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1.
DNA Cell Biol ; 38(10): 1048-1055, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433200

RESUMO

DNA condensed agents can improve the transfection efficiency of the cationic liposome delivery system. However, various condensed agents have distinct transfection efficiency and cellular cytotoxicity. The object of this study was to screen the optimal agents with the high transfection efficiency and low cytotoxicity from four polymer compressive materials, polyethylenimine (PEI), chitosan, poly-l-lysine (PLL), and spermidine. DNA was precompressed with these four agents and then combined to cationic liposomes. Subsequently, the entrapment and transfection efficiency of the obtained complexes were investigated. Finally, the particle sizes, cytotoxicity, and endocytosis fashion of these copolymers (Lipo-PEI, Lipo-chitosan, Lipo-PLL, and Lipo-spermidine) were examined. It was found that these four copolymers had significantly lower cytotoxicity and higher transfection efficiency (45.5%, 42.4%, 36.8%, and 47.4%, respectively) than those in the control groups. The transfection efficiency of Lipo-PEI and Lipo-spermidine copolymers were better than the other two copolymers. In 293T cells, nystatin significantly inhibited the transfection efficiency of Lipo-PEI-DNA and Lipo-spermidine-DNA (51.88% and 46.05%, respectively), which suggest that the endocytosis pathway of Lipo-spermidine and Lipo-PEI copolymers was probably caveolin dependent. Our study indicated that these dual-degradable copolymers especially liposome-spermidine copolymer could be used as the potential biocompatible gene delivery carriers.


Assuntos
Quitosana/química , Lipossomos/química , Polietilenoimina/química , Polilisina/química , Espermidina/química , Transfecção/métodos , Cátions , Caveolina 1/genética , Caveolina 1/metabolismo , Quitosana/metabolismo , Colesterol/química , Colesterol/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Células HEK293 , Humanos , Lipossomos/metabolismo , Nistatina/farmacologia , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/metabolismo , Polietilenoimina/metabolismo , Polilisina/metabolismo , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Espermidina/metabolismo
2.
Nat Commun ; 10(1): 2350, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138815

RESUMO

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.


Assuntos
Aciltransferases/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Endocitose/genética , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/crescimento & desenvolvimento , Quinases Ativadas por p21/metabolismo
3.
Biomed Res Int ; 2019: 1495130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31073519

RESUMO

Objective: With the development of exosomes studies increased around the whole world. Our present study was aimed to investigate the global status and trends in exosomes field. Methods: Publications related to exosomes studies from 1994 to 2017 were retrieved from the Web of Science database. The data source was studied and indexed by using bibliometric methodology. For visualized study, VOS viewer software was used to conduct bibliographic coupling analysis, coauthorship analysis, cocitation analysis, and cooccurrence analysis and to analyze the publication trend in exosomes research. Results: A total of 4960 publications were included. The relative research interests and number of publications were increasing per year globally. The USA made the highest contributions to the global research with the most citations, the highest H-index, and the most total link strength, while Sweden had the highest average citation per item. The journal PLOS ONE had the highest publication number. The Natl Canc Ctr was the most contributive institutions. Studies could be divided into three clusters: mechanism study, in vivo study, and in vitro study. Conclusions: The efforts should be put into mechanism studies, predicted to be the next hot spots in exosomes studies.


Assuntos
Endocitose/genética , Exossomos/genética , Revisão da Pesquisa por Pares , Bases de Dados Factuais , Exossomos/ultraestrutura , Vesículas Extracelulares/genética , Humanos , Publicações , Suécia
4.
Methods Mol Biol ; 1945: 141-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945245

RESUMO

ML-Rules is a rule-based language for multi-level modeling and simulation. ML-Rules supports dynamic nesting of entities and applying arbitrary functions on entity attributes and content, as well as for defining kinetics of reactions. This allows describing and simulating complex cellular dynamics operating at different organizational levels, e.g., to combine intra-, inter-, and cellular dynamics, like the proliferation of cells, or to include compartmental dynamics like merging and splitting of mitochondria or endocytosis. The expressiveness of the language is bought with additional efforts in executing ML-Rules models. Therefore, various simulators have been developed from which the user and automatic procedures can select. The experiment specification language SESSL facilitates design, execution, and reuse of simulation experiments. The chapter illuminates the specific features of ML-Rules as a rule-based modeling language, the implications for an efficient execution, and shows ML-Rules at work.


Assuntos
Biologia Computacional/métodos , Modelos Biológicos , Software , Algoritmos , Proliferação de Células/genética , Simulação por Computador , Endocitose/genética , Humanos , Cinética , Redes e Vias Metabólicas/genética , Mitocôndrias/genética
5.
Curr Genet ; 65(4): 1071-1080, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30953125

RESUMO

AP2 is a heterotetrameric clathrin adaptor complex that owns important roles in vesicle generation and cargo recognition. Cell-wall integrity (CWI) pathway is essential for fungal development, virulence, and adaptation to environment stresses. To date, the relationship between AP2 and CWI is largely unknown in phytopathogenic fungi. In this study, we identified the adaptor complex FgAP2 in Fusarium graminearum. The biological function analysis showed that FgAP2 complex contains FgAP2α, FgAP2ß, FgAP2σ, and FgAP2µ, and the subunit FgAP2µ, which is required for hyphal growth, conidiation, CWI, and virulence. Yeast two-hybrid showed that FgAP2µ interacts with the CWI sensor FgWsc2B. Consistently, western blotting analysis revealed that FgWsc2B positively regulates phosphorylation of FgMgv1, the MAP kinase of CWI. Moreover, the FgWsc2B deletion mutant exhibited defects in hyphal growth, virulence, and response to CWI damaging agents. Taken together, our data indicated that FgAP2µ is involved in CWI and virulence via interacting with FgWsc2B in F. graminearum.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Parede Celular/genética , Endocitose/genética , Fusarium/genética , Parede Celular/microbiologia , Proteínas Fúngicas/genética , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/patogenicidade , Pressão Osmótica , Fosforilação , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidade , Estresse Fisiológico/genética , Virulência/genética
6.
J Exp Clin Cancer Res ; 38(1): 174, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023336

RESUMO

BACKGROUND: Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC. METHODS: The changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on ß2-adrenoceptor (ß2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and ß2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that ß2AR colocalized with the recycling endosome marker Rab11, which indicated ß2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth. RESULTS: The microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited ß2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and ß2AR expression. Interestingly, EHD1 interacted with ß2AR and played a novel and critical role in ß2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or ß2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and ß2AR expression in patient specimens. CONCLUSION: Collectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via ß2AR signaling and highlight a potential target for antiangiogenic therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neovascularização Patológica/genética , Receptores Adrenérgicos beta 2/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas de Transporte Vesicular/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Proliferação de Células/genética , Endocitose/genética , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Microscopia Confocal , Neovascularização Patológica/patologia , Prognóstico , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rab de Ligação ao GTP/genética
7.
Appl Microbiol Biotechnol ; 103(12): 4859-4868, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025075

RESUMO

Actin-regulating kinase (Ark) plays an important role in controlling endocytosis, which has been shown to be involved in the development and virulence of several fungal pathogens. However, it remains unclear whether Ark1 is required for the development and pathogenicity of an entomopathogenic fungus. Here, MrArk1 (MAA_03415), a homologue of yeast Ark1, was characterized in the insect pathogenic fungus, Metarhizium robertsii. Disruption of MrArk1 led to defects in endocytosis and a marked reduction (58%) in conidiation capacity. The reduced conidiation level was accompanied by repression of several key conidiation-related genes, including brlA, abaA, and wetA. Additionally, the deletion mutant showed a significant decrease in its tolerance to heat shock, but not to UV-B irradiation. Bioassays demonstrated attenuated virulence for the deletion mutant against Galleria mellonella via normal cuticle infection, accompanied by suppressed appressorium formation and reduced transcript levels of several genes involved in cuticle penetration. Taken together, our results indicate that MrArk1 is involved in the heat tolerance, sporulation, and virulence of M. robertsii, and thus is an important factor for sustaining the fungal potential against insect pests.


Assuntos
Endocitose/genética , Metarhizium/genética , Metarhizium/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Animais , Bioensaio , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Cinesina/genética , Cinesina/metabolismo , Lepidópteros/microbiologia , Metarhizium/crescimento & desenvolvimento , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Virulência
8.
Science ; 364(6435): 57-62, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948546

RESUMO

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfatidilserinas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/metabolismo , Endocitose/genética , Regulação da Expressão Gênica de Plantas , Gravitropismo/genética , Ácidos Indolacéticos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfatidilserinas/farmacologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Imagem Individual de Molécula
9.
Science ; 363(6431)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30846568

RESUMO

Commensal bacteria influence host physiology, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB) are transferred into intestinal epithelial cells (IECs) through adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. This process transfers microbial cell wall-associated proteins, including an antigen that stimulates mucosal T helper 17 (TH17) cell differentiation, into the cytosol of IECs in a cell division control protein 42 homolog (CDC42)-dependent manner. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB and decreased epithelial antigen acquisition, with consequent loss of mucosal TH17 cells. Our findings demonstrate direct communication between a resident gut microbe and the host and show that under physiological conditions, IECs acquire antigens from commensal bacteria for generation of T cell responses to the resident microbiota.


Assuntos
Antígenos de Bactérias/imunologia , Endocitose/imunologia , Microbioma Gastrointestinal/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Mucosa Intestinal/imunologia , Linfócitos Intraepiteliais/imunologia , Células Th17/imunologia , Animais , Bactérias/imunologia , Endocitose/genética , Homeostase/genética , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Simbiose , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/fisiologia
10.
PLoS Genet ; 15(1): e1007914, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689641

RESUMO

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Desmossomos/genética , Endocitose/genética , Animais , Adesão Celular/genética , Linhagem Celular , Desmogleínas/genética , Desmogleínas/metabolismo , Epiderme/metabolismo , Humanos , Junções Intercelulares/genética , Queratinócitos/metabolismo , Camundongos
11.
PLoS One ; 14(1): e0210223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682048

RESUMO

The small GTPases, Rab5 and Rab7, are key regulators at multiple stages of the endocytic/endolysosomal pathway, including fusion and maturation of endosomes. In yeast, Vps21p (Rab5 homolog) recruits a GEF for Rab7 and activates the downstream Ypt7p (Rab7 homolog) on endosomal membrane. Although the model of this sequential activation from Vps21p to Ypt7p in the endocytic pathway has been established, activation mechanism of Ypt7p in the Vps21p-independent pathway has not been completely clarified. Here we show that Ypt7p is activated and mediates vacuolar fusion in cells lacking all yeast Rab5 genes, VPS21, YPT52, and YPT53. We also demonstrate that deletion of both VPS21 and YPT7 genes cause severe defect in the AP-3 pathway as well as the CPY pathway although the AP-3 pathway is mostly intact in each vps21Δ or ypt7Δ mutant. Interestingly, in vps21Δ ypt7Δ mutant cargos trafficked via the VPS or endocytic pathway accumulate beside nucleus whereas cargo trafficked via the AP-3 pathway disperse in the cytosol. These findings suggest that Ypt7p is activated and plays a Rab5-independent role in the AP-3-mediated pathway.


Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Membrana Celular/genética , Citosol/metabolismo , Endocitose/genética , Endossomos/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fusão de Membrana/genética , Fenótipo , Transdução de Sinais/genética , Vacúolos/genética , Vacúolos/metabolismo
12.
Hum Mol Genet ; 28(4): 584-597, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30335140

RESUMO

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.


Assuntos
Proteínas de Transporte/genética , Anormalidades Craniofaciais/genética , Epilepsia/genética , Deformidades Congênitas da Mão/genética , Perda Auditiva Neurossensorial/genética , Deficiência Intelectual/genética , Unhas Malformadas/genética , Convulsões/genética , Sequência de Aminoácidos/genética , Animais , Anormalidades Craniofaciais/fisiopatologia , Modelos Animais de Doenças , Endocitose/genética , Epilepsia/fisiopatologia , Exoma/genética , Regulação da Expressão Gênica , Deformidades Congênitas da Mão/fisiopatologia , Haploinsuficiência , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Deficiência Intelectual/fisiopatologia , Proteínas de Membrana , Camundongos , Mutação , Unhas Malformadas/fisiopatologia , Proteínas do Tecido Nervoso , Plasticidade Neuronal/genética , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Convulsões/fisiopatologia
13.
Nano Lett ; 19(1): 8-18, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30335394

RESUMO

The nanoparticle (nano)-cell membrane interface is one of the most important interactions determining the fate of nanoparticles (NPs), which can stimulate a series of biological events, allowing theranostic and other biomedical applications. So far, there remains a lack of knowledge about the mechanisms governing the nanoparticle-cell membrane interface, especially the impact of ligand exchange, in which molecules on the nanosurface become replaced with components of the cell membrane, resulting in unique interfacial phenomena. Herein, we describe a family of gold nanoparticles (AuNPs) of the same core size (∼13 nm core), modified with 12 different kinds of surface ligands, and the effects of their exchangeable ligands on both nanoparticle-supported lipid bilayers (SLBs) and nanoparticle-natural cell membrane interfaces. The ligands are categorized according to their molecular weight, charge, and bonding modes (physisorption or chemisorption). Importantly, we found that, depending on the adsorption affinity and size of ligand molecules, physisorbed ligands on the surface of NPs can be exchanged with lipid molecules. At a ligand exchange-dominated interface, the AuNPs typically aggregated into an ordered monolayer in the lipid bilayers, subsequently affecting cell membrane integrity, NP uptake efficiency, and the NP endocytosis pathways. These findings advance our understanding of the underlying mechanisms of the biological effects of nanoparticles from a new point of view and will aid in the design of novel, safe, and effective nanomaterials for biomedicine.


Assuntos
Adsorção , Membrana Celular/química , Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Membrana Celular/efeitos dos fármacos , Endocitose/genética , Ouro/química , Ligantes , Nanopartículas Metálicas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Propriedades de Superfície
14.
Mol Plant Microbe Interact ; 32(4): 437-451, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30451565

RESUMO

The actin cytoskeleton and actin-coupled endocytosis are conserved cellular processes required for the normal growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. We have previously shown that actin regulating kinase MoArk1 regulates actin dynamics and endocytosis to play a key role in virulence of the fungus. To understand the underlying mechanism, we have characterized the actin-binding protein MoAbp1 that interacts with MoArk1 from M. oryzae. The ΔMoabp1 mutant exhibited delayed endocytosis and defects in growth, host penetration, and invasive growth. Consistent with its putative function associated with actin-binding, MoAbp1 regulates the localization of actin patches and plays a role in MoArk1 phosphorylation. In addition, MoAbp1 interacts with MoCap (adenylyl cyclase-associated protein) affecting its normal patch localization pattern and the actin protein MoAct1 through its conserved domains. Taken together, our results support a notion that MoAbp1 functions as a protein scaffold linking MoArk1, MoCap1, and MoAct1 to regulate actin cytoskeleton dynamics critical in growth and pathogenicity of the blast fungus.


Assuntos
Citoesqueleto de Actina , Endocitose , Magnaporthe , Virulência , Citoesqueleto de Actina/metabolismo , Endocitose/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Proteínas dos Microfilamentos/metabolismo , Oryza/microbiologia , Virulência/genética
15.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30467237

RESUMO

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.


Assuntos
Proteína 2 Relacionada a Actina/genética , Cílios/genética , Neuropeptídeos/genética , Monoéster Fosfórico Hidrolases/genética , Actinas/genética , Linhagem Celular , Movimento Celular/genética , Centrossomo/metabolismo , Cílios/metabolismo , Quinase 5 Dependente de Ciclina/genética , Endocitose/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microtúbulos/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Fosforilação , Ligação Proteica , Proteoma/genética
16.
Int J Mol Sci ; 19(12)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501045

RESUMO

The glutamate (Glu) N-methyl-d-aspartate (NMDA) receptor (NMDAR) plays a critical role in synaptic communication given mainly by its ionotropic function that permeates Ca2+. This in turn activates calmodulin that triggers CaMKII, MAPK, CREB, and PI3K pathways, among others. However, NMDAR signaling is more complex. In the last two decades several groups have shown that the NMDAR also elicits flux-independent signaling (f-iNMDARs). It has been demonstrated that agonist (Glu or NMDA) or co-agonist (Glycine or d-Serine) bindings initiate intracellular events, including conformational changes, exchange of molecular interactions, release of second messengers, and signal transduction, that result in different cellular events including endocytosis, LTD, cell death, and neuroprotection, among others. Interestingly, f-iNMDARs has also been observed in pathological conditions and non-neuronal cells. Here, the molecular and cellular events elicited by these flux-independent actions (non-canonical or metabotropic-like) of the NMDAR are reviewed. Considering the NMDAR complexity, it is possible that these flux-independent events have a more relevant role in intracellular signaling that has been disregarded for decades. Moreover, considering the wide expression and function of the NMDAR in non-neuronal cells and other tissues beyond the nervous system and some evolutionary traits, f-iNMDARs calls for a reconsideration of how we understand the biology of this complex receptor.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Endocitose/genética , Endocitose/fisiologia , Humanos , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
17.
Nutrients ; 10(11)2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30400219

RESUMO

Macropinocytosis is a regulated form of endocytosis that mediates the nonselective uptake of nutrients to support growth under nutrient-deprived conditions. KRAS-mutant cancer cells upregulate macropinocytosis to import extracellular proteins, which subsequently undergo proteolytic degradation in the lysosome. Although transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and function, its role in the degradation of extracellular protein from macropinocytosis in KRAS-mutant cells has not previously been elucidated. In this study, we investigated the role of TFEB in the recovery of macropinocytosis-mediated mTORC1 activity and cell growth under nutrient depletion. Mouse embryonic fibroblasts (MEFs) expressing KrasG12D and KRAS-mutant human cancer cells took up markedly higher levels of tetramethylrhodamine (TMR)-dextran than the corresponding wild-type cells. siRNA-mediated inhibition of TFEB did not influence extracellular TMR-dextran uptake, but significantly attenuated lysosomal degradation of extracellular protein. Bovine serum albumin (BSA) treatment restored p-S6K levels and cell proliferation suppressed by leucine deprivation, and these effects were blocked by siTFEB. Collectively, our results show that TFEB plays a role in macropinocytosis-mediated KRAS-mutant cell growth under nutrient deprivation by promoting lysosomal degradation of extracellular proteins.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Endocitose/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endocitose/genética , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno , Rodaminas/metabolismo , Soroalbumina Bovina/farmacologia
18.
J Biol Chem ; 293(49): 18890-18902, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30291144

RESUMO

Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.


Assuntos
Dendritos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Neuropeptídeos/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Sítios de Ligação , Células COS , Moléculas de Adesão Celular/metabolismo , Dendritos/genética , Endocitose/genética , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Ratos
19.
Annu Rev Cell Dev Biol ; 34: 111-136, 2018 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-30296391

RESUMO

The plasma membrane of eukaryotic cells is not a simple sheet of lipids and proteins but is differentiated into subdomains with crucial functions. Caveolae, small pits in the plasma membrane, are the most abundant surface subdomains of many mammalian cells. The cellular functions of caveolae have long remained obscure, but a new molecular understanding of caveola formation has led to insights into their workings. Caveolae are formed by the coordinated action of a number of lipid-interacting proteins to produce a microdomain with a specific structure and lipid composition. Caveolae can bud from the plasma membrane to form an endocytic vesicle or can flatten into the membrane to help cells withstand mechanical stress. The role of caveolae as mechanoprotective and signal transduction elements is reviewed in the context of disease conditions associated with caveola dysfunction.


Assuntos
Cavéolas/metabolismo , Membrana Celular/genética , Vesículas Transportadoras/genética , Cavéolas/química , Cavéolas/patologia , Membrana Celular/química , Endocitose/genética , Humanos , Transdução de Sinais/genética , Estresse Mecânico , Relação Estrutura-Atividade , Vesículas Transportadoras/química
20.
Fungal Genet Biol ; 121: 29-45, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30240788

RESUMO

Snf7 is the core subunit protein of the yeast endosomal sorting complex required for transport (ESCRT) complex, which plays important roles in endocytosis and autophagy. In this study, we characterized MoSnf7 in Magnaporthe oryzae, a homolog of yeast Snf7, the core protein of ESCRT-III subcomplex. Like Snf7, MoSnf7 also localizes next to the vacuoles. Deletion of MoSNF7 resulted in significant decrease in vegetative growth and pathogenicity. Further analyses of ΔMosnf7 mutants showed that they were defective in endocytosis, sexual and asexual development, turgor pressure maintenance of appressorium at hyphal tips, and cell wall integrity. Additional assays for the localization and degradation of GFP-MoAtg8 in ΔMosnf7 mutants showed that they were defective in autophagy pathway. Based on the roles of yeast Snf7 in endocytosis and autophagy, we propose that the decreased vegetative growth and pathogenicity of ΔMosnf7 rice blast fungus M. oryzae, was partly due to the conservative roles of MoSnf7 in vesicle trafficking and autophagy pathway.


Assuntos
Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/genética , Esporos Fúngicos/genética , Autofagia/genética , Parede Celular/metabolismo , Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas Fúngicas/genética , Magnaporthe/patogenicidade , Oryza/genética , Doenças das Plantas/microbiologia , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/crescimento & desenvolvimento
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