RESUMO
Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.
RESUMO
During metastasis, tumor cells invade through the basement membrane and intravasate into blood vessels and then extravasate into distant organs to establish metastases. Here, we report a critical role of a transmembrane serine protease fibroblast activation protein (FAP) in tumor metastasis. Expression of FAP and TWIST1, a metastasis driver, is significantly correlated in several types of human carcinomas, and FAP is required for TWIST1-induced breast cancer metastasis to the lung. Mechanistically, FAP is localized at invadopodia and required for invadopodia-mediated extracellular matrix degradation independent of its proteolytic activity. Live cell imaging shows that association of invadopodia precursors with FAP at the cell membrane promotes the stabilization and growth of invadopodia precursors into mature invadopodia. Together, our study identified FAP as a functional target of TWIST1 in driving tumor metastasis via promoting invadopodia-mediated matrix degradation and uncovered a proteolytic activity-independent role of FAP in stabilizing invadopodia precursors for maturation.
Assuntos
Neoplasias da Mama , Podossomos , Humanos , Feminino , Podossomos/metabolismo , Linhagem Celular Tumoral , Peptídeo Hidrolases/metabolismo , Invasividade Neoplásica/patologia , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Fibroblastos/metabolismo , Matriz Extracelular/metabolismo , Melanoma Maligno CutâneoRESUMO
Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.
Assuntos
Actinas , Neutrófilos , Grânulos Citoplasmáticos , Exocitose , Gelatinases , Humanos , Inflamação , Proteínas dos MicrofilamentosRESUMO
Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (â¼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci â¼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.
Assuntos
Citoesqueleto de Actina , Actinas , Membrana Eritrocítica , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Espectrina/metabolismoRESUMO
The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns-/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies.Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein.
Assuntos
Autofagia Mediada por Chaperonas , Cistinose , Dineínas do Citoplasma , Proteína 2 de Membrana Associada ao Lisossomo , Autofagia , Cistina/metabolismo , Cistinose/genética , Cistinose/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Homeostase , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismoRESUMO
Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.
Assuntos
Armadilhas Extracelulares/metabolismo , Macroautofagia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , NADPH Oxidases/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Calmodulina/metabolismo , Modelos Animais de Doenças , Espaço Intracelular/metabolismo , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/deficiência , Ativação de Neutrófilo , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Salmonella typhimurium/fisiologiaRESUMO
Neutrophils are short-lived cells after isolation. The analysis of neutrophil vesicular trafficking requires rapid and gentle handling. Recently developed super-resolution microscopy technologies have generated unparalleled opportunities to help understand the molecular mechanisms regulating neutrophil vesicular trafficking, exocytosis, and associated functions at the molecular level. Here, we describe super-resolution and total internal reflection fluorescence (TIRF) microscopy approaches for the analysis of vesicular trafficking and associated functions of primary neutrophils.
Assuntos
Exocitose/genética , Microscopia de Fluorescência/métodos , Neutrófilos/ultraestrutura , Cultura Primária de Células/métodos , Movimento Celular/genética , Humanos , Neutrófilos/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1ß release. In contrast, we report that although N-GSDMD is required for IL-1ß secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1ß via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.
Assuntos
Membrana Celular/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neutrófilos/metabolismo , Organelas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/genética , Caspase 1/metabolismo , Permeabilidade da Membrana Celular/genética , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Transporte Proteico , Piroptose/genéticaRESUMO
Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolution was an extensive loss of nonessential genes from the protomitochondrial endosymbiont genome and transfer of some of the essential endosymbiont genes to the host nucleus. We have developed an approach to recapitulate various aspects of endosymbiont genome minimization using a synthetic system consisting of Escherichia coli endosymbionts within host yeast cells. As a first step, we identified a number of E. coli auxotrophs of central metabolites that can form viable endosymbionts within yeast cells. These studies provide a platform to identify nonessential biosynthetic pathways that can be deleted in the E. coli endosymbionts to investigate the evolutionary adaptations in the host and endosymbiont during the evolution of mitochondria.
Assuntos
Escherichia coli/metabolismo , Saccharomyces cerevisiae/metabolismo , Simbiose , Escherichia coli/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Simbiose/genéticaRESUMO
Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.
Assuntos
Quimiotaxia/imunologia , Guanosina Trifosfato/imunologia , Proteínas de Membrana/imunologia , Neuropeptídeos/imunologia , Neutrófilos/imunologia , Pseudópodes/imunologia , Proteínas de Transporte Vesicular/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Quimiotaxia/genética , Guanosina Trifosfato/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neutrófilos/patologia , Pseudópodes/genética , Pseudópodes/patologia , Proteínas de Transporte Vesicular/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.
Assuntos
Bioengenharia/métodos , Mitocôndrias/genética , Simbiose/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Evolução Biológica , Escherichia coli/genética , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tiamina/metabolismoRESUMO
The molecular mechanisms that regulate late endosomal maturation and function are not completely elucidated, and direct evidence of a calcium sensor is lacking. Here we identify a novel mechanism of late endosomal maturation that involves a new molecular interaction between the tethering factor Munc13-4, syntaxin 7, and VAMP8. Munc13-4 binding to syntaxin 7 was significantly increased by calcium. Colocalization of Munc13-4 and syntaxin 7 at late endosomes was demonstrated by high-resolution and live-cell microscopy. Munc13-4-deficient cells show increased numbers of significantly enlarged late endosomes, a phenotype that was mimicked by the fusion inhibitor chloroquine in wild-type cells and rescued by expression of Munc13-4 but not by a syntaxin 7-binding-deficient mutant. Late endosomes from Munc13-4-KO neutrophils show decreased degradative capacity. Munc13-4-knockout neutrophils show impaired endosomal-initiated, TLR9-dependent signaling and deficient TLR9-specific CD11b up-regulation. Thus we present a novel mechanism of late endosomal maturation and propose that Munc13-4 regulates the late endocytic machinery and late endosomal-associated innate immune cellular functions.
Assuntos
Endossomos/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Qa-SNARE/metabolismo , Receptor Toll-Like 9/fisiologia , Animais , Antígeno CD11b/metabolismo , Cálcio/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas R-SNARE/metabolismo , Transdução de SinaisRESUMO
The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.
Assuntos
Endocitose , Endossomos/metabolismo , Exocitose , Proteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Livres de Patógenos Específicos , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns(-/-) mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns(-/-) mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Autofagia , Cistinose/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Cistina/metabolismo , Cistinose/genética , Cistinose/fisiopatologia , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genéticaRESUMO
Increased expression of adenylyl cyclase VI has beneficial effects on the heart, but strategies that increase cAMP production in cardiac myocytes usually are harmful. Might adenylyl cyclase VI have beneficial effects unrelated to increased beta-adrenergic receptor-mediated signaling? We previously reported that adenylyl cyclase VI reduces cardiac phospholamban expression. Our focus in the current studies is how adenylyl cyclase VI influences phospholamban phosphorylation. In cultured cardiac myocytes, increased expression of adenylyl cyclase VI activates Akt by phosphorylation at serine 473 and threonine 308 and is associated with increased nuclear phospho-Akt. Activated Akt phosphorylates phospholamban, a process that does not require beta-adrenergic receptor stimulation or protein kinase A activation. These previously unrecognized signaling events would be predicted to promote calcium handling and increase contractile function of the intact heart independently of beta-adrenergic receptor activation. We speculate that phospholamban phosphorylation, through activation of Akt, may be an important mechanism by which adenylyl cyclase VI increases the function of the failing heart.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Regulação Enzimológica da Expressão Gênica , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenilil Ciclases/genética , Animais , Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/enzimologia , Núcleo Celular/genética , Células Cultivadas , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Camundongos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismoRESUMO
Chemotactic responsiveness is crucial to neutrophil recruitment to sites of infection. During chemotaxis, highly divergent cytoskeletal programs are executed at the leading and trailing edge of motile neutrophils. The Rho family of small GTPases plays a critical role in cell migration, and recent work has focused on elucidating the specific roles played by Rac1, Rac2, Cdc42, and Rho during cellular chemotaxis. Rac GTPases regulate actin polymerization and extension of the leading edge, whereas Rho GTPases control myosin-based contraction of the trailing edge. Rac and Rho signaling are thought to crosstalk with one another, and previous research has focused on mutual inhibition of Rac and Rho signaling during chemotaxis. Indeed, polarization of neutrophils has been proposed to involve the activity of a negative feedback system where Rac activation at the front of the cell inhibits local Rho activation, and vice versa. Using primary human neutrophils and neutrophils derived from a Rac1/Rac2-null transgenic mouse model, we demonstrate here that Rac1 (and not Rac2) is essential for Rho and myosin activation at the trailing edge to regulate uropod function. We conclude that Rac plays both positive and negative roles in the organization of the Rhomyosin "backness" program, thereby promoting stable polarity in chemotaxing neutrophils.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Miosinas/metabolismo , Neuropeptídeos/metabolismo , Neutrófilos/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Polaridade Celular , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Cadeias Leves de Miosina/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução Genética , Proteínas rac de Ligação ao GTP/deficiência , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Using a purpose-designed experimental model, we have defined new, statistically significant, differences in gene expression between heavily and weakly metastatic human breast cancer cell populations, in vivo and in vitro. The differences increased under selection pressures designed to increase metastatic proficiency. Conversely, the expression signatures of primary tumors generated by more aggressive variants, and their matched metastases in the lungs and lymph nodes, all tended to converge. However, the few persisting differences among these selectively enriched malignant growths in the breast, lungs, and lymph nodes were highly statistically significant, implying potential mechanistic involvement of the corresponding genes. The evidence that has emerged from the current work indicates that selective enhancement of metastatic proficiency by serial transplantation co-purifies a subliminal gene expression pattern within the tumor cell population. This signature most likely includes genes participating in metastasis pathogenesis, and we document manageable numbers of candidates for this role. The findings also suggest that metastasis to at least two different organs occurs through closely similar genetic mechanisms.
Assuntos
Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Metástase Linfática/genética , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transplante HeterólogoRESUMO
We investigated alpha1-antichymotrypsin (ACT) gene expression in xenograft tumors generated by two isogenic human breast cancer cell lines derived from the same parent, MDA-MB-435, which display opposite metastatic behaviors. Microarray and real-time PCR experiments showed an overexpression of this serine protease inhibitor in the metastatic tumors (M-4A4T) and its derived metastases (M4-Mets) compared with the weakly metastatic tumors (NM-2C5T), and its release into the blood was confirmed by western-blotting. However, functional assays in vivo using genetically engineered tumor cells demonstrated that ACT up-regulation was not, by itself, responsible for the metastatic phenotype. We also made observations that ACT gene regulation was sensitive to tumor-host interactions: inoculation of these lines into the mouse mammary gland greatly increased ACT production and accentuated the intrinsic difference observed when they are cultured in vitro. Sensitivity of tumor cells to their environment was further analyzed by in vitro experiments, which demonstrated that a purified ECM environment and soluble components from normal host mammary cells were both able to significantly promote ACT expression. In addition, we took advantage of the xenogeneic nature of the model to measure ACT expression by the host cells (mouse) and the tumor cells (human) within the neoplasm using species-specific primers in real-time PCR experiments. It was found that the presence of tumor cells, irrespective of their metastatic capabilities, induced local ACT production by host cells at the primary and secondary tumor sites. Thus, this work indicates that there is a specific association of ACT overexpression with the metastatic phenotype in our breast cancer metastasis model. Moreover, because of the xenogeneic nature of our system, we were able to provide evidence of tumor-host reciprocal regulation of ACT production.
Assuntos
Neoplasias da Mama/patologia , Transplante Heterólogo/fisiologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Serpinas/genéticaRESUMO
A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Substâncias de Crescimento/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Citoesqueleto/metabolismo , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Humanos , Immunoblotting , Células Jurkat , Peptídeos/química , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Tempo , Células Tumorais Cultivadas , Tirosina/metabolismo , Quinases Ativadas por p21 , Domínios de Homologia de srcRESUMO
The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.