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1.
J Clin Immunol ; 42(7): 1535-1544, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35767111

RESUMEN

Mutations in the ARPC1B isoform component of human actin-related protein 2/3 complex have been recently associated with an inborn error of immunity characterized by combined immunodeficiency, allergies, autoinflammation, and platelet abnormalities. Currently, indications on the management of this novel disease and information on its outcome are lacking. We report the first case series of 7 children with a homozygous mutation in ARPC1B gene who underwent allogeneic-HSCT (allo-HSCT). All patients presented an early clinical onset, characterized by recurrent infections, failure to thrive and gastrointestinal bleeding episodes complicated with neonatal hemorrhagic enteritis in 3 cases, and macrophage activating syndrome in 2. Allo-HSCT was performed at the median age of 1.83 years after a myeloablative conditioning regimen in all cases. Engraftment occurred in all patients with full donor chimerism in 6 out of 7. The clinical course after engraftment was uneventful in 3 out of 7 children; 2 patients developed a grade 1-2 acute graft-versus-host disease (GvHD), and 1 patient a grade 1 chronic-GvHD. JC virus-related progressive multifocal leukoencephalopathy was diagnosed in one patient 13 months after haploidentical-HSCT and successfully managed with donor-derived viral-specific T-cell infusion. Only one patient had a fatal outcome 3 months after HSCT because of sepsis, after veno-occlusive disease, and transplant-associated microangiopathy. At a median follow-up of 19 months (range 3-110), 6 out of 7 patients are alive and disease-free. The severity of the clinical phenotype at diagnosis and the high survival rate, with limited transplant-related morbidity, strongly support the indication to allo-HSCT for patients with this diagnosis.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Trasplante de Células Madre Hematopoyéticas , Humanos , Recién Nacido , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Supervivencia sin Enfermedad , Enfermedad Injerto contra Huésped , Acondicionamiento Pretrasplante , Lactante , Quimera por Trasplante
2.
Cell Rep ; 36(1): 109318, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-34233185

RESUMEN

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.


Asunto(s)
Forma de la Célula , Imagenología Tridimensional , Células Asesinas Naturales/citología , Linfocitos T/citología , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Adolescente , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Exocitosis/efectos de los fármacos , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Masculino , Compuestos de Organoselenio/farmacología , Compuestos de Organosilicio/farmacología , Análisis de la Célula Individual , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Tionas/farmacología , Uracilo/análogos & derivados , Uracilo/farmacología , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/metabolismo
3.
Front Immunol ; 12: 678030, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34135903

RESUMEN

The actin-related protein (ARP) 2/3 complex, essential for organizing and nucleating branched actin filaments, is required for several cellular immune processes, including cell migration and granule exocytosis. Recently, genetic defects in ARPC1B, a subunit of this complex, were reported. Mutations in ARPC1B result in defective ARP2/3-dependent actin filament branching, leading to a combined immunodeficiency with severe inflammation. In vitro, neutrophils of these patients showed defects in actin polymerization and chemotaxis, whereas adhesion was not altered under static conditions. Here we show that under physiological flow conditions human ARPC1B-deficient neutrophils were able to transmigrate through TNF-α-pre-activated endothelial cells with a decreased efficiency and, once transmigrated, showed definite impairment in subendothelial crawling. Furthermore, severe locomotion and migration defects were observed in a 3D collagen matrix and a perfusable vessel-on-a-chip model. These data illustrate that neutrophils employ ARP2/3-independent steps of adhesion strengthening for transmigration but rely on ARP2/3-dependent modes of migration in a more complex multidimensional environment.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/genética , Mutación , Neutrófilos/inmunología , Enfermedades de Inmunodeficiencia Primaria/inmunología , Migración Transendotelial y Transepitelial/inmunología , Actinas/química , Estudios de Casos y Controles , Adhesión Celular/genética , Células Cultivadas , Quimiotaxis/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Infiltración Neutrófila/genética , Polimerizacion , Enfermedades de Inmunodeficiencia Primaria/sangre , Enfermedades de Inmunodeficiencia Primaria/genética
5.
PLoS Biol ; 17(10): e3000500, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31652255

RESUMEN

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/genética , Endocitosis/genética , Regulación Fúngica de la Expresión Génica , Mecanotransducción Celular , Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/ultraestructura , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Fenómenos Biomecánicos , Clatrina/deficiencia , Clatrina/genética , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética
6.
J Allergy Clin Immunol ; 143(6): 2296-2299, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30771411
7.
J Immunol ; 199(12): 4036-4045, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29127144

RESUMEN

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott-Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott-Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/genética , Plaquetas/patología , Mutación del Sistema de Lectura , Síndromes de Inmunodeficiencia/genética , Linfopoyesis/genética , Linfocitos T/patología , Trombopoyesis/genética , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Preescolar , Codón sin Sentido , Consanguinidad , Resultado Fatal , Humanos , Lactante , Masculino , Complejos Multiproteicos , Linaje , Polimerizacion , Recombinación V(D)J , Síndrome de Wiskott-Aldrich/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética
8.
Nat Commun ; 8: 14816, 2017 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-28368018

RESUMEN

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Trastornos de las Plaquetas Sanguíneas/metabolismo , Plaquetas/metabolismo , Inflamación/patología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/patología , Plaquetas/ultraestructura , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Forma de la Célula , Susceptibilidad a Enfermedades , Fibrinógeno/farmacología , Técnicas de Inactivación de Genes , Humanos , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Megacariocitos/patología , Mutación/genética , Vasculitis/patología , Síndrome de Wiskott-Aldrich/patología
11.
J Neurosci ; 33(14): 6081-92, 2013 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-23554489

RESUMEN

Despite evidence for a strong genetic contribution to several major psychiatric disorders, individual candidate genes account for only a small fraction of these disorders, leading to the suggestion that multigenetic pathways may be involved. Several known genetic risk factors for psychiatric disease are related to the regulation of actin polymerization, which plays a key role in synaptic plasticity. To gain insight into and test the possible pathogenetic role of this pathway, we designed a conditional knock-out of the Arp2/3 complex, a conserved final output for actin signaling pathways that orchestrates de novo actin polymerization. Here we report that postnatal loss of the Arp2/3 subunit ArpC3 in forebrain excitatory neurons leads to an asymmetric structural plasticity of dendritic spines, followed by a progressive loss of spine synapses. This progression of synaptic deficits corresponds with an evolution of distinct cognitive, psychomotor, and social disturbances as the mice age. Together, these results point to the dysfunction of actin signaling, specifically that which converges to regulate Arp2/3, as an important cellular pathway that may contribute to the etiology of complex psychiatric disorders.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Espinas Dendríticas/patología , Trastornos Mentales/genética , Plasticidad Neuronal/fisiología , Neuronas/patología , Sinapsis/patología , Actinas/metabolismo , Animales , Animales Recién Nacidos , Proteínas Bacterianas/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Espinas Dendríticas/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos , Conducta Exploratoria/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/patología , Proteínas Luminiscentes/genética , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratones , Ratones Transgénicos , Plasticidad Neuronal/genética , Fotoblanqueo , Canales de Potasio/genética , Canales de potasio activados por Sodio , Proteínas/genética , Proteínas/metabolismo , ARN no Traducido , Receptores de Glutamato/metabolismo , Reflejo de Sobresalto/genética , Transducción de Señal/genética , Conducta Social , Sinapsis/genética , Factores de Tiempo
12.
Mol Biol Cell ; 21(20): 3529-39, 2010 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-20739464

RESUMEN

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.


Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Células Musculares/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Electroforesis en Gel Bidimensional , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Células Musculares/citología , Células Musculares/efectos de los fármacos , Monoéster Fosfórico Hidrolasas , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Transferrina/metabolismo , Xenopus
13.
Mol Cell Biol ; 26(16): 6185-96, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16880528

RESUMEN

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Fenotipo , Transposasas/genética , Trofoblastos/patología , Actinas/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Citoesqueleto/ultraestructura , Elementos Transponibles de ADN/genética , Prueba de Complementación Genética , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional , Mutación/genética , Trofoblastos/citología , Trofoblastos/ultraestructura
14.
Mol Biol Cell ; 17(6): 2581-91, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16597702

RESUMEN

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Dictyostelium/fisiología , Seudópodos/fisiología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Interferencia de ARN , Familia de Proteínas del Síndrome de Wiskott-Aldrich/deficiencia , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética
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