RESUMO
Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.
Assuntos
Perda Auditiva Neurossensorial , Células-Tronco Pluripotentes Induzidas , Mutação , Humanos , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Linhagem Celular , Feminino , Transativadores/genética , Diferenciação Celular , Criança , CariótipoRESUMO
We report the establishment of a human induced pluripotent stem cell (iPSC) line from a 54-year-old male patient with an A1555G mutation in the mitochondrial 12S ribosomal RNA gene (MTRNR1), associated with sensorineural hearing loss. The established iPSC line expressed stemness markers or undifferentiated state markers. We also demonstrated the capacity of the cells to differentiate into the three germ layers, suggesting its pluripotency and utility in the pathological study of sensorineural hearing loss and drug screening for ear disorders.
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DNA Mitocondrial , Células-Tronco Pluripotentes Induzidas , Mutação , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , DNA Mitocondrial/genética , Masculino , Pessoa de Meia-Idade , Diferenciação Celular , Linhagem Celular , RNA Ribossômico/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Perda Auditiva/genética , Perda Auditiva/patologiaRESUMO
Mammalian auditory hair cells transduce sound-evoked traveling waves in the cochlea into nerve stimuli, which are essential for hearing function. Pillar cells located between the inner and outer hair cells are involved in the formation of the tunnel of Corti, which incorporates outer-hair-cell-driven fluid oscillation and basilar membrane movement, leading to the fine-tuned frequency-specific perception of sounds by the inner hair cells. However, the detailed molecular mechanism underlying the development and maintenance of pillar cells remains to be elucidated. In this study, we examined the expression and function of brain-specific angiogenesis inhibitor 3 (Bai3), an adhesion G-protein-coupled receptor, in the cochlea. We found that Bai3 was expressed in hair cells in neonatal mice and pillar cells in adult mice, and, interestingly, Bai3 knockout mice revealed the abnormal formation of pillar cells, with the elevation of the hearing threshold in a frequency-dependent manner. Furthermore, old Bai3 knockout mice showed the degeneration of hair cells and spiral ganglion neurons in the basal turn. The results suggest that Bai3 plays a crucial role in the development and/or maintenance of pillar cells, which, in turn, are necessary for normal hearing function. Our results may contribute to understanding the mechanisms of hearing loss in human patients.
Assuntos
Cóclea , Audição , Proteínas de Membrana , Proteínas do Tecido Nervoso , Animais , Camundongos , Encéfalo , Cóclea/metabolismo , Células Ciliadas Auditivas Externas , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas de Membrana/genéticaRESUMO
Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type â SGNs (SGN-â ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-â s even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.
Assuntos
Neurônios , Gânglio Espiral da Cóclea , Animais , Camundongos , Gânglio Espiral da Cóclea/metabolismo , Neurônios/metabolismo , Apoptose/fisiologia , Transmissão Sináptica/fisiologia , Camundongos Knockout , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
We report the establishment of two human induced pluripotent stem cell (iPSC) lines from individuals without auditory disorders. Extensive audiometry tests were performed to confirm normal hearing. The generated iPSC lines expressed pluripotency genes and showed differentiation capability into the three germ layers. The iPSC lines will be used as controls for pathological analysis and drug screening for ear disorders.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genéticaRESUMO
Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs. Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors. Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1. Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.
RESUMO
Mammalian auditory hair cells are not spontaneously replaced. Their number and coordinated polarization are fairly well-maintained and both these factors might be essential for the cochlear amplifier. Cell cycle regulation has critical roles in regulating appropriate cell size and cell number. However, little is known about the physiological roles of the Hippo pathway, which is one of the most important signaling cascades that regulates cell growth, differentiation, and regenerative capacity in the cochlear sensory epithelium. Herein, we investigated the in vivo role of the large tumor suppressor 1 (LATS1), an essential kinase in the Hippo/yes-associated protein pathway, in the cochlea using the LATS1 knockout mice. LATS1 was expressed in hair cells and supporting cells. It was strongly expressed on the surface of the cuticular plate of the organ of Corti. We found that LATS1 knockout caused congenital hearing loss due to the irregular orientation and slightly reduced number of hair cells, whereas the number of supporting cells remained unchanged. On the surface of the hair cells, the kinocilium and stereocilia were dispersed during and after morphogenesis. However, the expression of the receptor-independent polarity regulators, such as Par3 or Gαi, was not affected. We concluded that LATS1 has an indispensable role in the maturation of mammalian auditory hair cells, but not in the development of the supporting cells, and thus, has a role in the hearing acquisition.
Assuntos
Cóclea/patologia , Perda Auditiva/congênito , Perda Auditiva/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Cóclea/metabolismo , Feminino , Deleção de Genes , Perda Auditiva/patologia , Masculino , Camundongos KnockoutRESUMO
OBJECTIVES: Autophagy is an intracellular housekeeping process that degrades cytoplasmic organelles, damaged molecules, and abnormal proteins or pathogens and is essential for normal hearing. Recent studies revealed the essential roles of autophagy in hearing and balance. The aim of this study was to evaluate the activation state of rapamycin-induced autophagy in cochlear outer sulcus cells (OSCs). METHODS: We used autophagy reporter transgenic mice expressing the green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) fusion protein and counted GFP-LC3 puncta in cochlear OSCs to evaluate the activation state of autophagy after oral administration of rapamycin. RESULTS: We observed basal level GFP-LC3 expression and an increase in the number of GFP-LC3 puncta in cochlear OSCs by oral administration of rapamycin. This increase was detected when the daily rapamycin intake was as low as 0.025 mg/kg, and it was dose dependent. The increased number of puncta was more at the basal turn than the apical turn. CONCLUSION: Oral intake of low-dose rapamycin activates autophagy in cochlear OSCs. LEVEL OF EVIDENCE: NA.
RESUMO
SLC26A4 is the second most frequent gene implicated in congenital hearing loss after GJB2 mutations. Here, we report the generation of induced pluripotent stem cells (iPSCs), from a patient who was carrying a homozygous c.919-2A>G variant in the SLC26A4 gene. This is the most common variant of SLC26A4 gene in the Chinese population and the second most prevalent one in other Asian countries. The established patient-derived iPSC displayed all the features of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.
Assuntos
Bócio Nodular/patologia , Perda Auditiva Neurossensorial/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Sítios de Splice de RNA/genética , Transportadores de Sulfato/genética , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Feminino , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
INTRODUCTION: Pendred syndrome is an autosomal-recessive disease characterized by congenital hearing loss and thyroid goiter. Previously, cell stress susceptibilities were shown to increase in patient-derived cells with intracellular aggregation using an in vitro acute cochlear cell model derived from patient-specific pluripotent stem (iPS) cells. Moreover, we showed that rapamycin can relieve cell death. However, studies regarding long-term cell survival without cell stressors that mimic the natural course of disease or the rational minimum concentration of rapamycin that prevents cell death are missing. METHODS: In this report, we first investigated the rational minimum concentration of rapamycin using patient-specific iPS cells derived-cochlear cells with three different conditions of acute stress. We next confirmed the effects of rapamycin in long-term cell survival and phenotypes by using cochlear cells derived from three different patient-derived iPS cells. RESULTS: We found that inner ear cells derived from Pendred syndrome patients are more vulnerable than those from healthy individuals during long-term culturing; however, this susceptibility was relieved via treatment with low-dose rapamycin. The slow progression of hearing loss in patients may be explained, in part, by the vulnerability observed in patient cells during long-term culturing. We successfully evaluated the rational minimum concentration of rapamycin for treatment of Pendred syndrome. CONCLUSION: Our results suggest that low-dose rapamycin not only decreases acute symptoms but may prevent progression of hearing loss in Pendred syndrome patients.
RESUMO
There is, at present, no curative treatment for genetic hearing loss. We have previously reported that transuterine gene transfer of wild type CONNEXIN30 (CX30) genes into otocysts in CX30-deleted mice could restore hearing. Cell transplantation therapy might be another therapeutic option, although it is still unknown whether stem cell-derived progenitor cells could migrate into mouse otocysts. Here, we show successful cell transplantation of progenitors of outer sulcus cell-like cells derived from human-derived induced pluripotent stem cells into mouse otocysts on embryonic day 11.5. The delivered cells engrafted more frequently in the non-sensory region in the inner ear of CX30-deleted mice than in wild type mice and survived for up to 1 week after transplantation. Some of the engrafted cells expressed CX30 proteins in the non-sensory region. This is the first report that demonstrates successful engraftment of exogenous cells in prenatal developing otocysts in mice. Future studies using this mouse otocystic injection model in vivo will provide further clues for developing treatment modalities for congenital hearing loss in humans.
Assuntos
Orelha Interna/embriologia , Embrião de Mamíferos/metabolismo , Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco , Animais , Contagem de Células , Células Cultivadas , Cóclea , Conexina 30 , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Most cells in tissues are polarized and usually have two distinct plasma membrane domains-an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin-Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a-knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.
Assuntos
Claudinas/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Claudinas/genética , Vesículas Citoplasmáticas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cães , Feminino , Expressão Gênica , Humanos , Rim/citologia , Rim/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos ICR , Microscopia de Fluorescência , Transporte Proteico , Junções Íntimas/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
A number of transcriptional pathways regulating fetal lung development are active during repair of the injured lung. We hypothesized that C/EBPalpha, a transcription factor critical for lung maturation, plays a role in protection of the alveolar epithelium following hyperoxic injury of the mature lung. Transgenic Cebpalpha(Delta/Delta) mice, in which Cebpalpha was conditionally deleted from Clara cells and type II cells after birth, were developed. While no pulmonary abnormalities were observed in the Cebpalpha(Delta/Delta) mice (7-8 wk old) under normal conditions, the mice were highly susceptible to hyperoxia. Cebpalpha(Delta/Delta) mice died within 4 days of exposure to 95% oxygen in association with severe lung inflammation, altered maturation of surfactant protein B and C, decreased surfactant lipid secretion, and abnormal lung mechanics at a time when all control mice survived. mRNA microarray analysis of isolated type II cells at 0, 2, and 24 h of hyperoxia demonstrated the reduced expression of number of genes regulating surfactant lipid and protein homeostasis, including Srebf, Scap, Lpcat1, Abca3, Sftpb, and Napsa. Genes influencing cell signaling or immune responses were induced in the lungs of Cebpalpha(Delta/Delta) mice. C/EBPalpha was required for the regulation of genes associated with surfactant lipid homeostasis, surfactant protein biosynthesis, processing and transport, defense response to stress, and cell redox homeostasis during exposure to hyperoxia. While C/EBPalpha did not play a critical role in postnatal pulmonary function under normal conditions, C/EBPalpha mediated protection of the lung during acute lung injury induced by hyperoxia.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Hiperóxia/fisiopatologia , Alvéolos Pulmonares/fisiologia , Mucosa Respiratória/fisiologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/mortalidade , Animais , Líquido da Lavagem Broncoalveolar , Deleção de Genes , Homeostase/fisiologia , Hiperóxia/metabolismo , Hiperóxia/mortalidade , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Oxigênio/toxicidade , Regiões Promotoras Genéticas/fisiologia , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismoRESUMO
Slp1 is a putative Rab27 effector protein and implicated in intracellular membrane transport; however, the precise tissue distribution and function of Slp1 protein remain largely unknown. In this study we investigated the tissue distribution of Slp1 in mice and found that Slp1 is abundantly expressed in the pancreas, especially in the apical region of pancreatic acinar cells. Slp1 interacted with Rab27B in vivo and both proteins were co-localized on zymogen granules. Morphological analysis of fasted Slp1 knockout mice showed an increased number of zymogen granules in the pancreatic acinar cells, indicating that Slp1 is part of the machinery of amylase secretion by the exocrine pancreas.
Assuntos
Amilases/metabolismo , Expressão Gênica , Proteínas de Membrana/metabolismo , Pâncreas Exócrino/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pâncreas Exócrino/citologia , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a-interacting proteins have been identified; however, only Munc13-4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin-like proteins (Slps): Slp1/JFC1, Slp2-a/exophilin4, Slp3-a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2-a are expressed in CTL. Isoforms of Slp2-a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2-a interact with Rab27a in CTL, and Slp2-a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2-a contains PEST-like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2-a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2-a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2-a or Slp1 fail to impair CTL-mediated killing of targets; however, overexpression of a dominant-negative construct consisting of the SHD of Slp2-a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2-a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2-a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.
Assuntos
Proteínas Sanguíneas/imunologia , Membrana Celular/metabolismo , Proteínas de Membrana/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Proteínas Sanguíneas/genética , Calpaína/genética , Calpaína/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologia , Proteínas rab27 de Ligação ao GTPRESUMO
Synaptotagmin-like protein (Slp) 2-a is a putative Rab27A/B-effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2-a protein and its functions remain largely unknown. In this study we used a specific anti-Slp2-a antibody to investigate the tissue distribution of Slp2-a in mice and found that Slp2-a is most abundantly expressed in mouse stomach. Co-immunoprecipitation experiments indicated that Slp2-a interacts with Rab27A/B in vivo. We also discovered that Slp2-a and Rab27A/B are predominantly localized at the apical region of gastric-surface mucous cells, where mucus granules are accumulated. Analysis of Slp2-a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric-surface mucous cells and reduction of mucus secretion by Slp2-a-deficient gastric primary cells. Based on these results, we propose that Slp2-a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.
Assuntos
Mucosa Gástrica/metabolismo , Proteínas de Membrana/metabolismo , Muco/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Mucosa Gástrica/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Recombinação Genética , Estômago/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Membrana Celular/metabolismo , Exocitose , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/genética , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/metabolismo , Sinaptotagminas , TransfecçãoRESUMO
Basic helix-loop-helix (bHLH) transcription factors are implicated in cell fate determination and differentiation in neurogenesis. We identified a novel chick bHLH transcription factor, NeuroAB. A phylogenetic tree prepared from bHLH sequences suggested that NeuroAB belongs to the BETA3 group in the Atonal-related protein family (ARPs). In situ hybridization and immunostaining indicated that NeuroAB is expressed predominantly in postmitotic bipolar cells and GABAergic amacrine cells in the retina. Reporter and DNA pull down assays indicated that NeuroAB functions as a transcriptional repressor by binding to the E-box sequence, and its activity is modulated by phosphorylation at a specific serine residue that fits the consensus phosphorylation site for glycogen synthase kinase 3beta (GSK3beta). Since members of the BETA3 group possess this consensus site, it is suggested that their activities are commonly regulated by GSK3beta or other kinases bearing the same substrate specificity. We found that the expression of GSK3beta is spatially and temporally regulated in the developing retina; its strong expression was observed in ganglion cells from E8 and a subset of amacrine cells from E12. These findings suggest that NeuroAB is involved in the maturation and maintenance of bipolar cells and GABAergic amacrine cells and regulation by GSK3beta plays an important role in retinogenesis.
Assuntos
Células Amácrinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Proteínas Repressoras/metabolismo , Retina/citologia , Células Amácrinas/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Embrião de Galinha/anatomia & histologia , Embrião de Galinha/fisiologia , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Proteínas Repressoras/classificação , Proteínas Repressoras/genética , Retina/embriologia , Retina/metabolismo , Transcrição GênicaRESUMO
Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598-5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca(2+)-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.
Assuntos
Cálcio/fisiologia , Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Glicoproteínas de Membrana/metabolismo , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Grânulos Citoplasmáticos/ultraestrutura , Exocitose/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Cinética , Aprendizagem , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Memória , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Células PC12 , Feocromocitoma , Cloreto de Potássio/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , SinaptotagminasRESUMO
Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic beta-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542-28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitro and in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218). How Slp4-a.Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a.Rab8 and Slp4-a.Rab27A complexes, but not Slp4-a.Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.