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1.
J Cell Biol ; 220(8)2021 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-34106209

RESUMEN

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Oxidorreductasas/metabolismo , Citoesqueleto de Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Cortactina/genética , Cortactina/metabolismo , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Microscopía Fluorescente , Oxidación-Reducción , Oxidorreductasas/genética , Virus Vaccinia/genética , Virus Vaccinia/metabolismo
2.
PLoS Genet ; 17(4): e1009512, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33872315

RESUMEN

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Síndrome de Wiskott-Aldrich/genética , Proteínas de Unión al GTP rho/genética , Citoesqueleto de Actina/genética , Proteína 2 Relacionada con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Apoptosis/genética , Citocromos c/genética , Daño del ADN/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Interferente Pequeño/genética , Proteína del Síndrome de Wiskott-Aldrich/genética
3.
FASEB J ; 35(5): e21521, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33811691

RESUMEN

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.


Asunto(s)
Movimiento Celular , Selectina E/metabolismo , Endotelio Vascular/fisiología , Uniones Intercelulares/fisiología , Neutrófilos/fisiología , Migración Transendotelial y Transepitelial , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Células Cultivadas , Selectina E/genética , Endotelio Vascular/citología , Humanos , Neutrófilos/citología , Seudópodos
4.
mBio ; 12(1)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33468693

RESUMEN

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.


Asunto(s)
Actinas/genética , Proteínas Bacterianas/genética , Chlamydia trachomatis/genética , Citoesqueleto/metabolismo , Chaperonas Moleculares/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/metabolismo , Citoesqueleto/microbiología , Citoesqueleto/ultraestructura , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Chaperonas Moleculares/metabolismo , Polimerizacion , Transducción de Señal , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo
5.
J Cell Physiol ; 235(9): 6127-6138, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-31975378

RESUMEN

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli-Sertoli junctions.


Asunto(s)
Proteína 3 Relacionada con la Actina/genética , Barrera Hematotesticular/metabolismo , Proteínas/genética , Proteína de la Zonula Occludens-1/genética , Citoesqueleto de Actina/genética , Uniones Adherentes/genética , Animales , Masculino , Ratones , Ocludina/genética , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Epitelio Seminífero/crecimiento & desarrollo , Epitelio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis/genética , Uniones Estrechas/genética
6.
J Cell Biochem ; 121(2): 1934-1944, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31637768

RESUMEN

PURPOSE: Long noncoding RNAs (lncRNAs) play an indispensable role in cancer progression. We aim at exploring the effect of AC009022.1 on colorectal cancer (CRC) development in this paper. METHODS: CRC tissues and matched normal tissues of 68 CRC patients were collected. HCT-116 and SW620 cells were transfected and grouped. The cell counting kit-8 assay, cell scratch test, and Transwell experiment were performed to sequentially detect cells proliferation, migration, and invasion. In vivo experiments were conducted. The Luciferase reporter gene assay was used. Gene expression was detected by quantitative reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: AC009022.1 expression was abnormally elevated in CRC tissues and cells. High AC009022.1 expression in CRC patients was significantly associated with poor prognosis. Compared with the siNC group, HCT-116 and SW620 cells of siAC009022.1 group exhibited much lower OD450 value (P < .001) and invasive cell number (P < .01), and significantly higher relative wound width (P < .01). Much lower subcutaneous tumor volume and weight were found in the siAC009022.1 group compared with siNC group (P < .001). In CRC cells, AC009022.1 directly inhibited miR-497-5p expression while miR-497-5p directly hindered ACTR3B expression. Compared with HCT-116 and SW620 cells of oe-AC009022.1 group, those of oe-AC009022.1 + miR-497-5p-mimics group and oe-AC009022.1 + siACTR3B group had obviously lower OD450 value (P < .001) and invasion cell number (P < .01), and markedly higher relative wound width (P < .01). CONCLUSIONS: AC009022.1 enhanced CRC cell proliferation, migration, and invasion by promoting ACTR3B expression via suppressing miR-497-5p.


Asunto(s)
Proteína 3 Relacionada con la Actina/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Largo no Codificante/genética , Proteína 3 Relacionada con la Actina/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Immunol Cell Biol ; 98(2): 93-113, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31698518

RESUMEN

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Movimiento Celular/genética , Linfocitos T Citotóxicos/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Actinas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño , Análisis de la Célula Individual , Linfocitos T Citotóxicos/citología , Pez Cebra
8.
J Clin Invest ; 129(7): 2807-2823, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-31063459

RESUMEN

Phosphorylation of Dynamin-related protein1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here we established the role of Drp1 Serine 600 (S600) phosphorylation on mitochondrial fission in vivo, and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus- but not cell type-dependent. Mechanistically, we showed that mitochondrial fission in high glucose conditions occurs through concomitant binding of phospho-Drp1S600 with mitochondrial fission factor (Mff) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo, and highlight the stimulus-specific consequences of Drp1S600 phosphorylation on mitochondrial dynamics.


Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Dinaminas/metabolismo , Mutación Missense , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Sustitución de Aminoácidos , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Dinaminas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Dinámicas Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación/genética
9.
Am J Med Genet B Neuropsychiatr Genet ; 180(3): 213-222, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30779416

RESUMEN

We previously identified bipolar disorder (BD) susceptibility loci on 8q24, 14q32, and 2q12-14 in a genome-wide nonparametric linkage screen in a Latino cohort. We now perform a fine mapping analysis using a dense map of additional SNPs to identify BD susceptibility genes within these regions. One thousand nine hundred and thirty-eight individuals with Latino ancestry (880 individuals with BD Type I or Schizoaffective, Bipolar Type) from 416 Latino pedigrees from the United States, Mexico, Costa Rica, and Guatemala were genotyped with 3,074 SNPs to provide dense coverage of the 8q24 (11.5 cM), 14q32 (7.5 cM), and 2q12-14 (6.5 cM) chromosomal loci. Single-marker association tests in the presence of linkage were performed using the LAMP software. The top linkage peak (rs7834818; LOD = 5.08, p = 3.30E - 5) and associated single marker (rs2280915, p = 2.70E - 12) were located within FBXO32 on 8q24. On chromosome 2, the top linkage peak (rs6750326; LOD = 5.06, p = 3.50E - 5) and associated single marker (rs11887088, p = 2.90E - 6) were located in intragenic regions near ACTR3 and DPP10. None of the additional markers in the region around chromosome 14q32 met significance levels for linkage or association. We identified six SNPs on 2q12-q14 and one SNP in FBXO32 on 8q24 that were significantly associated with BD in this Latino cohort.


Asunto(s)
Trastorno Bipolar/genética , Cromosomas Humanos Par 2/genética , Trastornos Psicóticos/genética , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Adulto , Trastorno Bipolar/psicología , Mapeo Cromosómico/métodos , Costa Rica , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Femenino , Ligamiento Genético/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Guatemala , Hispánicos o Latinos/genética , Humanos , Escala de Lod , Masculino , México , Persona de Mediana Edad , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Trastornos Psicóticos/psicología , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Estados Unidos
10.
Toxicol Lett ; 295: 277-287, 2018 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-29981920

RESUMEN

There are reports of fluorochloridone (FLC)-induced male reproductive toxicity, but the underlying toxicological mechanisms remain unknown. In this study, we looked at how FLC exposure affected the integrity of the blood-testis barrier (BTB) and the Sertoli cell barrier and studied the molecular mechanisms. Male rats received gavage administration of FLC (30 mg/kg/d) for 14 consecutive days with sample collection at the 7th and 14th day; and primary cultured Sertoli cells were treated with 0-10 µM FLC in vitro for 24 h. Our in vivo findings revealed that FLC exposure caused time-dependent testicular injuries, sperm quality decrease as well as adverse changes in BTB integrity, F-actin organization, and expressions of claudin-11 and Arp3. In Sertoli cells isolated from FLC-treated rat testis, Sertoli cell barrier tightness was increased. In Sertoli cells in vitro exposed to FLC, abnormal changes in the barrier permeability were also observed, and the protein expressions of occludin, claudin-11, ZO-1, connexin-43, and Arp3 were significantly decreased in a dose- and time-dependent manner. Furthermore, the FLC-induced adverse changes in Sertoli cell barrier and F-actin were partly alleviated by the induction of Arp3 overexpression. In conclusion, our findings revealed that FLC perturbed BTB/Sertoli cell barrier function through Arp3-mediated F-actin disorganization.


Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Contaminantes Ocupacionales del Aire/toxicidad , Barrera Hematotesticular/efectos de los fármacos , Pirrolidinonas/toxicidad , Reproducción/efectos de los fármacos , Células de Sertoli/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Proteína 3 Relacionada con la Actina/genética , Animales , Barrera Hematotesticular/metabolismo , Barrera Hematotesticular/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Masculino , Permeabilidad , Ratas Sprague-Dawley , Medición de Riesgo , Células de Sertoli/metabolismo , Células de Sertoli/patología , Transducción de Señal/efectos de los fármacos , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Factores de Tiempo
11.
J Cell Biol ; 217(9): 3255-3266, 2018 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-29945904

RESUMEN

Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.


Asunto(s)
Actinas/metabolismo , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Células 3T3 , Proteína 3 Relacionada con la Actina/genética , Animales , Axonema/fisiología , Cuerpos Basales/metabolismo , Línea Celular , Activación Enzimática/fisiología , Regulación de la Expresión Génica/fisiología , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Polimerizacion , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Familia-src Quinasas/metabolismo
12.
Dev Biol ; 428(1): 215-223, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28602951

RESUMEN

Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Genetic studies in Caenorhabditis elegans have identified signaling cascades that activate CED-10/Rac1 GTPase and promote actin cytoskeletal rearrangement during apoptotic cell engulfment. However, the molecular connection between CED-10 activation and actin reorganization remains elusive. Here, we provide evidence that CED-10 binds to the Arp2/3 nucleation promoting factor WASP; CED-10 recruits WASP and Arp2/3 to apoptotic cell corpses in the phagocytes. The loss of WASP and Arp2/3 impaired cell corpse engulfment. Furthermore, we uncover that a WASP-activating factor SEM-5/GRB2 functions in the phagocytes to promote cell corpse clearance. Together, our results suggest CED-10 reorganizes the actin cytoskeleton by recruiting the WASP-Arp2/3 actin nucleation factors during apoptotic cell engulfment.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Apoptosis/fisiología , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Fagocitosis/genética , Proteínas de Unión al GTP rac/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Activación Enzimática/genética , Proteína Adaptadora GRB2/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/genética
13.
Biol Cell ; 109(4): 162-166, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28186323

RESUMEN

Arpin is an Arp2/3 inhibitory protein, which decreases the protrusion lifetime and hence directional persistence in the migration of diverse cells. Arpin is activated by the small GTPase Rac, which controls cell protrusion, thus closing a negative feedback loop that renders the protrusion intrinsically unstable. Because of these properties, it was proposed that Arpin might play a role in directed migration, where directional persistence has to be fine-tuned. We report here, however, that Arpin-depleted tumour cells and Arpin knock-out Dictyostelium amoeba display no obvious defect in chemotaxis. These results do not rule out a potential role of Arpin in other systems, but argue against a general role of Arpin in chemotaxis.


Asunto(s)
Proteínas Portadoras/metabolismo , Quimiotaxis/fisiología , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Animales , Dictyostelium/metabolismo , Humanos
14.
Med Sci Monit ; 23: 695-703, 2017 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-28170369

RESUMEN

BACKGROUND The human LMO2 gene was first cloned from an acute T lymphocytic leukemia patient; it is primarily expressed in hematopoietic and vascular endothelial systems, and functions as a pivotal transcriptional regulator during embryonic hematopoiesis and angiogenesis. However, some recent reports indicated that LMO2 is widely expressed in many tissues and tumors, predominantly in cytoplasm, and revealed complicated functions on tumor behaviors in a variety of cancer types. As an adaptor molecule, binding partners and function details of LMO2 in these solid tumors need to be further investigated. MATERIAL AND METHODS In this study, we used yeast two-hybrid method to screen potential LMO2 interacting partners, MBP-pulldown, and co-immunoprecipitation assay to confirm protein-protein interactions, and confocal microscopy to reveal the subcellular localization of relevant proteins and actin cytoskeleton changes in relevant cells. RESULTS We found that ARP3 and profilin1 were 2 binding partners of LMO2, primarily in cytoplasm. LMO2. Functionally, LMO2 mediated the assembly of a complex including ARP3, profilin1, and actin monomer, increased actin monomer binding to profilin1, and promoted lamellipodia/filopodia formation in basal-type breast cancer cells. CONCLUSIONS Our data indicate a novel functional mechanism of LMO2 in facilitating the delivery of actin monomers to the branched microfilament and increasing lamellipodia/filopodia formation in basal-type breast cancer cells, suggesting a cancer-promoting role of LMO2 in a subtype-dependent manner and its potential as a subtype-specific biomarker for clinical treatment of breast cancers.


Asunto(s)
Proteína 3 Relacionada con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/patología , Proteínas con Dominio LIM/metabolismo , Neoplasias Basocelulares/patología , Profilinas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Seudópodos/metabolismo , Proteína 3 Relacionada con la Actina/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Femenino , Células HEK293 , Humanos , Proteínas con Dominio LIM/genética , Neoplasias Basocelulares/genética , Neoplasias Basocelulares/metabolismo , Profilinas/genética , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Seudópodos/genética , Seudópodos/patología , Transfección , Técnicas del Sistema de Dos Híbridos
15.
Biochim Biophys Acta Mol Cell Res ; 1864(3): 527-545, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27974247

RESUMEN

Throughout spermatogenesis, two important processes occur at late stage VIII of the seminiferous epithelial cycle in the rat testis: preleptotene spermatocytes commence entry into the adluminal compartment and step 19 spermatids release from the seminiferous epithelium. Presently, it is not clear how these processes, which involve extensive restructuring of unique Sertoli-Sertoli and Sertoli-germ cell junctions, are mediated. We aimed to determine whether annexin A2 (ANXA2), a Ca2+-dependent and phospholipid-binding protein, participates in cell junction dynamics. To address this, in vitro and in vivo RNA interference studies were performed on prepubertal Sertoli cells and adult rat testes. The endpoints of Anxa2 knockdown were determined by immunoblotting, morphological analyses, fluorescent immunostaining, and barrier integrity assays. In the testis, ANXA2 localized to the Sertoli cell stalk, with specific staining at the blood-testis barrier and the concave (ventral) surface of elongated spermatids. ANXA2 also bound actin when testis lysates were used for immunoprecipitation. Anxa2 knockdown was found to disrupt the Sertoli cell/blood-testis barrier in vitro and in vivo. The disruption in barrier function was substantiated by changes in the localization of claudin-11, zona occludens-1, N-cadherin, and ß-catenin. Furthermore, Anxa2 knockdown resulted in spermiation defects caused by a dysfunction of tubulobulbar complexes, testis-specific actin-rich ultrastructures that internalize remnant cell junction components prior to spermiation. Additionally, there were changes in the localization of several tubulobulbar complex component proteins, including actin-related protein 3, cortactin, and dynamin I/II. Our results indicate that ANXA2 is critical for the integrity of the blood-testis barrier and the timely release of spermatids.


Asunto(s)
Anexina A2/genética , Barrera Hematotesticular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatogénesis/genética , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Animales , Anexina A2/antagonistas & inhibidores , Anexina A2/metabolismo , Barrera Hematotesticular/crecimiento & desarrollo , Cadherinas/genética , Cadherinas/metabolismo , Claudinas/genética , Claudinas/metabolismo , Cortactina/genética , Cortactina/metabolismo , Dinamina I/genética , Dinamina I/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , Uniones Intercelulares/genética , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Epitelio Seminífero/citología , Epitelio Seminífero/crecimiento & desarrollo , Epitelio Seminífero/metabolismo , Células de Sertoli/citología , Transducción de Señal , Espermátides/crecimiento & desarrollo , Espermátides/ultraestructura , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Espermatocitos/ultraestructura , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
16.
Cell Adh Migr ; 11(5-6): 447-463, 2017 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-27791462

RESUMEN

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.


Asunto(s)
Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Lamina Tipo A/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/genética , Adhesión Celular/fisiología , Células Cultivadas , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Fibroblastos , Humanos , Interleucina-6/metabolismo , Lamina Tipo A/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteoma/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Cicatrización de Heridas/fisiología
17.
J Neurosci ; 36(49): 12393-12411, 2016 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-27927957

RESUMEN

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT: The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization complex Arp2/3 is reduced in dysbindin-deficient cells, thus affecting actin-dependent phenotypes in two cellular compartments where dysbindin resides, endosomes and presynapses. Our studies indicate that a central cellular structure affected by schizophrenia susceptibility loci is the actin cytoskeleton, an organelle necessary for synaptic function in the presynaptic and postsynaptic compartment.


Asunto(s)
Proteína 3 Relacionada con la Actina/genética , Angiopoyetinas/genética , Proteínas Portadoras/genética , Proteínas Asociadas a la Distrofina/genética , Lectinas/genética , Esquizofrenia/genética , Sinapsis , Actinas/genética , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Células Cultivadas , Citoesqueleto/genética , Drosophila melanogaster , Disbindina , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Polimerizacion , Proteoma
18.
Proc Natl Acad Sci U S A ; 113(43): E6610-E6619, 2016 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-27791032

RESUMEN

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."


Asunto(s)
Proteínas de Capping de la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Dictyostelium/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Protozoarias/genética , Seudópodos/metabolismo , Proteínas de Capping de la Actina/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Quimiotaxis/genética , Secuencia Conservada , Dictyostelium/genética , Dictyostelium/ultraestructura , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Ratones , Mutación , Fosforilación , Pinocitosis/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismo , Seudópodos/genética , Seudópodos/ultraestructura , Alineación de Secuencia , Transducción de Señal
19.
Nat Commun ; 7: 12226, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27417392

RESUMEN

Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína 2 Relacionada con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
20.
J Exp Bot ; 67(18): 5325-5337, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27473572

RESUMEN

Gravitropism is vital for shaping directional plant growth in response to the forces of gravity. Signals perceived in the gravity-sensing cells can be converted into biochemical signals and transmitted. Sedimentation of amyloplasts in the columella cells triggers asymmetric auxin redistribution in root tips, leading to downward root growth. The actin cytoskeleton is thought to play an important role in root gravitropism, although the molecular mechanism has not been resolved. DISTORTED1 (DIS1) encodes the ARP3 subunit of the Arabidopsis Actin-Related Protein 2/3 (ARP2/3) complex, and the ARP3/DIS1 mutant dis1-1 showed delayed root curvature after gravity stimulation. Microrheological analysis revealed that the high apparent viscosity within dis1-1 central columella cells is closely associated with abnormal movement trajectories of amyloplasts. Analysis using a sensitive auxin input reporter DII-VENUS showed that asymmetric auxin redistribution was reduced in the root tips of dis1-1, and the actin-disrupting drug Latrunculin B increased the asymmetric auxin redistribution. An uptake assay using the membrane-selective dye FM4-64 indicated that endocytosis was decelerated in dis1-1 root epidermal cells. Treatment and wash-out with Brefeldin A, which inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, showed that cycling of the auxin-transporter PIN-FORMED (PIN) proteins to the plasma membrane was also suppressed in dis1-1 roots. The results reveal that ARP3/DIS1 acts in root gravitropism by affecting amyloplast sedimentation and PIN-mediated polar auxin transport through regulation of PIN protein trafficking.


Asunto(s)
Proteína 3 Relacionada con la Actina/fisiología , Proteínas de Arabidopsis/fisiología , Gravitropismo/fisiología , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/fisiología , Raíces de Plantas/fisiología , Plastidios/fisiología , Proteína 3 Relacionada con la Actina/genética , Actinas/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Gravitropismo/genética , Microscopía Confocal , Plastidios/genética
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