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1.
Nat Mater ; 22(5): 644-655, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36581770

RESUMEN

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.


Asunto(s)
Actinas , Neoplasias , ADN , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Citosol/metabolismo , Transducción de Señal
2.
Cell ; 134(1): 135-47, 2008 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-18614017

RESUMEN

The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.


Asunto(s)
Movimiento Celular , Endocitosis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismo , Actinas/metabolismo , Animales , Línea Celular Tumoral , Clatrina/metabolismo , Embrión de Mamíferos/citología , Embrión no Mamífero/citología , Endosomas/metabolismo , Células Germinativas/citología , Células Germinativas/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Ratones , Pez Cebra
3.
Small ; 18(17): e2106097, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35344274

RESUMEN

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 , Pronóstico
4.
Eur Phys J E Soft Matter ; 45(5): 50, 2022 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-35604494

RESUMEN

The connection between the properties of a cell tissue and those of the single constituent cells remains to be elucidated. At the purely mechanical level, the degree of rigidity of different cellular components, such as the nucleus and the cytoplasm, modulates the interplay between the cell inner processes and the external environment, while simultaneously mediating the mechanical interactions between neighboring cells. Being able to quantify the correlation between single-cell and tissue properties would improve our mechanobiological understanding of cell tissues. Here we develop a methodology to quantitatively extract a set of structural and motility parameters from the analysis of time-lapse movies of nuclei belonging to jammed and flocking cell monolayers. We then study in detail the correlation between the dynamical state of the tissue and the deformation of the nuclei. We observe that the nuclear deformation rate linearly correlates with the local divergence of the velocity field, which leads to a non-invasive estimate of the elastic modulus of the nucleus relative to the one of the cytoplasm. We also find that nuclei belonging to flocking monolayers, subjected to larger mechanical perturbations, are about two time stiffer than nuclei belonging to dynamically arrested monolayers, in agreement with atomic force microscopy results. Our results demonstrate a non-invasive route to the determination of nuclear relative stiffness for cells in a monolayer.


Asunto(s)
Núcleo Celular , Citoplasma , Módulo de Elasticidad , Microscopía de Fuerza Atómica/métodos
5.
Soft Matter ; 17(13): 3550-3559, 2021 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-33346771

RESUMEN

The accurate quantification of cellular motility and of the structural changes occurring in multicellular aggregates is critical in describing and understanding key biological processes, such as wound repair, embryogenesis and cancer invasion. Current methods based on cell tracking or velocimetry either suffer from limited spatial resolution or are challenging and time-consuming, especially for three-dimensional (3D) cell assemblies. Here we propose a conceptually simple, robust and tracking-free approach for the quantification of the dynamical activity of cells via a two-step procedure. We first characterise the global features of the collective cell migration by registering the temporal stack of the acquired images. As a second step, a map of the local cell motility is obtained by performing a mean squared amplitude analysis of the intensity fluctuations occurring when two registered image frames acquired at different times are subtracted. We successfully apply our approach to cell monolayers undergoing a jamming transition, as well as to monolayers and 3D aggregates that exhibit a cooperative unjamming-via-flocking transition. Our approach is capable of disentangling very efficiently and of assessing accurately the global and local contributions to cell motility.


Asunto(s)
Imagenología Tridimensional , Movimiento Celular , Movimiento (Física)
6.
Nat Mater ; 18(11): 1252-1263, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31332337

RESUMEN

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/metabolismo
7.
Immunity ; 35(3): 388-99, 2011 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-21835647

RESUMEN

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.


Asunto(s)
Proteínas de Capping de la Actina/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas del Citoesqueleto/inmunología , Células Dendríticas/inmunología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Presentación de Antígeno , Movimiento Celular/inmunología , Proliferación Celular , Células Cultivadas , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Dermatitis por Contacto/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología
10.
Nat Mater ; 16(5): 587-596, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28135264

RESUMEN

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.


Asunto(s)
Endocitosis , Epitelio/metabolismo , Fenómenos Biomecánicos , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Proteínas de Unión al GTP rab5/metabolismo
11.
EMBO Rep ; 17(7): 1061-80, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27255086

RESUMEN

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.


Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Aparato de Golgi/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Biomarcadores de Tumor , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Endosomas/metabolismo , Exocitosis , Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Invasividad Neoplásica , Pronóstico , Transporte de Proteínas , Proteolisis , Recurrencia , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al GTP rab/genética
13.
Biomed Pharmacother ; 177: 116991, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38906021

RESUMEN

Macropinocytosis is a cellular process that enables cells to engulf extracellular material, such as nutrients, growth factors, and even whole cells. It is involved in several physiological functions as well as pathological conditions. In cancer cells, macropinocytosis plays a crucial role in promoting tumor growth and survival under nutrient-limited conditions. In particular KRAS mutations have been identified as main drivers of macropinocytosis in pancreatic, breast, and non-small cell lung cancers. We performed a high-content screening to identify inhibitors of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC)-derived cells, aiming to prevent nutrient scavenging of PDAC tumors. The screening campaign was conducted in a well-known pancreatic KRAS-mutated cell line (MIAPaCa-2) cultured under nutrient deprivation and using FITC-dextran to precisely quantify macropinocytosis. We assembled a collection of 3584 small molecules, including drugs approved by the Food and Drug Administration (FDA), drug-like molecules against molecular targets, kinase-targeted compounds, and molecules designed to hamper protein-protein interactions. We identified 28 molecules that inhibited macropinocytosis, with potency ranging from 0.4 to 29.9 µM (EC50). A few of them interfered with other endocytic pathways, while 11 compounds did not and were therefore considered specific "bona fide" macropinocytosis inhibitors and further characterized. Four compounds (Ivermectin, Tyrphostin A9, LY2090314, and Pyrvinium Pamoate) selectively hampered nutrient scavenging in KRAS-mutated cancer cells. Their ability to impair albumin-dependent proliferation was replicated both in different 2D cell culture systems and 3D organotypic models. These findings provide a new set of compounds specifically targeting macropinocytosis, which could have therapeutic applications in cancer and infectious diseases.


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pinocitosis , Pinocitosis/efectos de los fármacos , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Mutación
14.
Nat Cell Biol ; 8(12): 1337-47, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17115031

RESUMEN

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Forma de la Célula , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Unión Proteica , Transporte de Proteínas , Seudópodos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo
16.
Nat Genet ; 31(2): 190-4, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11992123

RESUMEN

Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer. In the absence of other genetic alterations, this deregulation results in lack of differentiation, hyperproliferation and apoptosis. The pRB protein acts as a transcriptional repressor by targeting the E2F transcription factors, whose functions are required for entry into S phase. Increased E2F activity can induce S phase in quiescent cells--this is a central element of most models for the development of cancer. We show that although E2F1 alone is not sufficient to induce S phase in diploid mouse and human fibroblasts, increased E2F1 activity can result in S-phase entry in diploid fibroblasts in which the p53-mediated G1 checkpoint is suppressed. In addition, we show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results indicate that, in addition to acting as an E2F-dependent transcriptional repressor, pRB is also required for the cells to retain the G1 checkpoint in response to unprogrammed proliferative signals.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Fase G1/genética , Proteína de Retinoblastoma/fisiología , Fase S/genética , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Fibroblastos/citología , Fibroblastos/fisiología , Fase G1/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Fase S/fisiología , Transducción de Señal/genética
17.
Cell Rep ; 42(8): 113001, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37590133

RESUMEN

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.


Asunto(s)
Mama , Seudópodos , Cicatrización de Heridas , Movimiento (Física)
18.
bioRxiv ; 2023 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-36747801

RESUMEN

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.

19.
Nat Cell Biol ; 7(10): 969-76, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16155590

RESUMEN

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.


Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Membrana Celular/metabolismo , Proteínas del Citoesqueleto , Receptores ErbB/metabolismo , Células HeLa , Humanos , Receptores de Transferrina/metabolismo , Vesículas Transportadoras/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Proteína de Unión al GTP cdc42/metabolismo
20.
PLoS Biol ; 7(6): e1000138, 2009 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-19564905

RESUMEN

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteínas del Citoesqueleto/metabolismo , Neuronas/efectos de los fármacos , Seudópodos/fisiología , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Axones/metabolismo , Axones/fisiología , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Interferencia de ARN , Ratas , Transfección
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