RESUMO
Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2(-/-) mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.
Assuntos
Autofagia , Canais de Cálcio/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Animais , Canais de Cálcio/genética , Homeostase , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Peptídeo Hidrolases/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismoRESUMO
Trimeric intracellular cation channels (TRIC) represents a novel class of trimeric intracellular cation channels. Two TRIC isoforms have been identified in both the human and the mouse genomes: TRIC-A, a subtype predominantly expressed in the sarcoplasmic reticulum (SR) of muscle cells, and TRIC-B, a ubiquitous subtype expressed in the endoplasmic reticulum (ER) of all tissues. Genetic ablation of either TRIC-A or TRIC-B leads to compromised K(+) permeation and Ca(2+) release across the SR/ER membrane, supporting the hypothesis that TRIC channels provide a counter balancing K(+) flux that reduces SR/ER membrane depolarization for maintenance of the electrochemical gradient that drives SR/ER Ca(2+) release. TRIC-A and TRIC-B seem to have differential functions in Ca(2+) signaling in excitable and nonexcitable cells. Tric-a(-/-) mice display defective Ca(2+) sparks and spontaneous transient outward currents in arterial smooth muscle and develop hypertension, in addition to skeletal muscle dysfunction. Knockout of TRIC-B results in abnormal IP3 receptor-mediated Ca(2+) release in airway epithelial cells, respiratory defects, and neonatal lethality. Double knockout mice lacking both TRIC-A and TRIC-B show embryonic lethality as a result of cardiac arrest. Such an aggravated lethality indicates that TRIC-A and TRIC-B share complementary physiological functions in Ca(2+) signaling in embryonic cardiomyocytes. Tric-a(-/-) and Tric-b(+/-) mice are viable and susceptible to stress-induced heart failure. Recent evidence suggests that TRIC-A directly modulates the function of the cardiac ryanodine receptor 2 Ca(2+) release channel, which in turn controls store-overload-induced Ca(2+) release from the SR. Thus, the TRIC channels, in addition to providing a countercurrent for SR/ER Ca(2+) release, may also function as accessory proteins that directly modulate the ryanodine receptor/IP3 receptor channel functions.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/fisiologia , Canais Iônicos/metabolismo , Retículo Sarcoplasmático/fisiologia , Animais , Homeostase/fisiologia , Humanos , Canais Iônicos/genética , CamundongosRESUMO
Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5-11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.
Assuntos
Degradação Associada com o Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Saco Vitelino/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Toxina da Cólera/metabolismo , Desenvolvimento Embrionário/genética , Endoderma/citologia , Endoderma/patologia , Estresse do Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Dobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno , alfa 1-Antitripsina/metabolismoRESUMO
The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.
Assuntos
Pressão Sanguínea/fisiologia , Sinalização do Cálcio/fisiologia , Canais Iônicos/biossíntese , Proteínas Musculares/biossíntese , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Expressão Gênica , Canais Iônicos/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologiaRESUMO
Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.
Assuntos
Caderinas/análise , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/genética , Linhagem Celular , Células Cultivadas , Fator de Transcrição GATA4/genética , Expressão Gênica , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.
Assuntos
Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Canais Iônicos/metabolismo , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Sinalização do Cálcio , Citoplasma/metabolismo , Coração/embriologia , Canais Iônicos/isolamento & purificação , Camundongos , Camundongos Knockout , Microssomos/metabolismo , Dados de Sequência Molecular , Permeabilidade , Potássio/metabolismo , Coelhos , Retículo Sarcoplasmático/metabolismoRESUMO
TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the endoplasmic reticulum (ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B (also referred to as TMEM38B) locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model; Tric-b deficiency deranges ER Ca2+ handling and thus reduces extracellular matrix (ECM) synthesis in osteoblasts, leading to poor mineralization. Here we report irregular cell death and insufficient ECM in long-bone growth plates from Tric-b-knockout embryos. In the knockout growth plate chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, activated PERK/eIF2α (PKR-like ER kinase/ eukaryotic initiation factor 2α) signaling seemed to inordinately alter gene expression to induce apoptosis-related proteins including CHOP (CCAAT/enhancer binding protein homologous protein) and caspase 12 in the knockout chondrocytes. Ca2+ imaging detected aberrant Ca2+ handling in the knockout chondrocytes; ER Ca2+ release was impaired, while cytoplasmic Ca2+ level was elevated. Our observations suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic states by compromising cellular Ca2+-handling and exacerbating ER stress response, leading to impaired ECM synthesis and accidental cell death.
Assuntos
Retículo Endoplasmático , Lâmina de Crescimento , Animais , Camundongos , Lâmina de Crescimento/metabolismo , Camundongos Knockout , Morte Celular , Retículo Endoplasmático/metabolismo , Transdução de Sinais , Estresse do Retículo Endoplasmático/genética , Canais Iônicos/metabolismoRESUMO
We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.
Assuntos
Técnicas Genéticas , Camundongos Transgênicos/genética , Plasmídeos/genética , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Interferência de RNA , RNA Interferente Pequeno/genética , Distribuição TecidualRESUMO
The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor â¼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.
Assuntos
Cálcio/metabolismo , Canais Iônicos/deficiência , Canais Iônicos/genética , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Transporte Biológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Coelhos , Retículo Sarcoplasmático/genéticaRESUMO
This study investigated the development of Ca²(+) signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca²(+) channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca²(+) concentration ([Ca²(+)](i)) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca²(+)](i) in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca²(+)](i) after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca²(+) -induced Ca²(+) release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.
Assuntos
Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Gastrulação/fisiologia , Salamandridae/embriologia , Salamandridae/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.
Assuntos
Envelhecimento/metabolismo , Sinalização do Cálcio , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes , Estimulação Elétrica , Homeostase , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/ultraestrutura , Pressão Osmótica , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Epêndima/citologia , Proteína Smad6/fisiologia , Proteína Smad7/fisiologia , Animais , Linhagem Celular , Cílios/fisiologia , Epêndima/fisiologia , Camundongos , Proteína Smad6/genética , Proteína Smad7/genéticaRESUMO
Claudin proteins are the major components of tight junctions connecting adjacent cells, where they regulate a variety of cellular activities. In the present paper we identified two Xenopus claudin5 genes (cldn5a and 5b), which are expressed early in the developing cardiac region. Precocious cldn5 expression was observed in explants of non-heart-forming mesoderm under inhibition of the canonical Wnt pathway. Cardiogenesis was severely perturbed by antisense oligonucleotides against cldn5 or by Cldn5 proteins lacking the cytoplasmic domain. Results of light- and electron-microscopic observations suggested that cldn5a and 5b are required for Xenopus heart tube formation through epithelialization of the precardiac mesoderm.
Assuntos
Coração/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus/genética , Xenopus/metabolismo , Animais , Claudina-5 , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/química , Xenopus/embriologia , Proteínas de Xenopus/químicaRESUMO
During vertebrate cardiac development, the heart tube formed by fusion of right and left presumptive cardiac mesoderms (PCMs) undergoes looping toward the right, resulting in an asymmetrical heart. Here, we examined the right and left PCMs with regard to heart-tube looping using right- and left-half newt embryos (Cynops pyrrhogaster ). In the half embryos, the rightward (normal) loop of the heart tube was formed from the left PCM, irrespective of the timing of its separation, while the leftward (reversed) loop of the heart tube was formed from the right PCM, separated by stage 18. In addition, the direction of the leftward loop was inverted to the rightward direction in right-half embryos bisected after stage 18. Incision or resection of the embryonic caudal region implicated interactions between the right and left sides of this region as crucial for inverting the direction of the heart-tube loop from leftward to rightward in the right-half embryos. In situ hybridization of CyNodal (Cynops nodal-related gene) suggested that the inversion of heart looping in the right-half embryos has no association with the CyNodal expression pattern. Based on these findings, we propose a mechanism for the rightward looping underlying normal amphibian cardiac development.
Assuntos
Lateralidade Funcional/fisiologia , Coração/embriologia , Organogênese/fisiologia , Salamandridae/embriologia , Animais , Embrião não Mamífero/cirurgia , Hibridização In Situ , Ligadura , Mesoderma/fisiologia , Modelos BiológicosRESUMO
The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.
Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Animais , Dimerização , Retículo Endoplasmático/ultraestrutura , Células HEK293 , Humanos , Luz , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/efeitos da radiação , Células NIH 3T3 , OptogenéticaRESUMO
The nodal and nodal-related genes play fundamental roles during deuterostome left-right axis formation. Several of these genes show left-sided expression in the lateral plate mesoderm and brain region. We have isolated the nodal-related gene, CyNodal, from Cynops pyrrhogaster. CyNodal mRNA is detected at the marginal zone and left side of several tissues. The left-sideness of CyNodal mRNA expression is highly conserved throughout vertebrate evolution. However, CyNodal mRNA expression shows little variation from the Xenopus nodal-related gene 1, in that CyNodal gene expression in the left lateral plate mesoderm shifts from posterior to anterior at least twice.
Assuntos
Padronização Corporal/genética , Diencéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Proteínas/genética , Salamandridae , Sequência de Aminoácidos , Animais , Padronização Corporal/fisiologia , Sequência Conservada , Cisteína/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Embrião não Mamífero , Evolução Molecular , Feminino , Hibridização In Situ , Dados de Sequência Molecular , Salamandridae/embriologia , Salamandridae/genética , Salamandridae/metabolismo , Homologia de Sequência de Aminoácidos , Coloração e RotulagemRESUMO
We isolated and characterized the Xenopus translocon-associated protein XTRAP-gamma, one of four subunits of the translocon-associated protein complex. TRAP has been proposed to aid the translocation of nascent polypeptides into the lumen of the endoplasmic reticulum, but this has not been demonstrated until now. XTRAP-gamma was specifically expressed in the pronephros tubules of Xenopus laevis from stage 25 during kidney development. Antisense morpholino oligonucleotide-mediated knockdown of XTRAP-gamma suppressed tubulogenesis and decreased expression of the pronephros marker genes Pax-2 and Wnt-4. XTRAP-gamma morpholinos also inhibited differentiation of the pronephros in activin/retinoic acid-treated animal caps. We conclude that XTRAP-gamma plays an important role in the process of pronephros differentiation.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Embrião não Mamífero/fisiologia , Rim/embriologia , Glicoproteínas de Membrana/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Peptídeos/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Ativinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Proteínas de Homeodomínio , Hibridização In Situ , Inibinas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Regulação para Cima , Proteínas de Xenopus/genéticaRESUMO
The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.
Assuntos
Osso e Ossos/metabolismo , Calcificação Fisiológica , Colágeno/metabolismo , Canais Iônicos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Cátions/metabolismo , Colágeno/química , Retículo Endoplasmático/metabolismo , Feminino , Fêmur/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Crânio/metabolismo , Microtomografia por Raio-XRESUMO
Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, ß-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Medicina Regenerativa/métodos , Estômago/fisiologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Gastrite Hipertrófica/genética , Gastrite Hipertrófica/metabolismo , Gastrite Hipertrófica/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Histamina/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Organogênese , Pepsinogênio A/metabolismo , Fenótipo , Esferoides Celulares , Estômago/citologia , Estômago/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation.