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1.
Histochem Cell Biol ; 159(4): 313-327, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36504003

RESUMO

Adipocyte differentiation is a sequential process involving increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte-specific gene expression, and accumulation of lipid droplets in the cytoplasm. Expression of the transcription factors involved is usually detected using canonical biochemical or biomolecular procedures such as Western blotting or qPCR of pooled cell lysates. While this provides a useful average index for adipogenesis for some populations, the precise stage of adipogenesis cannot be distinguished at the single-cell level, because the heterogenous nature of differentiation among cells limits the utility of averaged data. We have created a classifier to sort cells, and used it to determine the stage of adipocyte differentiation at the single-cell level. We used a machine learning method with microscopic images of cell stained for PPARγ and lipid droplets as input data. Our results show that the classifier can successfully determine the precise stage of differentiation. Stage classification and subsequent model fitting using the sequential reaction model revealed the action of pioglitazone and rosiglitazone to be promotion of transition from the stage of increased PPARγ expression to the next stage. This indicates that these drugs are PPARγ agonists, and that our classifier and model can accurately estimate drug action points and would be suitable for evaluating the stage/state of individual cells during differentiation or disease progression. The incorporation of both biochemical and morphological information derived from immunofluorescence image of cells and so overcomes limitations of current models.


Assuntos
Adipogenia , PPAR gama , Diferenciação Celular , Adipócitos , Gotículas Lipídicas , Aprendizado de Máquina
2.
Int J Mol Sci ; 21(17)2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32872628

RESUMO

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Cianobactérias/metabolismo , Fotorreceptores Microbianos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Biliverdina/química , Cor , Células HeLa , Humanos , Luz , Homologia de Sequência
3.
Phys Chem Chem Phys ; 20(5): 2982-2985, 2018 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-29022027

RESUMO

In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.


Assuntos
DNA/química , Ressonância Magnética Nuclear Biomolecular , RNA/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Microscopia Confocal , Estreptolisinas/química , Estreptolisinas/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(10): E1067-76, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25713368

RESUMO

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic ß-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.


Assuntos
Trifosfato de Adenosina/biossíntese , Proteínas de Transporte/antagonistas & inibidores , Cisteína/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Animais , Linhagem Celular , Secreção de Insulina , Camundongos , Hormônios Tireóideos , Proteínas de Ligação a Hormônio da Tireoide
5.
PLoS Pathog ; 11(3): e1004747, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25742138

RESUMO

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments.  On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.


Assuntos
Brucelose/imunologia , Endorribonucleases/imunologia , Interações Hospedeiro-Parasita/fisiologia , Proteínas Serina-Treonina Quinases/imunologia , Resposta a Proteínas não Dobradas/imunologia , Proteínas de Transporte Vesicular/imunologia , Western Blotting , Brucella abortus/fisiologia , Brucelose/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/microbiologia , Retículo Endoplasmático/patologia , Endorribonucleases/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Vacúolos/metabolismo , Vacúolos/microbiologia , Vacúolos/patologia , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral/fisiologia
6.
Biochim Biophys Acta ; 1853(10 Pt A): 2592-609, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25962623

RESUMO

Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Brefeldina A/farmacologia , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/genética , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/genética
7.
Genes Cells ; 20(4): 340-57, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25736587

RESUMO

Forkhead box O1 (FOXO1) is an important target for insulin. It is widely accepted that insulin-induced phosphorylation of FOXO1 by Akt leads to its nuclear exclusion and results in the inhibition of FOXO1-mediated transcription of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1 (PCK1) in hepatocytes. However, many results that contradict this model have accumulated. Here, we provide a new mechanism for insulin-dependent repression of FOXO1-mediated transcription. We showed insulin-induced translocation of endogenous Ser256-phosphorylated FOXO1, which is essential for regulation of FOXO1-mediated transcription, from nuclear speckles to the nuclear periphery. This insulin-dependent translocation of FOXO1 regulated transcriptional repression of PCK1 concomitant with the formation of the FOXO1-euchromatic histone-lysine N-methyltransferase2 (EHMT2) complex and histone modifications of the PCK1 promoter region. Notably, our results suggest that FOXO1 uses nucleoporin 98 kDa NUP98 for this transcriptional regulation. These results provide a new insight into various FOXO1-mediated transcriptional regulation and FOXO1-mediated essential biological pathways.


Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Proteína Forkhead Box O1 , Células Hep G2 , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Insulina/metabolismo , Carioferinas/metabolismo , Metilação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Serina/metabolismo , Fatores de Processamento de Serina-Arginina , Proteína Exportina 1
8.
Genes Cells ; 19(7): 565-81, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24889144

RESUMO

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.


Assuntos
Células Epiteliais/metabolismo , Proteína 2 com Domínio MARVEL/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Receptores de Lipoproteínas/metabolismo , Junções Íntimas/metabolismo , Animais , Antracenos/farmacologia , Apigenina/farmacologia , Linhagem Celular , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Nitrilas , Fosforilação , Serina/metabolismo , Tirfostinas/farmacologia
9.
FEBS Open Bio ; 14(4): 695-720, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38425293

RESUMO

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/ß-catenin pathway, with significant downregulation of both Ser 675-phosphorylated ß-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and ß-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/ß-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.


Assuntos
Neoplasias Colorretais , Proteína Forkhead Box M1 , Humanos , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , beta Catenina/metabolismo , Via de Sinalização Wnt/genética , Neoplasias Colorretais/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
10.
J Neurosci ; 32(41): 14227-41, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23055492

RESUMO

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT(1A)R in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons.


Assuntos
Dendritos/fisiologia , Regiões de Interação com a Matriz/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Receptor 5-HT1A de Serotonina/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Células Cultivadas , Dendritos/genética , Marcação de Genes/métodos , Humanos , Regiões de Interação com a Matriz/genética , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular/genética
11.
Biochim Biophys Acta ; 1823(4): 861-75, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22326427

RESUMO

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.


Assuntos
Compartimento Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Bioensaio , Transporte Biológico/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Toxina da Cólera/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Ativadores de Enzimas/farmacologia , Técnicas de Silenciamento de Genes , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Proteínas do Envelope Viral/metabolismo
12.
FEBS J ; 290(12): 3221-3242, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36705569

RESUMO

The M2 isoform of pyruvate kinase (PKM2) is abundantly expressed in various cancer cells and associated with tumorigenesis, tumour proliferation and tumour progression. However, the role of PKM2 in these oncological processes is not fully understood. In the present study, we depleted PKM2 expression using RNA interference (RNAi), which induced apoptotic cell death and was accompanied by the downregulation of GM130, giantin, and p115 in HeLa and ME-180 cervical cancer cells. The decreased expression of these proteins caused structural and functional disturbances in the Golgi apparatus, which manifested as the dispersion of the Golgi apparatus and delayed anterograde trafficking from the ER to the Golgi. The transcription factor, TFE3, which functions in the Golgi stress response, was responsible for the expression of GM130, giantin, and p115 that maintained the integrity of the organelle under normal growth conditions. In PKM2-knockdown cells, the translation of TFE3 was markedly reduced. Knockdown of TFE3 by RNAi resulted in the downregulation of GM130, giantin, and p115, dispersion of the Golgi apparatus, and apoptotic cell death, similar to those observed following PKM2 knockdown. Conversely, the exogenous expression of TFE3 in PKM2 knockdown cells partially mitigated the aforementioned effects. We also demonstrated that PKM2 bound to the 5' UTR on TFE3 mRNA and promoted translation. This study is the first to identify a new function for PKM2, which activates the basal Golgi stress response to maintain the integrity of the Golgi apparatus through the translation of TFE3 and promote cancer cell survival.


Assuntos
Proteínas de Membrana , Neoplasias do Colo do Útero , Humanos , Feminino , Proteínas de Membrana/metabolismo , Neoplasias do Colo do Útero/genética , Células HeLa , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo
13.
Stem Cell Reports ; 18(3): 688-705, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36764297

RESUMO

In addition to increasing ß-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.


Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Smad
14.
Biochim Biophys Acta ; 1813(5): 784-801, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21277337

RESUMO

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.


Assuntos
Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Peróxido de Hidrogênio/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Bactérias/metabolismo , Bioensaio , Transporte Biológico/efeitos dos fármacos , Toxina da Cólera/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Proteínas Luminescentes/metabolismo , Estrutura Terciária de Proteína , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
15.
J Cell Sci ; 123(Pt 19): 3215-25, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20807802

RESUMO

Glycogen synthase kinase ß (GSK3ß) phosphorylates many substrates in mammalian cells, and functions in many physiological processes. We observed that GSK3ß knockdown by siRNA perturbed both Golgi morphology in HeLa cells and the anterograde transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) from the trans-Golgi network (TGN) to prelysosomal compartments (PLC), diverting it to the exocytic pathway. Moreover, we demonstrate that a portion of GSK3ß was localized to the TGN through the Golgi peripheral protein p230 and that this localization regulated CLASP2 phosphorylation. Our results also show that GSK3ß knockdown resulted in accumulation of CLASP2 at microtubule plus ends at the cell periphery. Our findings support the hypothesis that GSK3ß at the TGN acts as a guide, activates exocytic transport, and redirects CI-M6PR from transport to the PLC into the exocytic pathway by regulating the affinity of CLASPs for microtubules.


Assuntos
Autoantígenos/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptor IGF Tipo 2/metabolismo , Membrana Celular/metabolismo , Exocitose/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Complexo de Golgi/genética , Células HeLa , Humanos , Lisossomos/metabolismo , Microtúbulos/metabolismo , Fosforilação , Transporte Proteico/genética , RNA Interferente Pequeno/genética
16.
Front Cell Dev Biol ; 10: 1027043, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36601537

RESUMO

"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.

18.
Sci Rep ; 11(1): 2900, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536479

RESUMO

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell-cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.


Assuntos
Técnicas Citológicas/métodos , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Endocitose , Células HeLa , Humanos , Fusão de Membrana
19.
iScience ; 24(7): 102724, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34337357

RESUMO

To infer a "live" protein network in single cells, we developed a novel Protein Localization and Modification-based Covariation Network (PLOM-CON) analysis method using a large set of quantitative data on the abundance (quantity), post-translational modification state (quality), and localization/morphological information of target proteins from microscope immunostained images. The generated network exhibited synchronized time-dependent behaviors of the target proteins to visualize how a live protein network develops or changes in cells under specific experimental conditions. As a proof of concept for PLOM-CON analysis, we applied this method to elucidate the role of actin scaffolds, in which actin fibers and signaling molecules accumulate and form membrane-associated protein condensates, in insulin signaling in rat hepatoma cells. We found that the actin scaffold in cells may function as a platform for glycogenesis and protein synthesis upon insulin stimulation.

20.
J Biol Chem ; 284(39): 26620-30, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19632984

RESUMO

The mammalian intracellular phospholipase A(1) (iPLA(1)) family consists of three members, iPLA(1)alpha/PA-PLA(1), iPLA(1)beta/p125, and iPLA(1)gamma/KIAA0725p. Although iPLA(1)beta has been implicated in organization of the ER-Golgi compartments, little is known about the physiological role of its closest paralog, iPLA(1)gamma. Here we show that iPLA(1)gamma mediates a specific retrograde membrane transport pathway between the endoplasmic reticulum (ER) and the Golgi complex. iPLA(1)gamma appeared to be localized to the cytosol, the cis-Golgi, and the ER-Golgi intermediate compartment (ERGIC). Time-lapse microscopy revealed that a population of GFP-iPLA(1)gamma was associated with transport carriers moving out from the Golgi complex. Knockdown of iPLA(1)gamma expression by RNAi did not affect the anterograde transport of VSVGts045 but dramatically delayed two types of Golgi-to-ER retrograde membrane transport; that is, transfer of the Golgi membrane into the ER in the presence of brefeldin A and delivery of cholera toxin B subunit from the Golgi complex to the ER. Notably, knockdown of iPLA(1)gamma did not impair COPI- and Rab6-dependent retrograde transports represented by ERGIC-53 recycling and ER delivery of Shiga toxin, respectively. Thus, iPLA(1)gamma is a novel membrane transport factor that contributes to a specific Golgi-to-ER retrograde pathway distinct from presently characterized COPI- and Rab6-dependent pathways.


Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfolipases A1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células CHO , Complexo I de Proteína do Envoltório/genética , Cricetinae , Cricetulus , Citosol/metabolismo , Citosol/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Fosfolipases A1/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transfecção , Proteínas rab de Ligação ao GTP/genética
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