RESUMEN
The endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB-EFA6B-ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B-ARF6 activity.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Movimiento Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Mesodermo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor 6 de Ribosilación del ADP , Línea Celular Tumoral , Polaridad Celular , Células HeLa , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas de la Membrana/genética , Mesodermo/citología , Proteínas del Tejido Nervioso/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.
Asunto(s)
Autofagosomas/enzimología , Autofagia , Neoplasias de la Mama/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Endocitosis , Endosomas/enzimología , Lisosomas/enzimología , Proteína de Unión al GTP rab2/metabolismo , Animales , Animales Modificados Genéticamente , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Humanos , Fusión de Membrana , Proteolisis , Interferencia de ARN , Transducción de Señal , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab2/genética , Proteínas de Unión a GTP rab7RESUMEN
The ability of cells to alter their phenotypic and morphological characteristics, known as cellular plasticity, is critical in normal embryonic development and adult tissue repair and contributes to the pathogenesis of diseases, such as organ fibrosis and cancer. The epithelial-to-mesenchymal transition (EMT) is a type of cellular plasticity. This transition involves genetic and epigenetic changes as well as alterations in protein expression and post-translational modifications. These changes result in reduced cell-cell adhesion, enhanced cell adhesion to the extracellular matrix, and altered organization of the cytoskeleton and of cell polarity. Among these modifications, loss of cell polarity represents the nearly invariable, distinguishing feature of EMT that frequently precedes the other traits or might even occur in their absence. EMT transforms cell morphology and physiology, and hence cell identity, from one typical of cells that form a tight barrier, like epithelial and endothelial cells, to one characterized by a highly motile mesenchymal phenotype. Time-resolved proteomic and phosphoproteomic analyses of cells undergoing EMT recently identified thousands of changes in proteins involved in many cellular processes, including cell proliferation and motility, DNA repair, and - unexpectedly - membrane trafficking (1). These results have highlighted a picture of great complexity. First, the EMT transition is not an all-or-none response but rather a gradual process that develops over time. Second, EMT events are highly dynamic and frequently reversible, involving both cell-autonomous and non-autonomous mechanisms. The net results is that EMT generates populations of mixed cells, with partial or full phenotypes, possibly accounting (at least in part) for the physiological as well as pathological cellular heterogeneity of some tissues. Endocytic circuitries have emerged as complex connectivity infrastructures for numerous cellular networks required for the execution of different biological processes, with a primary role in the control of polarized functions. Thus, they may be relevant for controlling EMT or certain aspects of it. Here, by discussing a few paradigmatic cases, we will outline how endocytosis may be harnessed by the EMT process to promote dynamic changes in cellular identity, and to increase cellular flexibility and adaptation to micro-environmental cues, ultimately impacting on physiological and pathological processes, first and foremost cancer progression.
