RESUMO
Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.
Assuntos
Região CA1 Hipocampal , Memória , Neurônios , Receptores CCR5 , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Memória/fisiologia , Rememoração Mental/fisiologia , Camundongos , Neurônios/metabolismo , Receptores CCR5/deficiência , Receptores CCR5/genética , Receptores CCR5/metabolismo , Fatores de TempoRESUMO
The inducible activation system is valuable for investigating spatiotemporal roles of molecules. A chemically inducible activation system for Fas (CD95/APO-1), which works efficiently to induce apoptosis and leads non-apoptotic pathways, has not yet been developed. Here, we engineered a rapamycin-induced dimerization system of Fas consisting of FKBP and FRB proteins. Treatment of rapamycin specifically induces cellular apoptosis. In neurons and cells with high c-FLIP expression, rapamycin-induced Fas activation triggered the activation of the non-apoptotic pathway components instead of cell death. Intracranial delivery of the system could be utilized to induce apoptosis of tumor cells upon rapamycin treatment. Our results demonstrate a novel inducible Fas activation system which operates with high efficiency and temporal precision in vitro and in vivo promising a potential therapeutic strategy.
Assuntos
Engenharia de Proteínas/métodos , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Gravidez , Ratos Sprague-Dawley , Proteína 1A de Ligação a Tacrolimo/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/genéticaRESUMO
Cell migration is controlled by various Ca(2+) signals. Local Ca(2+) signals, in particular, have been identified as versatile modulators of cell migration because of their spatiotemporal diversity. However, little is known about how local Ca(2+) signals coordinate between the front and rear regions in directionally migrating cells. Here, we elucidate the spatial role of local Ca(2+) signals in directed cell migration through combinatorial application of an optogenetic toolkit. An optically guided cell migration approach revealed the existence of Ca(2+) sparklets mediated by L-type voltage-dependent Ca(2+) channels in the rear part of migrating cells. Notably, we found that this locally concentrated Ca(2+) influx acts as an essential transducer in establishing a global front-to-rear increasing Ca(2+) gradient. This asymmetrical Ca(2+) gradient is crucial for maintaining front-rear morphological polarity by restricting spontaneous lamellipodia formation in the rear part of migrating cells. Collectively, our findings demonstrate a clear link between local Ca(2+) sparklets and front-rear coordination during directed cell migration.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Movimento Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Optogenética/métodos , Células Endoteliais da Veia Umbilical Humana/citologia , HumanosRESUMO
Probing protein-protein interactions in living cells is crucial for understanding the protein functions and developing drugs. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time through live-cell imaging. A repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. The potential and utility of our approach was demonstrated by tracking the rapamycin-mediated interaction between FKBP12-rapamycin binding (FRB) domain and a FK506-binding protein (FKBP) and their localization by live-cell imaging. The present approach can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells.
Assuntos
Proteínas Luminescentes/metabolismo , Técnicas de Sonda Molecular , Mapeamento de Interação de Proteínas/métodos , Frações Subcelulares/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Tacrolimo/metabolismo , Sondas Moleculares/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteína Vermelha FluorescenteRESUMO
The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Exotoxinas/metabolismo , Espaço Intracelular/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Toxinas Shiga/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exotoxinas/química , Exotoxinas/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Toxinas Shiga/química , Toxinas Shiga/genética , Fatores de Virulência/química , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.
Assuntos
Optogenética , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Humanos , Pseudópodes/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Axônios/metabolismoRESUMO
In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to the formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from the Vax1AA/AA mouse optic stalk (OS) and grow slowly to arrive at the hypothalamus at a late stage. The RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas after failing to access the midline, resulting in reduced visual acuity and abnormal oculomotor responses. Overall, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in proper visuomotor responses.
Assuntos
Neuropeptídeos , Quiasma Óptico , Camundongos , Animais , Quiasma Óptico/metabolismo , Células Ganglionares da Retina , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Proteínas de Homeodomínio/metabolismoRESUMO
Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.
Assuntos
Células-Tronco Neurais , Animais , Proliferação de Células , Hipocampo , Mamíferos , Camundongos , Neurogênese , Transdução de SinaisRESUMO
Despite efforts to visualize the spatio-temporal dynamics of single messenger RNAs, the ability to precisely control their function has lagged. This study presents an optogenetic approach for manipulating the localization and translation of specific mRNAs by trapping them in clusters. This clustering greatly amplified reporter signals, enabling endogenous RNA-protein interactions to be clearly visualized in single cells. Functionally, this sequestration reduced the ability of mRNAs to access ribosomes, markedly attenuating protein synthesis. A spatio-temporally resolved analysis indicated that sequestration of endogenous ß-actin mRNA attenuated cell motility through the regulation of focal-adhesion dynamics. These results suggest a mechanism highlighting the indispensable role of newly synthesized ß-actin protein for efficient cell migration. This platform may be broadly applicable for use in investigating the spatio-temporal activities of specific mRNAs in various biological processes.
Assuntos
Optogenética/métodos , Biossíntese de Proteínas , RNA Mensageiro , Actinas/biossíntese , Actinas/genética , Animais , Proteína 9 Associada à CRISPR , Movimento Celular/genética , Células Cultivadas , Células HeLa , Humanos , Luz , Camundongos , Células NIH 3T3 , Ratos Sprague-DawleyRESUMO
Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.
Assuntos
Canais de Cálcio/metabolismo , Criptocromos/metabolismo , Tecnologia de Fibra Óptica , Optogenética , Molécula 1 de Interação Estromal/metabolismo , Animais , Astrócitos , Encéfalo/metabolismo , Cálcio/metabolismo , Criptocromos/química , Criptocromos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Neurônios/metabolismo , Alinhamento de Sequência , Molécula 1 de Interação Estromal/química , VigíliaRESUMO
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Monoméricas de Ligação ao GTP/análise , Optogenética/métodos , Transdução de Sinais , Análise de Célula Única/métodos , Animais , Técnicas Biossensoriais/instrumentação , Espinhas Dendríticas/metabolismo , Embrião de Mamíferos , Feminino , Células HeLa , Hipocampo/citologia , Humanos , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Optogenética/instrumentação , Técnicas de Cultura de Órgãos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Análise de Célula Única/instrumentação , Técnicas Estereotáxicas , Imagem com Lapso de TempoRESUMO
Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.
Assuntos
Actinas/metabolismo , Receptor trkB/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Feminino , Luz , Neuritos/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Optogenética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Cells employ signaling pathways to make decisions in response to changes in their immediate environment. Transforming growth factor beta (TGF-ß) is an important growth factor that regulates many cellular functions in development and disease. Although the molecular mechanisms of TGF-ß signaling have been well studied, our understanding of this pathway is limited by the lack of tools that allow the control of TGF-ß signaling with high spatiotemporal resolution. Here, we developed an optogenetic system (optoTGFBRs) that enables the precise control of TGF-ß signaling in time and space. Using the optoTGFBRs system, we show that TGF-ß signaling can be selectively and sequentially activated in single cells through the modulation of the pattern of light stimulations. By simultaneously monitoring the subcellular localization of TGF-ß receptor and Smad2 proteins, we characterized the dynamics of TGF-ß signaling in response to different patterns of blue light stimulations. The spatial and temporal precision of light control will make the optoTGFBRs system as a powerful tool for quantitative analyses of TGF-ß signaling at the single cell level.
Assuntos
Luz , Optogenética/métodos , Receptores de Fatores de Crescimento Transformadores beta , Transdução de Sinais/genética , Proteína Smad2 , Fator de Crescimento Transformador beta , Células HeLa , Humanos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Although microRNAs have emerged as key regulators in diverse cellular processes, the roles of microRNAs are poorly understood in human embryonic stem cells (hESCs) during differentiation into specialized cell types. In this study, we used a microRNA array with 799 human microRNA probes to examine the expression profiles of microRNAs in hESCs during differentiation into endodermal and mesodermal lineages in vitro. Among the microRNAs analyzed, 7 and 20 microRNAs were enriched in the developmental process of hESCs into mesodermal and endodermal lineages, respectively. In particular, the expression levels of miR-200 family, which is known to regulate the epithelial to mesenchymal transition (EMT), gradually increased in hESCs during differentiation into hepatocytes while they gradually decreased during differentiation into vascular endothelial cells. Downregulation of ZEB1, a direct target of miR-200 family, and E-CADHERIN, a target protein of ZEB1, was observed in hESCs during differentiation into endodermal and mesodermal lineages, respectively. These results indicate that miR-200 family has an important role in determining the cell fate between endodermal and mesodermal lineages from the pluripotent state.
RESUMO
Protein homo-oligomerization is an important molecular mechanism in many biological processes. Therefore, the ability to control protein homo-oligomerization allows the manipulation and interrogation of numerous cellular events. To achieve this, cryptochrome 2 (CRY2) from Arabidopsis thaliana has been recently utilized for blue light-dependent spatiotemporal control of protein homo-oligomerization. However, limited knowledge on molecular characteristics of CRY2 obscures its widespread applications. Here, we identify important determinants for efficient cryptochrome 2 clustering and introduce a new CRY2 module, named ''CRY2clust'', to induce rapid and efficient homo-oligomerization of target proteins by employing diverse fluorescent proteins and an extremely short peptide. Furthermore, we demonstrate advancement and versatility of CRY2clust by comparing against previously reported optogenetic tools. Our work not only expands the optogenetic clustering toolbox but also provides a guideline for designing CRY2-based new optogenetic modules.Cryptochrome 2 (CRY2) from A. thaliana can be used to control light-dependent protein homo-oligomerization, but the molecular mechanism of CRY2 clustering is not known, limiting its application. Here the authors identify determinants of CRY2 clustering and engineer fusion partners to modulate clustering efficiency.
Assuntos
Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Corantes Fluorescentes/química , Optogenética/métodos , Proteínas de Arabidopsis/genética , Análise por Conglomerados , Criptocromos/genética , Células HeLa , Humanos , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
FGFR1 is a member of the fibroblast growth factor family, which controls diverse cellular functions such as cell proliferation, migration, and differentiation. OptoFGFR1, an optogenetic method to modulate the FGFR signaling pathway with light by utilizing PHR domain of cryptochrome2 and cytoplasmic region of FGFR1, enabled light-guided activation of FGFR to study its effects on downstream signaling pathway and during diverse biological processes such as cell migration. Here, we describe about optogenetic and microscopic methods to spatiotemporally manipulate FGFR signaling in a single cell or group of cells using confocal microscope and LED array.
Assuntos
Criptocromos/metabolismo , Optogenética/métodos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Western Blotting/métodos , Técnicas de Cultura de Células/métodos , Criptocromos/genética , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia Confocal/métodos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transfecção/métodosRESUMO
We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping. The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells. We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.
Assuntos
Optogenética/métodos , Espalhamento de Radiação , Transdução de Sinais , Crânio/metabolismo , Animais , Cálcio/metabolismo , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Fibroblast growth factor receptors (FGFRs) regulate diverse cellular behaviors that should be exquisitely controlled in space and time. We engineered an optically controlled FGFR (optoFGFR1) by exploiting cryptochrome 2, which homointeracts upon blue light irradiation. OptoFGFR1 can rapidly and reversibly control intracellular FGFR1 signaling within seconds by illumination with blue light. At the subcellular level, localized activation of optoFGFR1 induced cytoskeletal reorganization. Utilizing the high spatiotemporal precision of optoFGFR1, we efficiently controlled cell polarity and induced directed cell migration. OptoFGFR1 provides an effective means to precisely control FGFR signaling and is an important optogenetic tool that can be used to study diverse biological processes both in vitro and in vivo.
Assuntos
Luz , Optogenética/métodos , Engenharia de Proteínas , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Polaridade Celular/efeitos da radiação , Criptocromos/química , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Análise Espaço-TemporalRESUMO
Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.
Assuntos
Fatores de Crescimento Neural/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Arabidopsis , Proteínas de Arabidopsis/genética , Linhagem Celular , Criptocromos/genética , Hipocampo/citologia , Humanos , Luz , Ratos , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismoRESUMO
AIM: Human embryonic stem cells (hESCs) are able to self-renew and differentiate into a variety of cell types. Although miRNAs have emerged as key regulators in the cellular process, a few studies have been reported about behaviors of miRNAs during differentiation of hESCs into a specialized cell type. Here, we demonstrate that different kinds of miRNAs may function in a lineage-specific manner during the differentiation of human embryonic stem cells (hESCs). METHODS: hESCs were induced to definitive endoderm (DE) cells and further differentiated to hepatocytes. The expression levels of miRNAs were examined in hESCs, DE cells, and hepatocytes by miRNA array using 799 human miRNA probes. RESULTS: Among 387 miRNAs significantly detected, 13 and 56 miRNAs were downregulated and upregulated during transition of hESCs to DE cells, respectively, while 30 and 92 miRNAs were downregulated and upregulated during differentiation of DE cells to hepatocytes, respectively. In particular, 5, 4, and 86 miRNAs were enriched in hESCs, DE cells, and hepatocytes, respectively. Quantitative RT-PCR represented that miR-512-3p, miR-512-5p and miR-520c-3p were enriched in hESCs, miR-9*, miR-205 and miR-375 in hESC-derived DE cells, and miR-10a, miR-122 and miR-21 in hESC-derived hepatocytes. Expression patterns of lineage-specific miRNAs in the liver tissue were similar to those of hESC-derived hepatocytes. CONCLUSION: The results indicate that different kinds of miRNAs may function in a lineage-specific manner during differentiation of hESCs into a specialized cell type.