeLa , Humanos , Complejo de la Endopetidasa Proteasomal/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid-liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.
Asunto(s)
Clatrina , Endocitosis , Biofisica , Membrana Celular/metabolismo , Clatrina/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, â¼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.
Asunto(s)
Receptores ErbB/química , Animales , Anisotropía , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Cinética , Ligandos , Ratones , Células 3T3 NIH , Unión Proteica , Proteínas Tirosina Quinasas/química , Transducción de Señal , Espectrometría de Fluorescencia/métodosRESUMEN
Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis/fisiología , Células Eucariotas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina/metabolismo , Animales , Células CHO , Cricetinae , Cisteína Endopeptidasas/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Ratones , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Transporte de Proteínas/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.
Asunto(s)
Endocitosis , Hierro/farmacología , Receptores de Transferrina/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Apoferritinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Células HeLa , Células Hep G2 , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transferrina/metabolismoRESUMEN
Targeting of membrane proteins to their proper destination requires specific mechanisms. Protein cargos are included in vesicles that bud off a donor organelle and ultimately fuse with a target organelle, where the cargos are delivered. Endocytosis of transmembrane receptors (e.g., receptor tyrosine kinases, RTKs) follows a common scheme that consists of an internalization reaction and a delivery step, during which cargos are transferred to an endosomal station to be either directed to the lysosome for degradation or recycled back to the cell surface. At each stage along the endocytic route, short motifs within protein cargos and/or post-translational modifications regulate transmembrane receptor sorting. In recent years, studies have shown that ubiquitination acts as a signal for the internalization and sorting of plasma membrane proteins. Here, we present an overview of ubiquitin's role as a 'signal' for intracellular trafficking and give examples of the multifaced mechanisms of ubiquitin-regulated RTK endocytosis.
Asunto(s)
Endocitosis/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ubiquitina/metabolismo , Animales , Membrana Celular/metabolismo , Endosomas/fisiología , Humanos , Transporte de Proteínas/fisiología , Transducción de Señal/fisiología , Ubiquitinación/fisiologíaRESUMEN
Mitotic progression is orchestrated by morphological and mechanical changes promoted by the coordinated activities of the microtubule (MT) cytoskeleton, the actin cytoskeleton and the plasma membrane (PM). MTs assemble the mitotic spindle, which assists sister chromatid separation, and contact the rigid and tensile actomyosin cortex rounded-up underneath the PM. Here, we highlight the dynamic crosstalk between MTs, actin and cell membranes during mitosis, and discuss the molecular connections between them. We also summarize recent views on how MT traction forces, the actomyosin cortex and membrane trafficking contribute to spindle positioning in isolated cells in culture and in epithelial sheets. Finally, we describe the emerging role of membrane trafficking in synchronizing actomyosin tension and cell shape changes with cell-substrate adhesion, cell-cell contacts and extracellular signalling events regulating proliferation.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/citología , Microtúbulos/metabolismo , Actomiosina/metabolismo , Animales , Transporte Biológico , Adhesión Celular , Comunicación Celular , Técnicas de Cultivo de Célula , Células Epiteliales/metabolismo , Humanos , Mitosis , Huso Acromático/metabolismoRESUMEN
The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a ß-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFß-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFß-dependent regulatory loops conferring cellular plasticity and invasive behavior.