RESUMEN
The plasma membrane separates the interior of cells from the outside environment. The membrane tension, defined as the force per unit length acting on a cross-section of membrane, regulates many vital biological processes. In this review, we summarize the first historical findings and the latest advances, showing membrane tension as an important physical parameter in cell biology. We also discuss how this parameter must be better integrated and we propose experimental approaches for key unanswered questions.
Asunto(s)
Membrana Celular/fisiología , Animales , Fenómenos Fisiológicos Celulares , Homeostasis , Humanos , Membrana Dobles de Lípidos , Presión OsmóticaRESUMEN
Biomimetic materials have long been the (he)art of bioengineering. They usually aim at mimicking in vivo conditions to allow in vitro culture, differentiation and expansion of cells. The past decade has witnessed a considerable amount of progress in soft lithography, bio-inspired micro-fabrication and biochemistry, allowing the design of sophisticated and physiologically relevant micro- and nano-environments. These systems now provide an exquisite toolbox with which we can control a large set of physicochemical environmental parameters that determine cell behavior. Bio-functionalized surfaces have evolved from simple protein-coated solid surfaces or cellular extracts into nano-textured 3D surfaces with controlled rheological and topographical properties. The mechanobiological molecular processes by which cells interact and sense their environment can now be unambiguously understood down to the single-molecule level. This Commentary highlights recent successful examples where bio-functionalized substrates have contributed in raising and answering new questions in the area of extracellular matrix sensing by cells, cell-cell adhesion and cell migration. The use, the availability, the impact and the challenges of such approaches in the field of biology are discussed.
Asunto(s)
Materiales Biomiméticos/farmacología , Señales (Psicología) , Ambiente , Animales , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , HumanosRESUMEN
Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.
RESUMEN
NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.
Asunto(s)
Movimiento Celular , Núcleo Celular/metabolismo , Neocórtex/citología , Neocórtex/embriología , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Aspergillus nidulans/metabolismo , Diferenciación Celular , Supervivencia Celular , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/metabolismo , Genes Dominantes/genética , Mitosis , Neocórtex/metabolismo , Proteínas Nucleares/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Células Madre/citología , Células Madre/metabolismo , Relación Estructura-ActividadRESUMEN
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endosomas/metabolismo , Helicobacter pylori/química , Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Glicosilfosfatidilinositoles/metabolismo , Células HeLa , Humanos , Transporte de ProteínasRESUMEN
Glioblastoma (GBM) cells invade the brain by following linear structures like blood vessel walls and white matter tracts by using specific motility modes. In this protocol, we describe two micropatterning techniques allowing recapitulation of these linear tracks in vitro: micro-contact printing and deep UV photolithography. We also detail how to maintain, transfect, and prepare human glioma propagating cells (hGPCs) for migration assays on linear tracks, followed by image acquisition and analysis, to measure key parameters of their motility. For complete details on the use and execution of this protocol, please refer to Monzo et al. (2016) and Monzo et al. (2021a).
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Encéfalo , Movimiento Celular , HumanosRESUMEN
Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.
Asunto(s)
Movimiento Celular/fisiología , Forminas/metabolismo , Glioblastoma/metabolismo , Invasividad Neoplásica/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proteínas Fetales/metabolismo , Glioblastoma/patología , Humanos , Proteínas de Microfilamentos/metabolismoRESUMEN
The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.
Asunto(s)
Actomiosina/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Actinas/metabolismo , Animales , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis/fisiología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Espectrina/genética , Estrés MecánicoRESUMEN
The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.
Asunto(s)
Proteínas Bacterianas/fisiología , Toxinas Bacterianas/metabolismo , Clatrina/fisiología , Endosomas/fisiología , Helicobacter pylori/fisiología , Pinocitosis/fisiología , Factores de Virulencia/fisiología , Proteína de Unión al GTP cdc42/fisiología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Caveolina 1/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Dinamina II/metabolismo , Humanos , Microdominios de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Transferrina/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab5/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis.
Asunto(s)
Citocinesis , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas/metabolismo , Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Anafase , Anticuerpos Monoclonales/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas del Citoesqueleto , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía por Video , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas/química , Proteínas/genética , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Telofase , Técnicas del Sistema de Dos Híbridos , Xantenos , Dominios Homologos srcRESUMEN
Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II-independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.
Asunto(s)
Adhesión Celular , Membrana Celular/fisiología , Movimiento Celular , Citoesqueleto/fisiología , Fibroblastos/fisiología , Mecanotransducción Celular , Seudópodos/fisiología , Actinas/genética , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Forma de la Célula , Células Cultivadas , Simulación por Computador , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Microscopía por Video , Modelos Biológicos , Miosina Tipo II/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Seudópodos/metabolismo , Estrés Mecánico , Factores de Tiempo , Transfección , Vinculina/metabolismoRESUMEN
Glioblastomas are extremely aggressive brain tumors with highly invasive properties. Brain linear tracks such as blood vessel walls constitute their main invasive routes. Here we analyze rat C6 and patient-derived glioma cell motility in vitro using micropatterned linear tracks to mimic blood vessels. On laminin-coated tracks (3-10 µm), these cells used an efficient saltatory mode of migration similar to their in vivo migration. This saltatory migration was also observed on larger tracks (50-400 µm in width) at high cell densities. In these cases, the mechanical constraints imposed by neighboring cells triggered this efficient mode of migration, resulting in the formation of remarkable antiparallel streams of cells along the tracks. This motility involved microtubule-dependent polarization, contractile actin bundles and dynamic paxillin-containing adhesions in the leading process and in the tail. Glioma linear migration was dramatically reduced by inhibiting formins but, surprisingly, accelerated by inhibiting Arp2/3. Protein expression and phenotypic analysis indicated that the formin FHOD3 played a role in this motility but not mDia1 or mDia2. We propose that glioma migration under confinement on laminin relies on formins, including FHOD3, but not Arp2/3 and that the low level of adhesion allows rapid antiparallel migration.
Asunto(s)
Neoplasias Encefálicas/patología , Ensayos de Migración Celular/métodos , Glioma/patología , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Fenómenos Biomecánicos , Neoplasias Encefálicas/irrigación sanguínea , Adhesión Celular , Recuento de Células , Movimiento Celular , Forminas , Glioblastoma/patología , Glioma/irrigación sanguínea , Humanos , Laminina/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Paxillin/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
In this chapter, we describe various approaches that allow us to study interactions between the small GTPase Rab4a and its two effectors, Rabip4 and CD2AP. Two complementary approaches, one using the yeast two-hybrid system and the other using a GST pull-down assay, are described. We document the studies of the localization of these proteins by cellular fractionation. Finally, we develop cellular imaging techniques to study the morphology of vesicular structures containing Rab4a. We show that the coexpression of Rab4a with its effectors affects Rab4a-containing structures, giving a clear indication of their interaction in the mammalian cellular context.
Asunto(s)
Endosomas/metabolismo , Proteínas/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto , Endosomas/ultraestructura , Glutatión/metabolismo , Nucleótidos de Guanina/metabolismo , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab4/aislamiento & purificaciónRESUMEN
In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.
Asunto(s)
Movimiento Celular/fisiología , Matriz Extracelular/fisiología , Fibronectinas/metabolismo , Células 3T3 , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Línea Celular Tumoral , Perros , Matriz Extracelular/metabolismo , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células de Riñón Canino Madin Darby , Ratones , Células 3T3 NIH , Células PC12 , Ratas , Transducción de Señal/fisiologíaRESUMEN
Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.
Asunto(s)
Dineínas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Células COS , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Complejo Dinactina , Dineínas/genética , Humanos , Cinetocoros/ultraestructura , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/genética , Fenotipo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Huso Acromático/metabolismoRESUMEN
The ability of gliomas to invade the brain limits the efficacy of standard therapies. In this study, we have examined glioma migration in living brain tissue by using two novel in vivo model systems. Within the brain, glioma cells migrate like nontransformed, neural progenitor cells-extending a prominent leading cytoplasmic process followed by a burst of forward movement by the cell body that requires myosin II. In contrast, on a two-dimensional surface, glioma cells migrate more like fibroblasts, and they do not require myosin II to move. To explain this phenomenon, we studied glioma migration through a series of synthetic membranes with defined pore sizes. Our results demonstrate that the A and B isoforms of myosin II are specifically required when a glioma cell has to squeeze through pores smaller than its nuclear diameter. They support a model in which the neural progenitor-like mode of glioma invasion and the requirement for myosin II represent an adaptation needed to move within the brain, which has a submicrometer effective pore size. Furthermore, the absolute requirement for myosin II in brain invasion underscores the importance of this molecular motor as a potential target for new anti-invasive therapies to treat malignant brain tumors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Miosina Tipo II/fisiología , Animales , Encéfalo/patología , Movimiento Celular , Electroforesis en Gel Bidimensional , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Modelos Biológicos , Miosina Tipo II/química , Trasplante de Neoplasias , Ratas , Ratas Sprague-DawleyRESUMEN
The small GTP-binding protein Rab4 has been involved in the recycling of alphavbeta3 integrins in response to platelet-derived growth factor (PDGF) stimulation suggesting a role for Rab4 in cell adhesion and migration. In this study, we explored the role of Rabip4 and Rabip4', two Rab4 effector proteins, in migration of NIH 3T3 fibroblasts. In these cells, Rabip4 and Rabip4', collectively named Rabip4s, were partially co-localized with the early endosomal marker EEA1. PDGF treatment re-distributed endogenous Rabip4s toward the cell periphery where they colocalized with F-actin. In cells expressing green fluorescent protein (GFP)-Rabip4 or GFP-Rabip4', constitutive appearance of GFP-Rabip4s at the cell periphery was accompanied by local increase in cortical F-actin in membrane ruffles at the leading edge. The expression of GFP-Rabip4 induced an increased migration compared with control cells expressing GFP alone, even in the absence of PDGF stimulation. On the contrary, in cells expressing a mutated form of Rabip4s unable to interact with Rab4, lack of typical leading edge was observed. Furthermore, PDGF treatment did not stimulate the migration of these cells. Furthermore, down-regulation of the expression of Rabip4s inhibited PDGF-stimulated cell migration. Endogenous Rabip4s were localized with alphav integrins at the leading edge following PDGF treatment, whereas in cells expressing GFP-Rabip4s, alphav integrins, together with GFP-Rabip4s, were constitutively localized at the leading edge. In contrast, reduction in Rabip4s expression levels using small interfering RNA was associated with impaired PDGF-induced translocation of alphav integrins toward the leading edge. Taken together, our data provide evidence that Rabip4s, possibly via their interaction with Rab4, regulate integrin trafficking and are involved in the migration of NIH 3T3 fibroblasts.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Unión al GTP rab4/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Movimiento Celular , ADN Complementario/metabolismo , Regulación hacia Abajo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Integrina alfaV/metabolismo , Ratones , Mutación , Células 3T3 NIH , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Isoformas de ProteínasRESUMEN
Insulin regulates glucose uptake in the adipocytes by modulating Glut 4 localization, a traffic pathway involving the endocytic small GTPases Rab4, Rab5, and RabThe expression of the Rab4 effector Rabip4 leads to a 30% increase in glucose uptake and Glut 4 translocation in the presence of insulin, without modifications in the basal condition. This effect was not due to modifications of Glut 4 expression or insulin signaling, suggesting that Rabip4 controls Glut 4 trafficking. We present evidence that Rabip4 defines a subdomain of early endosomes and that Rabip4 is redistributed to the plasma membrane by insulin. Rabip4 is mostly absent from structures positive for early endosome antigen 1, Rab11 or transferrin receptors and from Glut 4 sequestration compartments. However, Rabip4 vesicles can be reached by internalized transferrin and Glut 4. Thus, Rabip4 probably defines an endocytic sorting platform for Glut 4 towards its sequestration pool. The expression of a form of Rabip4 unable to bind Rab4 does not modify basal and insulin-induced glucose transport. However, it induces an increase in the amount of Glut 4 at the plasma membrane and perturbs Glut 4 traffic from endosomes towards its sequestration compartments. These observations suggest that the uncoupling between Rabip4 and Rab4 induces the insertion of Glut 4 molecules that are unable to transport glucose into the plasma membrane.
Asunto(s)
Adipocitos/metabolismo , Endocitosis , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Células 3T3-L1 , Animales , Compartimento Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Microscopía Confocal , Modelos Biológicos , Mutación , Fracciones Subcelulares/metabolismo , Transferrina/metabolismo , Proteínas de Unión al GTP rab4/genéticaRESUMEN
The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4. We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS-positive vesicles. However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.