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1.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

2.
ACS Chem Biol ; 15(6): 1650-1661, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32315152

RESUMO

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.

3.
J Anim Ecol ; 89(7): 1735-1746, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32227334

RESUMO

Rate of colour change and background matching capacity are important functional traits for avoiding predation and hiding from prey. Acute changes in environmental temperature are known to impact the rate at which animals change colour, and therefore may affect their survival. Many ectotherms have the ability to acclimate performance traits such as locomotion, metabolic rate and growth rate with changes in seasonal temperature. However, it remains unclear how other functional traits that are directly linked to behaviour and survival respond to long-term changes in temperature (within an individual's lifetime). We assessed whether the rate of colour change is altered by long-term changes in temperature (seasonal variation) and if rate of colour change can acclimate to seasonal thermal conditions. We used an intertidal rock-pool goby Bathygobius cocosensis, to test this and exposed individuals to representative seasonal mean temperatures (16 or 31°C, herein referred to cold- and warm-exposed fish respectively) for 9 weeks and then tested their rate of luminance change when placed on white and black backgrounds at acute test temperatures 16 and 31°C. We modelled rate of luminance change using the visual sensitives of a coral trout Plectropmus leopardus to determine how well gobies matched their backgrounds in terms of luminance contrast to a potential predator. After exposure to long-term seasonal conditions, the warm-exposed fish had faster rates of luminance change and matched their background more closely when tested at 31 than at 16°C. Similarly, the cold-exposed fish had faster rates of luminance change and matched their backgrounds more closely at 16°C than at 31°C. This demonstrates that rate of luminance change can be adjusted to compensate for long-term changes in seasonal temperature. This is the first study to show that animals can acclimate rate of colour change for background matching to seasonal thermal conditions. We also show that rapid changes in acute temperature reduce background matching capabilities. Stochastic changes in climate are likely to affect the frequency of predator-prey interactions which may have substantial knock-on effects throughout ecosystems.

4.
Fungal Genet Biol ; 138: 103365, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32145317

RESUMO

Green fluorescent protein (GFP) and its counterparts are modern molecular biology research tools indispensable in many experimental systems. Within fungi, researchers studying Saccharomyces cerevisiae and other model ascomycetes have access to a wide variety of fluorescent proteins. Unfortunately, many of these tools have not crossed the phylum divide into the Basidiomycota, where only GFP S65T, Venus, Ds-Red, and mCherry are currently available. To address this, we searched the literature for potential candidates to be expressed in the human fungal pathogen Cryptococcus neoformans and identified a suite of eight more modern fluorescent proteins that span the visible spectrum. A single copy of each fluorophore was heterologously expressed in Safe Haven 1 and their fluorescence intensities compared in this encapsulated yeast. mTurquoise2, mTFP1, Clover, mNeonGreen, mRuby3, and Citrine were highly visible under the microscope, whereas Superfolder GFP and mMaroon1 were not. Expressed fluorophores did not impact growth or virulence as demonstrated by an in vitro spotting assay and murine inhalation model, respectively.

5.
ACS Chem Biol ; 14(9): 2071-2087, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390185

RESUMO

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.


Assuntos
Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Cíclicos/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Sequência de Aminoácidos , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/toxicidade , Sequências Hélice-Alça-Hélice , Humanos , Bicamadas Lipídicas/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/toxicidade , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/síntese química , Proteínas Supressoras de Tumor/toxicidade
6.
Philos Trans R Soc Lond B Biol Sci ; 374(1765): 20180151, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30966999

RESUMO

Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental sampling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling. Pathogen-mediated activation of surface and endosomal Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, using multiple forms of imaging and microscopy, we track membrane-associated, fluorescently-tagged Rab8a expressed in live macrophages, using a variety of cell markers to demonstrate Rab8a localization and its enrichment on early macropinosomes. Production of a novel biosensor and its use for quantitative FRET analysis in live cells, pinpoints macropinosomes as the site for TLR-induced activation of Rab8a. We have previously shown that TLR signalling, cytokine outputs and macrophage programming are regulated by the GTPase Rab8a with PI3 Kγ as its effector. Finally, we highlight another effector, the phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, suggesting that Rab8a may operate on multiple levels to modulate phosphoinositides in macropinosomes. These findings extend our understanding of macropinosomes as regulatory compartments for innate immune function in macrophages. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.


Assuntos
Macrófagos/fisiologia , Pinocitose/fisiologia , Transdução de Sinais/fisiologia , Receptores Toll-Like/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Camundongos , Células RAW 264.7 , Receptores Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(13): 6341-6350, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30846555

RESUMO

Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.


Assuntos
Imunidade Inata/efeitos dos fármacos , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/metabolismo , Zinco/toxicidade , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética
8.
J Cell Biol ; 217(11): 3873-3885, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30150290

RESUMO

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.


Assuntos
Actinas/metabolismo , Estruturas da Membrana Celular/metabolismo , Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/genética , Animais , Sistemas CRISPR-Cas , Estruturas da Membrana Celular/genética , Deleção de Genes , Lipopolissacarídeos/farmacologia , Camundongos , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/genética
9.
Sci Immunol ; 3(26)2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143554

RESUMO

Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria (Salmonella ΔsifA and Citrobacter rodentium) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Salmonella ΔsifA challenge increases bacterial burden in wild-type but not in Casp11-/- and Gsdmd -/- mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Caspases/imunologia , Armadilhas Extracelulares/imunologia , Inflamassomos/imunologia , Neutrófilos/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Morte Celular , Citrobacter rodentium , Citosol/imunologia , Citosol/microbiologia , Feminino , Humanos , Lipopolissacarídeos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Salmonella/imunologia , Salmonella enterica
10.
J Biol Chem ; 292(11): 4411-4422, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28130450

RESUMO

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling. With CRISPR/Cas9-mediated gene editing to stably knock out and recover Rab8a in macrophage cell lines, we match Akt signaling profiles with cytokine outputs, confirming that Rab8a is a novel regulator of the Akt/mammalian target of rapamycin (mTOR) pathway downstream of multiple TLRs. Upon developing a Rab8a activation assay, we show that TLR3 and 9 agonists also activate Rab8a. Live-cell imaging reveals that Rab8a is first recruited to the plasma membrane and dorsal ruffles, but it is retained during collapse of ruffles to form macropinosomes enriched for phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), suggesting that the macropinosome is the location where Rab8a is active. We pinpoint macropinosomes as the sites for Rab8-mediated biasing of inflammatory signaling responses via inducible production of anti-inflammatory cytokines. Thus, Rab8a and PI3Kγ are positioned in multiple TLR pathways, and this signaling axis may serve as a pharmacologically tractable target during infection and inflammation.


Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Citocinas/imunologia , Macrófagos/imunologia , Receptores Toll-Like/imunologia , Proteínas rab de Ligação ao GTP/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/análise , Fosfatos de Fosfatidilinositol/imunologia , Células RAW 264.7 , Transdução de Sinais , Receptores Toll-Like/análise , Proteínas rab de Ligação ao GTP/análise
11.
Methods Mol Biol ; 1519: 201-214, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27815881

RESUMO

The process of phagocytosis is crucial for fighting infection in innate immunity, for maintaining homeostasis through clearing cell debris, and for tissue remodeling in development. Here we describe two semi-automated image-based assays for the quantitative characterization of the early stages of phagocytosis and pathogen entry. A feature of these assays is the ability to detect and assess molecules or agents that subtly affect the stages both before and after cup closure or internalization.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Fagocitose , Animais , Células Cultivadas , Humanos , Macrófagos/microbiologia , Camundongos , Salmonella/fisiologia
12.
Clin Transl Immunology ; 5(4): e71, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27195114

RESUMO

The surface of mammalian cells offers an interface between the cell interior and its surrounding milieu. As part of the innate immune system, macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens, debris or dead cells. Their highly dynamic and constantly moving cell surface has extensions such as lamellipodia, filopodia and dorsal ruffles that help detect pathogens. Dorsal ruffles give rise to macropinosomes for rapid, high volume non-selective fluid sampling, receptor internalisation and plasma membrane turnover. Ruffles can also generate phagocytic cups for the receptor-mediated uptake of pathogens or particles. The membrane lipids, actin cytoskeleton, receptors and signalling proteins that constitute these cell surface domains are discussed. Although the cell surface is designed to counteract pathogens, many bacteria, viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival. Nevertheless, these features offer important potential for developing vaccines, drugs and preventative measures to help fight infection.

13.
Traffic ; 17(9): 1014-26, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27219021

RESUMO

Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines; these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized. Here we establish potential roles for APPL1 and 2 signaling adaptors as regulators of LPS/TLR4-induced signaling, transcription, and cytokine secretion. APPL1 and 2 are differentially localized to distinct signaling-competent membrane domains on the surface and in endocytic compartments of LPS-activated macrophages. By depleting cells of each adaptor respectively we show separate and opposing functions for APPL1 and 2 in Akt and MAPK signaling. Specifically, APPL2 has a dominant role in nuclear translocation of NF-KB p65 and it serves to constrain the secretion of pro- and anti-inflammatory cytokines. The APPLs, and in particular APPL2, are thus revealed as adaptors with important capacity to modulate inflammatory responses mounted by LPS/TLR4 during infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Citocinas/imunologia , Macrófagos/imunologia , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Citocinas/metabolismo , Imunidade Inata , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Transfecção
14.
PLoS One ; 11(1): e0147179, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26812621

RESUMO

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes.


Assuntos
Hidroxicolesteróis/metabolismo , Gotículas Lipídicas/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Cromatografia em Camada Delgada , Agonismo Inverso de Drogas , Metabolismo dos Lipídeos , Lipídeos/análise , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Esteroide Hidroxilases/antagonistas & inibidores , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Regulação para Cima
15.
Methods Cell Biol ; 130: 1-18, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26360024

RESUMO

Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.


Assuntos
Endossomos/ultraestrutura , Macrófagos/ultraestrutura , Animais , Membrana Celular , Endossomos/metabolismo , Meia-Vida , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Fagossomos/ultraestrutura , Transporte Proteico , Células RAW 264.7 , Transferrina/metabolismo
16.
Nat Commun ; 5: 4407, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25022365

RESUMO

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Fosfatidilinositol 3-Quinases/genética , Ligação Proteica , Células RAW 264.7 , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética , Receptor 4 Toll-Like/genética , Proteínas rab de Ligação ao GTP/genética
17.
J Theor Biol ; 358: 102-21, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-24882792

RESUMO

A key problem in the biological sciences is to be able to reliably estimate model parameters from experimental data. This is the well-known problem of parameter identifiability. Here, methods are developed for biologists and other modelers to design optimal experiments to ensure parameter identifiability at a structural level. The main results of the paper are to provide a general methodology for extracting parameters of linear models from an experimentally measured scalar function - the transfer function - and a framework for the identifiability analysis of complex model structures using linked models. Linked models are composed by letting the output of one model become the input to another model which is then experimentally measured. The linked model framework is shown to be applicable to designing experiments to identify the measured sub-model and recover the input from the unmeasured sub-model, even in cases that the unmeasured sub-model is not identifiable. Applications for a set of common model features are demonstrated, and the results combined in an example application to a real-world experimental system. These applications emphasize the insight into answering "where to measure" and "which experimental scheme" questions provided by both the parameter extraction methodology and the linked model framework. The aim is to demonstrate the tools' usefulness in guiding experimental design to maximize parameter information obtained, based on the model structure.


Assuntos
Modelos Lineares , Modelos Biológicos
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