RESUMO
Melanin is an organic material biosynthesized from tyrosine in pigment-producing cells. The present study reports a simple method to generate tailored functional materials in mammalian cells by chemically fabricating intracellular melanin. Our approach exploits synthetic tyrosine derivatives to hijack the melanin biosynthesis pathway in pigment-producing cells. Its application was exemplified by synthesizing and using a paramagnetic tyrosine derivative, m-YR, which endowed melanoma cells with responsiveness to external magnetic fields. The mechanical force generated by the magnet-responsive melanin forced the cells to elongate and align parallel to the magnetic power lines. Critically, even non-pigment cells were similarly remote-controlled by external magnetic fields once engineered to express tyrosinase and treated with m-YR, suggesting the versatility of the approach. The present methodology may potentially provide a new avenue for mechanobiology and magnetogenetic studies and a framework for magnetic control of specific cells.
Assuntos
Melaninas , Monofenol Mono-Oxigenase , Animais , Fenômenos Magnéticos , Mamíferos/metabolismo , Melaninas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Tirosina/metabolismoRESUMO
The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 µm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição HES-1/antagonistas & inibidores , Animais , Antineoplásicos/química , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proibitinas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.
Assuntos
Proteínas Luminescentes/genética , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Ondas Encefálicas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Potenciais Pós-Sinápticos Excitadores , Transferência Ressonante de Energia de Fluorescência , Hipocampo/citologia , Hipocampo/fisiologia , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Neurônios/classificação , Optogenética , Células PC12 , Monoéster Fosfórico Hidrolases/química , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging.
Assuntos
Proteínas Luminescentes/metabolismo , Animais , Linhagem Celular Tumoral , Fenômenos Eletrofisiológicos , Cinética , Proteínas Luminescentes/genética , Microeletrodos , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Vermelha FluorescenteRESUMO
The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC(50) of 10 mum and 2 nm, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.
Assuntos
Sistema Nervoso Central/metabolismo , Neurotensina/química , Receptores de Neurotensina/química , Receptores de Neurotensina/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Western Blotting , Encéfalo/metabolismo , Células CHO , Células COS , Linhagem Celular , Cricetinae , DNA Complementar/metabolismo , Densitometria , Dimerização , Relação Dose-Resposta a Droga , Epitopos/química , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Concentração Inibidora 50 , Ligantes , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Piperidinas/farmacologia , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Medula Espinal/metabolismo , Fatores de Tempo , Distribuição Tecidual , TransfecçãoRESUMO
Platelet-activating factor (PAF) is a phospholipid with potent and diverse physiological actions, particularly as a mediator of inflammation. We have reported previously that mutant G protein-coupled receptors (GPCRs) affect the functional properties of coexpressed wild-type human PAF receptor (hPAFR) (Le Gouill, C., Parent, J. L., Caron, C. A., Gaudreau, R., Volkov, L., Rola-Pleszczynski, M., and Stankova, J. (1999) J. Biol. Chem. 274, 12548-12554). Increasing evidence suggests that dimerization of GPCRs may play an important role in the regulation of their biological activity. Additional data have also suggested that dimerization may be important in the subsequent internalization of the delta-opioid receptor. To investigate the specific role of dimerization in the internalization process of GPCRs, we generated a fusion protein of hPAFR and bacterial DNA gyrase B (GyrB), dimerized through the addition of coumermycin. We found that dimerization potentiates PAF-induced internalization of hPAFR-GyrB in Chinese hamster ovary cells stably expressing c-Myc-hPAFR-GyrB. Coumermycin-driven dimerization was also sufficient to induce an agonist-independent sequestration process in an arrestin- and clathrin-independent manner. Moreover, the protein kinase C inhibitors staurosporine and GF109203X blocked the coumermycin-induced desensitization of hPAFR-GyrB, suggesting the implication of protein kinase C in the molecular mechanism mediating the agonist-independent desensitization of the receptor. Taken together, these findings suggest a novel mechanism of GPCR desensitization and internalization triggered by dimerization.