RESUMEN
Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.
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Adhesiones Focales , Aparato de Golgi , Humanos , Células CACO-2 , Citoesqueleto de Actina , Movimiento Celular , Proteínas de Transporte VesicularRESUMEN
The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity.
RESUMEN
Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ.
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Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular , Perros , Humanos , Unión ProteicaRESUMEN
The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.
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Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos/metabolismo , Movimiento Celular/fisiología , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Miosina Tipo II/metabolismo , Ovalbúmina/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Células de la Médula Ósea , Catepsinas/genética , Catepsinas/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Masculino , Ratones , Técnicas Analíticas Microfluídicas , Miosina Tipo II/genéticaRESUMEN
Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.
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Calcio/metabolismo , Movimiento Celular/fisiología , Células Dendríticas/inmunología , Espacio Extracelular/inmunología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miosina Tipo II/metabolismo , Animales , Polaridad Celular , Cartilla de ADN/genética , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Immunoblotting , Ratones , Microscopía Fluorescente , Microscopía por Video , Fosforilación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Dendritic cells (DCs) constitute a complex cell population that resides in both peripheral tissues and lymphoid organs. Their major function in tissues is to patrol their environment in search of danger-associated antigens to transport to lymph nodes and present to T lymphocytes. This process constitutes the first step of the adaptive immune response and relies on specific DC properties, including a high endocytic capacity as well as efficient motility in confined three-dimensional environments. Although cell motility has been widely studied, little is known on how the geometric characteristics of the environment influence DC migration and function. In this review, we give an overview of the basic physical principles and molecular mechanisms that control DC migration under confinement and discuss how such mechanisms impact the environment-patrolling capacity of DCs.
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Movimiento Celular/inmunología , Células Dendríticas/fisiología , Animales , Antígenos/inmunología , HumanosRESUMEN
The actin-binding protein filamins (FLNs) are major organizers of the actin cytoskeleton. They control the elasticity and stiffness of the actin network and provide connections with the extracellular microenvironment by anchoring transmembrane receptors to the actin filaments. Although numerous studies have revealed the importance of FLN levels, relatively little is known about the regulation of its stability in physiological relevant settings. Here, we show that the ASB2α cullin 5-ring E3 ubiquitin ligase is highly expressed in immature dendritic cells (DCs) and is down-regulated after DC maturation. We further demonstrate that FLNs are substrates of ASB2α in immature DCs and therefore are not stably expressed in these cells, whereas they exhibit high levels of expression in mature DCs. Using ASB2 conditional knockout mice, we show that ASB2α is a critical regulator of cell spreading and podosome rosette formation in immature DCs. Furthermore, we show that ASB2(-/-) immature DCs exhibit reduced matrix-degrading function leading to defective migration. Altogether, our results point to ASB2α and FLNs as newcomers in DC biology.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Movimiento Celular/genética , Células Dendríticas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Células Dendríticas/metabolismo , Filaminas , Técnicas de Silenciamiento del Gen , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Células Progenitoras de Granulocitos y Macrófagos/fisiología , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Células 3T3 NIH , Proteínas Supresoras de la Señalización de Citocinas , Transfección , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migration. Important parameters such as degree of adhesion, degree of confinement, mechanical properties, and geometry can be varied independently of each other. The device is fully compatible with almost any type of light microscopy and the simple geometry makes automated analysis very easy to perform, which allows screening strategy. The chapters is divided into five parts describing the design of different types of migration chambers, the fabrication of a mold by photolithography, the assembly of the chamber, the loading of cells, and finally the imaging on live or fixed cells.
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Ensayos de Migración Celular/métodos , Microfluídica/métodos , Imagen de Lapso de Tiempo/métodos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Técnicas de Cultivo de Célula , Ensayos de Migración Celular/instrumentación , Movimiento Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/fisiología , Ratones , Microfluídica/instrumentación , Microscopía Fluorescente , Silicio/química , Análisis de la Célula Individual , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Dendritic cells (DCs) need to migrate in the interstitial environment of peripheral tissues to reach secondary lymphoid organs and initiate a suitable immune response. Whether and how inflamed tissues instruct DCs to emigrate is not fully understood. In this study, we report the unexpected finding that the epithelial-derived cytokine TSLP triggers chemokinesis of resting primary human DCs in a cell-autonomous manner. TSLP induced the polarization of both microtubule and actin cytoskeletons and promoted DC 3-dimensional migration in transwell as well as in microfabricated channels that mimic the confined environment of peripheral tissues. TSLP-induced migration relied on the actin-based motor myosin II and was inhibited by blebbistatin. Accordingly, TSLP triggered the redistribution of phosphorylated myosin II regulatory light chain to the actin cortex, indicating that TSLP induces DC migration by promoting actomyosin contractility. Thus, TSLP produced by epithelial cells in inflamed tissue has a critical function in licensing DCs for cell-autonomous migration. This indicates that cytokines can directly trigger cell migration, which has important implications in immune physiopathology and vaccine design.
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Citocinas/inmunología , Células Dendríticas/citología , Movimiento Celular , Células Cultivadas , Citoesqueleto/inmunología , Citoesqueleto/ultraestructura , Células Dendríticas/inmunología , Humanos , Técnicas Analíticas Microfluídicas , Miosina Tipo II/inmunología , Miosina Tipo II/ultraestructura , Linfopoyetina del Estroma TímicoRESUMEN
The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.
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Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica/métodos , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Filaminas , Humanos , Leucemia Mieloide/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Talina/genética , Talina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation.
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Proteínas Contráctiles/metabolismo , Leucemia/patología , Proteínas de Microfilamentos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Actinas/metabolismo , Adhesión Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proteínas Contráctiles/genética , Filaminas , Humanos , Leucemia/tratamiento farmacológico , Proteínas de Microfilamentos/genética , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de la Señalización de Citocinas/genética , Tretinoina/farmacologíaRESUMEN
Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critical to develop new treatments for blood diseases and to manipulate stem cells. Despite the identification of many players in hematopoiesis, the molecular mechanisms controlling hematopoietic differentiation remain poorly understood. Due to a number of recent findings, the targeting of regulators of hematopoiesis to proteasomal degradation might be an important step in control of this developmental program.
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Neoplasias Hematológicas/metabolismo , Hematopoyesis , Sistema Hematopoyético/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas/metabolismo , Dominios RING Finger/fisiología , Proteínas Supresoras de Tumor/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate.