RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited treatment methods. Long non-coding RNAs (lncRNAs) have been found involved in tumorigenic and progression. The present study revealed that LINC01133, a fewly reported lncRNA, was one of 16 hub genes that could predict PDAC patients' prognosis. LINC01133 was over-expressed in PDAC tumors compared to adjacent pancreas and could promote PDAC proliferation and metastasis in vitro and in vivo, as well as inhibit PDAC apoptosis. LINC01133 expression positively correlated to secreted phosphoprotein 1 (SPP1) expression, leading to an enhanced epithelial-mesenchymal transition (EMT) process. LINC01133 bound with actin-related protein 3 (Arp3), the complex reduced SPP1 mRNA degradation which increased SPP1 mRNA level, ultimately leading to PDAC proliferation. This research revealed a novel mechanism of PDAC development and provided a potential prognosis indicator that may benefit PDAC patients.
Asunto(s)
Proteína 3 Relacionada con la Actina , Carcinoma Ductal Pancreático , Proliferación Celular , Transición Epitelial-Mesenquimal , Osteopontina , Neoplasias Pancreáticas , ARN Largo no Codificante , Animales , Femenino , Humanos , Masculino , Ratones , Proteína 3 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Apoptosis , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos BALB C , Ratones Desnudos , Osteopontina/metabolismo , Osteopontina/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Pronóstico , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes, including cell migration and membrane trafficking. Cortactin is important for actin branch stabilization, but the mechanism by which this occurs is unclear. Given this, we determined the structure of vertebrate cortactin-stabilized Arp2/3 actin branches using cryogenic electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site, rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3. It also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter-filament subunits. The preference of cortactin for activated Arp3 explains its retention at the actin branch and accounts for its synergy with other nucleation-promoting factors in regulating branched actin network dynamics.
Asunto(s)
Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Cortactina , Cortactina/metabolismo , Cortactina/química , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/química , Actinas/metabolismo , Actinas/química , Citoesqueleto de Actina/metabolismo , Animales , Microscopía por Crioelectrón , Modelos Moleculares , Humanos , Unión Proteica , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Cortactina , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Cortactina/metabolismo , Actinas/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.
Asunto(s)
Semen , Transducción de Señal , Animales , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Semen/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Espermatogénesis/fisiología , Citoesqueleto de Actina/metabolismo , Barrera Hematotesticular/metabolismo , Mamíferos/metabolismoRESUMEN
The actin 2/3 complex (Arp2/3) regulates actin polymerization and nucleation of actin filaments, is associated with cell motility, and has been shown to play a key role in the invasion and migration of cancer cells. nucleation-promoting factor (NPF) such as N-WASP (neural-WASP famly verprolin-homologous protein family), WAVE (WASP famly verprolin-homologous protein family), and WASH (WASP and Scar homologue) undergo conformational changes upon receipt of multiple upstream signals including Rho family GTPases, cdc42 (Cell division control protein 42 homolog), and phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5 P2) to bind and activate the Arp2/3 complex. Once activated, the Arp2/3 complex forms actin-based membrane protrusions necessary for cancer cells to acquire an invasive phenotype. Therefore, how to influence the invasion and migration of cancer cells by regulating the activity of the Arp2/3 complex has attracted great research interest in recent years. Several studies have explored the effects of phosphorylation modifications of cortactin and several NPFs (Nucleation Promoting Factor) including N-WASP and WAVE on the activity of the Arp2/3 complex and ultimately on cancer cell invasiveness, and have attempted to suggest new strategies for antiinvasive therapy as a result. Other studies have highlighted the potential of targeting genes encoding partial or complete proteins of the Arp2/3 complex as a therapeutic strategy to prevent cancer cell invasion and metastasis. This article reviews the role of the Arp2/3 complex in the development, invasion, and metastasis of different types of cancer and the mechanisms regulating the activity of the Arp2/3 complex.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Neoplasias , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease.
Asunto(s)
Actinas , Familia de Proteínas del Síndrome de Wiskott-Aldrich , Animales , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
BACKGROUND: Hereditary actin-related protein 2/3 complex subunit 1B deficiency is characterized clinically by ear, skin, and lung infections, bleeding, eczema, food allergy, asthma, skin vasculitis, colitis, arthritis, short stature, and lymphadenopathy. OBJECTIVE: We aimed to describe the clinical, laboratory, and genetic features of six patients from four Mexican families. METHODS: We performed exome sequencing in patients of four families with suspected actinopathy, collected their data from medical records, and reviewed the literature for reports of other patients with actin-related protein 2/3 complex subunit 1B deficiency. RESULTS: Six patients from four families were included. All had recurrent infections, mainly bacterial pneumonia, and cellulitis. A total of 67% had eczema whereas 50% had food allergies, failure to thrive, hepatomegaly, and bleeding. Eosinophilia was found in all; 84% had thrombocytopenia, 67% had abnormal-size platelets and anemia. Serum levels of IgG, IgA, and IgE were highly increased in most; IgM was normal or low. T cells were decreased in 67% of patients, whereas B and NK cells were increased in half of patients. Two of the four probands had compound heterozygous variants. One patient was successfully transplanted. We identified 28 other patients whose most prevalent features were eczema, recurrent infections, failure to thrive, bleeding, diarrhea, allergies, vasculitis, eosinophilia, platelet abnormalities, high IgE/IgA, low T cells, and high B cells. CONCLUSION: Actin-related protein 2/3 complex subunit 1B deficiency has a variable and heterogeneous clinical spectrum, expanded by these cases to include keloid scars and Epstein-Barr virus chronic hepatitis. A novel deletion in exon 8 was shared by three unrelated families and might be the result of a founder effect.
Asunto(s)
Eccema , Eosinofilia , Infecciones por Virus de Epstein-Barr , Vasculitis , Humanos , Proteína 2 Relacionada con la Actina , Actinas , Insuficiencia de Crecimiento , Herpesvirus Humano 4 , Inmunoglobulina A , Inmunoglobulina E , Reinfección , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
Spermatogenesis is a complicated process that includes spermatogonia differentiation, spermatocytes meiosis, spermatids spermiogenesis and final release of spermatozoa. Actin-related protein 3 (Arp3) and epidermal growth factor receptor pathway substrate 8 (Eps8) are two actin binding proteins that regulate cell adhesion in seminiferous tubules during mammalian spermatogenesis. However, the functions of these two proteins during spermatogenesis in nonmammalian species, especially Crustacea, are still unknown. Here, we cloned es-Arp3 and es-Eps8 from the testis of Chinese mitten crab Eriocheir sinensis. es-Arp3 and es-Eps8 were located in spermatocytes, spermatids and spermatozoa. Knockdown of es-Arp3 and es-Eps8 in vivo caused morphological changes to seminiferous tubules including delayed spermatozoa release, shedding of germ cells and vacuoles. Filamentous-actin (F-actin) filaments network was disorganized due to deficiency of es-Arp3 and es-Eps8. Accompanying this, four junctional proteins (α-catenin, ß-catenin, pinin and ZO1) displayed abnormal expression levels as well as penetrating biotin signals in seminiferous tubules. We also used the Arp2/3 complex inhibitor CK666 to block es-Arp3 activity and supported es-Arp3 knockdown results. In summary, our study demonstrated for the first time that es-Arp3 and es-Eps8 are important for spermatogenesis via regulating microfilament-mediated cell adhesion in Eriocheir sinensis.
Asunto(s)
Barrera Hematotesticular , Espermatogénesis , Animales , Masculino , Proteína 3 Relacionada con la Actina/metabolismo , Barrera Hematotesticular/metabolismo , Espermatogénesis/fisiología , Testículo , Espermátides , Túbulos Seminíferos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Mamíferos/metabolismoRESUMEN
Obesity is a major health crisis in the modern society. Studies have shown that the consumption of a high-fat diet (HFD) induces hypothalamic inflammation and leptin resistance, which consequently favours body mass gain. Actin related protein 2/3 complex subunit 1 (ARPC1B), an actin-binding protein, is highly expressed in immune cells. Recent studies have shown that ARPC1B has a certain anti-inflammatory effect. While ARPC1B expression is decreased in the hypothalamus of mice fed a HFD, the role of ARPC1B in HFD-induced obesity remains unclear. Thus, we investigated whether ARPC1B up-regulation in the hypothalamic arcuate nucleus (ARC) could inhibit the development of obesity. Herein, ARPC1B overexpression lentiviral particles were stereotaxically injected into the ARC of male C57BL/6J mice (7 weeks old) fed with HFD. Overexpression of ARPC1B in the hypothalamic ARC attenuated HFD-induced ARC inflammation, reduced body-weight gain and feed efficiency. Furthermore, up-regulation of ARC ARPC1B improved the glucose tolerance and reduced subcutaneous/epididymal fat mass accumulation, which decreased the serum total cholesterol, serum triglyceride and leptin levels. In addition, upon ARPC1B overexpression in the hypothalamic ARC, intraperitoneal injection of leptin increased the phosphorylation level of signal transducer and activator of transcription 3 (STAT3), an important transcription factor for leptin's action, in the ARC of obese mice. Accordingly, we suggest that up-regulation of ARPC1B in the hypothalamic ARC may improve the HFD-induced hypothalamic inflammation and leptin resistance. Our findings demonstrate that ARPC1B is a promising target for the treatment of diet-induced obesity.
Asunto(s)
Dieta Alta en Grasa , Leptina , Animales , Masculino , Ratones , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/farmacología , Proteína 3 Relacionada con la Actina/metabolismo , Núcleo Arqueado del Hipotálamo , Hipotálamo/metabolismo , Inflamación/metabolismo , Leptina/genética , Leptina/metabolismo , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Regulación hacia ArribaRESUMEN
Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Arabidopsis , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Proteína 2 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.
Asunto(s)
Actomiosina , Condensados Biomoleculares , Caenorhabditis elegans , Oocitos , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animales , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Emulsiones/química , Emulsiones/metabolismo , Oocitos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Mouse embryonic stem cells (mESCs), a model for differentiation into primed epiblast-like cells (EpiLCs), have revealed transcriptional and epigenetic control of early embryonic development. The control and significance of morphological changes, however, remain less defined. We show marked changes in morphology and actin architectures during differentiation that depend on Arp2/3 complex but not formin activity. Inhibiting Arp2/3 complex activity pharmacologically or genetically does not block exit from naive pluripotency, but attenuates increases in EpiLC markers. We find that inhibiting Arp2/3 complex activity delays formative pluripotency and causes globally defective lineage specification as indicated by RNA sequencing, with significant effects on TBX3-depedendent transcriptional programs. We also identify two previously unreported indicators of mESC differentiation, namely, MRTF and FHL2, which have inverse Arp2/3 complex-dependent nuclear translocation. Our findings on Arp2/3 complex activity in differentiation and the established role of formins in EMT indicate that these two actin nucleators regulate distinct modes of epithelial plasticity.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Animales , Diferenciación Celular/genética , Linaje de la Célula , Estratos Germinativos , Ratones , Células Madre Embrionarias de Ratones , Células Madre PluripotentesRESUMEN
When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes.
Asunto(s)
Actinas/fisiología , Leucocitos/fisiología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Animales , Fenómenos Biomecánicos/fisiología , Línea Celular , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica/fisiología , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
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/metabolismoRESUMEN
The blood-brain barrier (BBB) is critical to maintaining central nervous system (CNS) homeostasis. However, the effects of microgravity (MG) on the BBB remain unclear. This study aimed to investigate the influence of simulated MG (SMG) on the BBB and explore its potential mechanism using a proteomic approach. Rats were tail-suspended to simulate MG for 21 days. SMG could disrupt the BBB, including increased oxidative stress levels, proinflammatory cytokine levels, and permeability, damaged BBB ultrastructure, and downregulated tight junctions (TJs) and adherens junctions (AJs) protein expression in the rat brain. A total of 554 differentially expressed proteins (DEPs) induced by SMG were determined based on the label-free quantitative proteomic strategy. The bioinformatics analysis suggested that DEPs were mainly enriched in regulating the cell-cell junction and cell-extracellular matrix biological pathways. The inhibited Ras-related C3 botulinum toxin substrate 1 (Rac1)/Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (Wave2)/actin-related protein 3 (Arp3) pathway and the decreased ratio of filamentous actin (F-actin) to globular actin contributed to BBB dysfunction induced by SMG. In the human brain microvascular endothelial cell (HBMECs), SMG increased the oxidative stress levels and proinflammatory cytokine levels, promoted apoptosis, and arrested the cell cycle phase. Expression of TJs and AJs proteins were downregulated and the distribution of F-actin was altered in SMG-treated HBMECs. The key role of the Rac1/Wave2/Arp3 pathway in BBB dysfunction was confirmed in HBMECs with a specific Rac1 agonist. This study demonstrated that SMG induced BBB dysfunction and revealed that Rac1/Wave2/Arp3 could be a potential signaling pathway responsible for BBB disruption under SMG. These results might shed a novel light on maintaining astronaut CNS homeostasis during space travel.
Asunto(s)
Proteína 3 Relacionada con la Actina/metabolismo , Barrera Hematoencefálica/patología , Regulación de la Expresión Génica , Proteoma/metabolismo , Simulación de Ingravidez/efectos adversos , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina , Animales , Barrera Hematoencefálica/metabolismo , Masculino , Proteoma/análisis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Uniones EstrechasRESUMEN
The trafficking of proteins such as aquaporin-2 (AQP2) in the exocytotic pathway requires an active actin cytoskeleton network, but the mechanism is incompletely understood. Here, we show that the actin-related protein (Arp)2/3 complex, a key factor in actin filament branching and polymerization, is involved in the shuttling of AQP2 between the trans-Golgi network (TGN) and the plasma membrane. Arp2/3 inhibition (using CK-666) or siRNA knockdown blocks vasopressin-induced AQP2 membrane accumulation and induces the formation of distinct AQP2 perinuclear patches positive for markers of TGN-derived clathrin-coated vesicles. After a 20°C cold block, AQP2 formed perinuclear patches due to continuous endocytosis coupled with inhibition of exit from TGN-associated vesicles. Upon rewarming, AQP2 normally leaves the TGN and redistributes into the cytoplasm, entering the exocytotic pathway. Inhibition of Arp2/3 blocked this process and trapped AQP2 in clathrin-positive vesicles. Taken together, these results suggest that Arp2/3 is essential for AQP2 trafficking, specifically for its delivery into the post-TGN exocytotic pathway to the plasma membrane.NEW & NOTEWORTHY Aquaporin-2 (AQP2) undergoes constitutive recycling between the cytoplasm and plasma membrane, with an intricate balance between endocytosis and exocytosis. By inhibiting the actin-related protein (Arp)2/3 complex, we prevented AQP2 from entering the exocytotic pathway at the post-trans-Golgi network level and blocked AQP2 membrane accumulation. Arp2/3 inhibition, therefore, enables us to separate and target the exocytotic process, while not affecting endocytosis, thus allowing us to envisage strategies to modulate AQP2 trafficking and treat water balance disorders.
Asunto(s)
Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Acuaporina 2/metabolismo , Exocitosis/fisiología , Riñón/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Membrana Celular/metabolismo , Endocitosis/fisiología , Células LLC-PK1 , Fosforilación , Transporte de Proteínas/fisiología , Ratas , PorcinosRESUMEN
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.
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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 , SeudópodosRESUMEN
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/genéticaRESUMEN
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.