RESUMEN
The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors regulate the stability of filaments generated by the Arp2/3 complex to maintain the dynamics and architecture of actin networks. In this review, we summarise recent studies on the molecular mechanisms governing the tuning of Arp2/3 complex nucleated actin filaments, which includes investigations using microfluidics and single-molecule imaging to reveal the mechanosensitivity, dissociation and regeneration of actin branches. We also discuss the high-resolution cryo-EM structure of cortactin bound to actin branches, as well as the differences and similarities between the stability of Arp2/3 complex nucleated branches and linear filaments. These new studies provide a clearer picture of the stabilisation of Arp2/3 nucleated filaments at the molecular level. We also identified gaps in our understanding of how different factors collectively contribute to the stabilisation of Arp2/3 complex-generated actin networks.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismoRESUMEN
Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Members of the Wiskott-Aldrich Syndrome protein (WASp) family activate Arp2/3 complex (actin-related proteins 2 and 3 complex) to form actin filament branches. The proline-rich domain (PRD) of WASp contributes to branching nucleation, and the PRD of budding yeast Las17 binds actin filaments [Urbanek AN et al. (2013) Curr Biol 23, 196-203]. Biochemical assays showed the recombinant PRD of fission yeast Schizosaccharomyces pombe Wsp1p binds actin filaments with micromolar affinity. Recombinant PRDs of both Wsp1p and Las17p slowed the elongation of actin filaments by Mg-ATP-actin monomers by half and slowed the spontaneous polymerization of Mg-ATP-actin monomers modestly. The affinity of PRDs of WASp-family proteins for actin filaments is high enough to contribute to the reported stimulation of actin filament branching by Arp2/3 complex.
Asunto(s)
Actinas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Polimerizacion , Prolina/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/análisis , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Renal transplantation is the preferred treatment of end stage renal disease, but allograft survival is limited by the development of interstitial fibrosis and tubular atrophy in response to various stimuli. Much effort has been put into identifying new protein markers of fibrosis to support the diagnosis. In the present work, we performed an in-depth quantitative proteomics analysis of allograft biopsies from 31 prevalent renal transplant patients and correlated the quantified proteins with the volume fraction of fibrosis as determined by a morphometric method. Linear regression analysis identified four proteins that were highly associated with the degree of interstitial fibrosis, namely Coagulation Factor XIII A chain (estimate 18.7, adjusted p < 0.03), Uridine Phosphorylase 1 (estimate 19.4, adjusted p < 0.001), Actin-related protein 2/3 subunit 2 (estimate 34.2, adjusted p < 0.05) and Cytochrome C Oxidase Assembly Factor 6 homolog (estimate -44.9, adjusted p < 0.002), even after multiple testing. Proteins that were negatively associated with fibrosis (p < 0.005) were primarily related to normal metabolic processes and respiration, whereas proteins that were positively associated with fibrosis (p < 0.005) were involved in catabolic processes, cytoskeleton organization and the immune response. The identified proteins may be candidates for further validation with regards to renal fibrosis. The results support the notion that cytoskeleton organization and immune responses are prevalent processes in renal allograft fibrosis.
Asunto(s)
Aloinjertos/patología , Trasplante de Riñón , Riñón/patología , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Adulto , Anciano , Biomarcadores/análisis , Factor XIII/análisis , Femenino , Fibrosis , Humanos , Enfermedades Renales/patología , Masculino , Persona de Mediana Edad , Proteómica , Uridina Fosforilasa/análisisRESUMEN
CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Citotoxicidad Inmunológica , Linfocitos T Citotóxicos/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Actinas/análisis , Antígenos CD8/análisis , Polaridad Celular , Transportador de Glucosa de Tipo 1/análisis , Células HEK293 , Humanos , Sinapsis Inmunológicas/fisiología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/análisisRESUMEN
Objectives We analyze the relationship between CD44, epidermal growth factor receptor (EGFR), and p16 expression in oral cavity and oropharyngeal cancers in a diverse population. We also describe whether particular patterns of staining are associated with progression-free survival and overall survival. Study Design Prospective study, single-blind to pathologist and laboratory technologist. Setting Hospital based. Subjects and Methods Immunohistochemistry, comprising gross staining and cellular expression, was performed and interpreted in a blinded fashion on 24 lip/oral cavity and 40 oropharyngeal cancer specimens collected between 2007 and 2012 from participants of a larger study. Information on overall survival and progression-free survival was obtained from medical records. Results Nineteen cases were clinically p16 positive, 16 of which were oropharyngeal. Oral cavity lesions were more likely to exhibit strong CD44 membrane staining ( P = .0002). Strong CD44 membrane and strong EGFR membrane and/or cytoplasmic staining were more common in p16-negative cancers ( P = .006). Peripheral/mixed gross p16 staining pattern was associated with worse survival than the universal staining on univariate and multivariate analyses ( P = .006, P = .030). This held true when combining gross and cellular localization for p16. For CD44, universal gross staining demonstrated poorer overall survival compared with the peripheral/mixed group ( P = .039). CD44 peripheral/mixed group alone and when combined with universal p16 demonstrated the best survival on multivariate analysis ( P = .010). Conclusion In a diverse population, systematic analysis applying p16, CD44, and EGFR gross staining and cellular localization on immunohistochemistry demonstrates distinct patterns that may have prognostic potential exceeding current methods. Larger studies are warranted to investigate these findings further.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/análisis , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/química , Receptores ErbB/análisis , Receptores de Hialuranos/análisis , Boca/química , Neoplasias Orofaríngeas/química , Anciano , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Boca/patología , Proteínas de Neoplasias/análisis , Neoplasias Orofaríngeas/mortalidad , Neoplasias Orofaríngeas/patología , Pronóstico , Estudios Prospectivos , Método Simple Ciego , Tasa de SupervivenciaRESUMEN
The small heat shock protein HSPB8 and its co-chaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi- and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far under-appreciated connection between selective autophagy and cellular morphodynamics that guide cell division.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Citocinesis , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/análisis , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Reguladoras de la Apoptosis/análisis , División Celular , Técnicas de Silenciamiento del Gen , Células HeLa , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Glaucoma targets a variety of different tissues located in both anterior (e.g., trabecular meshwork) and posterior (e.g., optic nerve head) ocular segments. The transmission of damage between these structures cannot be simply ascribed to intraocular pressure increase. Recent experimental findings provide evidence for the involvement of molecular mediators including proteins and microRNAs. Aqueous humor protein composition is characteristically altered during glaucoma progression. Immunohistochemistry analyses indicate that proteins characterizing glaucomatous aqueous humor are released by damaged trabecular meshwork. This feature incudes (a) Nestin, involved in stem cell recruitment and glial cell activation; (b) A Kinase anchor protein, released as consequence of mitochondrial damage and Rho activation establishing cell shape and motility; (c) Actin related protein 2/3 complex, involved in actin polymerization and cell shape maintenance. As established both in vitro and in glaucomatous aqueous humor, trabecular meshwork cells damaged by oxidative stress release extracellular microRNAs inducing glial cell activation, an established pathogenic mechanism in neurodegenerative diseases. Released microRNAs include miR-21 (apoptosis), miR-450 (cell aging, maintenance of contractile tone), miR-107 (Nestin expression, apoptosis), miR-149 (endothelia and extracellular matrix homeostasis). Experimental evidences indicate that the uveoscleral pathway, via suprachoroidal space, can provide a potential route of access from the anterior region to the posterior segment of the eye and could represent the path followed by biologic mediators to reach the inner layer of the peripapillary retina and transmit damage signals from the anterior to posterior segment during glaucoma course.
Asunto(s)
Segmento Anterior del Ojo/patología , Glaucoma/patología , MicroARNs/análisis , Segmento Posterior del Ojo/patología , Proteínas de Anclaje a la Quinasa A/análisis , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Animales , Humor Acuoso/química , Humanos , Proteínas de la Membrana/análisis , Modelos Moleculares , Nestina/análisisRESUMEN
Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.
Asunto(s)
Actinas/metabolismo , Interacciones Huésped-Parásitos , Plasmodium berghei/fisiología , Multimerización de Proteína , Toxoplasma/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Animales , Línea Celular , Citoplasma/química , HumanosRESUMEN
During HIV-1 infection, distinct biological phenotypes are observed between R5 and X4 HIV-1 strains with respect to pathogenicity and tropism. In this study, temporal changes of the expression levels of the complete human transcriptome, representing 47,000 well-characterized human transcripts, were monitored in the first 24 h during HIV-1 R5 and X4 exposition in resting primary CD4(+) T cells. We provide evidence that R5 viruses modulate, to a greater extent than X4 viruses, the level of mRNA of the resting CD4(+) T cells. Indeed, modulation of the TCR signaling and the actin organization involving the WAVE/ABI complex and the ARP2/3 complex appeared to be associated with R5 exposition. The data suggest that the ability of R5 viruses to modulate TCR-mediated actin polymerization and signaling creates a favorable environment for CD4(+) T cell activation after TCR stimulation and may partly explain why R5 is the primary strain observed early in the natural infection process.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Perfilación de la Expresión Génica , VIH-1/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Actinas/análisis , Complejo CD3/análisis , Linfocitos T CD4-Positivos/química , Células Cultivadas , Humanos , Quinasa de Cadena Ligera de Miosina/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Familia de Proteínas del Síndrome de Wiskott-Aldrich/análisisRESUMEN
OBJECTIVE: To study the modulation of Jianjining Recipe (JJNR), a traditional Chinese compound herbal medicine for invigorating spleen and kidney on differential protein expression in spleen of rats with experimental autoimmune myasthenia gravis (EAMG). METHODS: EAMG rats were randomly divided into four groups: untreated group, JJNR-treated group, Qiangji Jianli capsule (QJJLC, a traditional Chinese compound herbal medicine)-treated group and prednisolone acetate (PA)-treated group. After therapeutic intervention with the above drugs for four consecutive weeks, the level of differential protein expression was analyzed by two-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Twelve differential proteins were identified by comparing EAMG rats and normal rats. The levels of allograft inflammatory factor-1, peroxiredoxin I and actin-related protein 2/3 complex subunit 5 were significantly regulated by JJNR (P<0.01). These proteins were closely associated with immune response and cell movement. CONCLUSION: The results suggest that there are differential protein expressions between EAMG rats and normal rats. Furthermore, as a Chinese medicine prescription with effect of invigorating spleen and kidney, JJNR can effectively regulate the levels of some EAMG-related protein expression.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Medicamentos Herbarios Chinos/uso terapéutico , Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Peroxirredoxinas/análisis , Complejo 2-3 Proteico Relacionado con la Actina/análisis , Animales , Medicamentos Herbarios Chinos/farmacología , Electroforesis en Gel Bidimensional , Masculino , Proteínas de Microfilamentos , Miastenia Gravis Autoinmune Experimental/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas LewRESUMEN
The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.