RESUMO
Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.
Assuntos
Encéfalo , Polaridade Celular , Camundongos Knockout , Neurônios , Vesículas Sinápticas , Proteínas rab de Ligação ao GTP , Animais , Polaridade Celular/fisiologia , Camundongos , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Neurônios/metabolismo , Feminino , Masculino , Vesículas Sinápticas/metabolismo , Encéfalo/metabolismo , Encéfalo/embriologia , Encéfalo/citologia , Células CultivadasRESUMO
Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.
Assuntos
Actinas , Proteínas de Transporte , Cílios , Actinas/metabolismo , Cílios/metabolismo , Ligação Proteica , Proteínas rab de Ligação ao GTP/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismoRESUMO
In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)-specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.
Assuntos
Encéfalo/patologia , Polaridade Celular , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Qb-SNARE/deficiência , Proteínas Qc-SNARE/deficiência , Animais , Apoptose , Encéfalo/fisiopatologia , Células COS , Caderinas/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Regulação para Baixo , Marcha , Camundongos Knockout , Neurogênese , Neurônios/patologia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE , Receptores Notch/metabolismo , Transdução de Sinais , Sintaxina 1/metabolismo , Vesículas Transportadoras/metabolismo , beta Catenina/metabolismoRESUMO
Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana/genética , Mitocôndrias , Canal de Ânion 2 Dependente de Voltagem/genética , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lipoilação , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
Intestinal epithelial cells (IECs) are not only responsible for the digestion and absorption of dietary substrates but also function as a first line of host defense against commensal and pathogenic luminal bacteria. Disruption of the epithelial layer causes malnutrition and enteritis. Rab6 is a small GTPase localized to the Golgi, where it regulates anterograde and retrograde transport by interacting with various effector proteins. Here, we generated mice with IEC-specific deletion of Rab6a (Rab6a∆IEC mice). While Rab6aΔIEC mice were born at the Mendelian ratio, they started to show IEC death, inflammation, and bleeding in the small intestine shortly after birth, and these changes culminated in early postnatal death. We further found massive lipid accumulation in the IECs of Rab6a∆IEC neonates. In contrast to Rab6a∆IEC neonates, knockout embryos did not show any of these abnormalities. Lipid accumulation and IEC death became evident when Rab6a∆IEC embryos were nursed by a foster mother, suggesting that dietary milk-derived lipids accumulated in Rab6a-deficient IECs and triggered IEC death. These results indicate that Rab6a plays a crucial role in regulating the lipid transport and maintaining tissue integrity.
Assuntos
Morte Celular , Células Epiteliais/patologia , Inflamação/patologia , Intestino Delgado/patologia , Lactação , Lipídeos/química , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Células Epiteliais/metabolismo , Feminino , Glicosilação , Inflamação/etiologia , Inflamação/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. Here, we show that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. We envision that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfatos de Fosfatidilinositol/genéticaRESUMO
Diet affects health through ingested calories and macronutrients, and macronutrient balance affects health span. The mechanisms regulating macronutrient-based diet choices are poorly understood. Previous studies had shown that NAD-dependent deacetylase sirtuin-1 (SIRT1) in part influences the health-promoting effects of caloric restriction by boosting fat use in peripheral tissues. Here, we show that neuronal SIRT1 shifts diet choice from sucrose to fat in mice, matching the peripheral metabolic shift. SIRT1-mediated suppression of simple sugar preference requires oxytocin signalling, and SIRT1 in oxytocin neurons drives this effect. The hepatokine FGF21 acts as an endocrine signal to oxytocin neurons, promoting neuronal activation and Oxt transcription and suppressing the simple sugar preference. SIRT1 promotes FGF21 signalling in oxytocin neurons and stimulates Oxt transcription through NRF2. Thus, neuronal SIRT1 contributes to the homeostatic regulation of macronutrient-based diet selection in mice.
Assuntos
Dieta , Fatores de Crescimento de Fibroblastos/metabolismo , Neurônios/metabolismo , Ocitocina/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Animais , Sequência de Bases , Comportamento de Escolha , Jejum , Feminino , Glucuronidase/metabolismo , Proteínas Klotho , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Ocitocina/genética , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , SacaroseRESUMO
The induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ), is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth.
Assuntos
Apoptose/efeitos dos fármacos , Cloroquina/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Trombina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Metástase Neoplásica , Proteínas Oncogênicas/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas rab de Ligação ao GTPRESUMO
VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.
Assuntos
Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/fisiologia , Proteínas R-SNARE/fisiologia , Trifosfato de Adenosina/biossíntese , Animais , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Homeostase , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout , Proteínas R-SNARE/deficiênciaRESUMO
The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Polaridade Celular , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Proteínas de Transporte/genética , Dinaminas/metabolismo , Células Epiteliais/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Mucosa Intestinal/ultraestrutura , Intestino Delgado/ultraestrutura , Lisossomos/enzimologia , Camundongos , Camundongos Knockout , Microvilosidades/enzimologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Organoides , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
Assuntos
Endossomos/enzimologia , Vírus do Nilo Ocidental/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Transporte Biológico , Chlorocebus aethiops , Endossomos/virologia , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Transporte Proteico , Vesículas Transportadoras/virologia , Células Vero , Proteínas Virais , Liberação de Vírus , Replicação ViralRESUMO
Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.
Assuntos
Actinas/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Chaperonina com TCP-1/fisiologia , Chaperoninas do Grupo II/fisiologia , Mucosa Intestinal/ultraestrutura , Microvilosidades/fisiologia , Tubulina (Proteína)/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Citoplasma/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Mucosa Intestinal/fisiologia , Microtúbulos/ultraestrutura , Microvilosidades/ultraestruturaRESUMO
Intestinal epithelial cells are highly polarized and exhibit a complex architecture with a columnar shape and a specialized apical surface supporting microvilli organized in a brush border. These microvilli are rooted in a dense meshwork of acto-myosin called the terminal web. We have shown recently that Drebrin E, an F-actin-binding protein, is a key protein for the organization of the terminal web and the brush border. Drebrin E is also required for the columnar cell shape of Caco2 cells (human colonic cells). Here, we found that the subcellular localization of several apical markers including dipeptidyl peptidase IV (DPPIV) was strikingly modified in Drebrin E-depleted Caco2 cells. Instead of being mostly present at the apical surface, these proteins are accumulated in an enlarged subapical compartment. Using known intracellular markers, we show by both confocal and electron microscopy that this compartment is related to lysosomes. We also demonstrate that the enrichment of DPPIV in this compartment originates from apical endocytosis and that depletion of Rab8a induces an accumulation of apical proteins in a similar compartment. Consistent with this, the phenotype observed in Drebrin E knock-down Caco2 cells shares some features with a pathology called microvillar inclusion disease (MVID) involving both Myosin Vb and Rab8a. Taken together, these results suggest that Drebrin E redirects the apical recycling pathway in intestinal epithelial cells to the lysosomes, demonstrating that Drebrin E is a key regulator in apical trafficking in Caco2 cells.
Assuntos
Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Neuropeptídeos/deficiência , Proteínas rab de Ligação ao GTP/metabolismo , Células CACO-2 , Polaridade Celular , Endocitose , Técnicas de Silenciamento de Genes , Humanos , Intestinos/citologia , Microvilosidades/genética , Microvilosidades/metabolismo , Neuropeptídeos/genética , Transporte Proteico , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca(2+) response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Células Dendríticas/imunologia , Eosinofilia Pulmonar/imunologia , Receptores Acoplados a Proteínas G/imunologia , Transferência Adotiva , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Cálcio/metabolismo , Movimento Celular , Células Dendríticas/patologia , Células Dendríticas/transplante , Feminino , Regulação da Expressão Gênica , Células Caliciformes/imunologia , Células Caliciformes/patologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Pulmão/imunologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Knockout , Ovalbumina , Eosinofilia Pulmonar/induzido quimicamente , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/patologia , Receptores CCR7/genética , Receptores CCR7/imunologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Equilíbrio Th1-Th2 , Células Th2/imunologia , Células Th2/patologiaRESUMO
Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.
Assuntos
Glucosilceramidas/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Globosídeos/biossíntese , Globosídeos/química , Globosídeos/metabolismo , Glucosilceramidas/química , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Rede trans-Golgi/metabolismoRESUMO
The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.
Assuntos
Endoderma/metabolismo , Mucosa Intestinal/metabolismo , Fosfatidilinositol 3-Quinases/genética , Vísceras/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Intestinos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Vísceras/embriologia , Vísceras/ultraestruturaRESUMO
The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.
Assuntos
Regulação da Expressão Gênica/fisiologia , Mutagênese Insercional/métodos , Neurônios/citologia , Neurônios/metabolismo , Animais , Toxinas Bacterianas , Southern Blotting , Clonagem Molecular , Técnicas de Silenciamento de Genes , Vetores Genéticos , Cariótipo , Células PC12 , Proteínas Citotóxicas Formadoras de Poros , RNA Interferente Pequeno , Ratos , Retroviridae , Fatores de TranscriçãoRESUMO
Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown as a receptor for protons. In the present study, we aimed to know whether OGR1 plays a role in insulin secretion and, if so, the manner in which it does. To this end, we created OGR1-deficient mice and examined insulin secretion activity in vivo and in vitro. OGR1 deficiency reduced insulin secretion induced by glucose administered ip, although it was not associated with glucose intolerance in vivo. Increased insulin sensitivity and reduced plasma glucagon level may explain, in part, the unusual normal glucose tolerance. In vitro islet experiments revealed that glucose-stimulated insulin secretion was dependent on extracellular pH and sensitive to OGR1; insulin secretion at pH 7.4 to 7.0, but not 8.0, was significantly suppressed by OGR1 deficiency and inhibition of G(q/11) proteins. Insulin secretion induced by KCl and tolbutamide was also significantly inhibited, whereas that induced by several insulin secretagogues, including vasopressin, a glucagon-like peptide 1 receptor agonist, and forskolin, was not suppressed by OGR1 deficiency. The inhibition of insulin secretion was associated with the reduction of glucose-induced increase in intracellular Ca(2+) concentration. In conclusion, the OGR1/G(q/11) protein pathway is activated by extracellular protons existing under the physiological extracellular pH of 7.4 and further stimulated by acidification, resulting in the enhancement of insulin secretion in response to high glucose concentrations and KCl.
Assuntos
Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Animais , Imuno-Histoquímica , Técnicas In Vitro , Secreção de Insulina , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/genéticaRESUMO
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6alpha plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6alpha are sensitive to ER stress. However, ATF6alpha-knockout mice show no apparent phenotype under normal growing conditions. In this report, we burdened mice with intraperitoneal injection of the ER stress-inducing reagent tunicamycin and found that wild-type mice were able to recover from the insult, whereas ATF6alpha-knockout mice exhibited liver dysfunction and steatosis. Thus, ATF6alpha-knockout mice accumulated neutral lipids in the liver such as triacylglycerol and cholesterol, which was ascribable to blockage of beta-oxidation of fatty acids caused by decreased mRNA levels of the enzymes involved in the process, suppression of very-low-density lipoprotein formation due to destabilized apolipoprotein B-100, and stimulation of lipid droplet formation resulting from transcriptional induction of adipose differentiation-related protein. Accordingly, the hepatocytes of tunicamycin-injected knockout mice were filled with many lipid droplets. These results establish links among ER stress, lipid metabolism, and steatosis.
Assuntos
Fator 6 Ativador da Transcrição/deficiência , Retículo Endoplasmático/patologia , Fígado Gorduroso/genética , Metabolismo dos Lipídeos/genética , Estresse Fisiológico/efeitos dos fármacos , Tunicamicina/farmacologia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apolipoproteína B-100/metabolismo , Peso Corporal/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Injeções Intraperitoneais , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Fígado/ultraestrutura , Testes de Função Hepática , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Fisiológico/imunologia , Tunicamicina/administração & dosagemRESUMO
Mechanisms of generation and maintenance of cell polarity have been investigated using various organisms and cell lines. During and after the establishment of cell polarity, polarized (vesicular) transport as well as cell-cell adhesion is essential. Here, I introduce each molecular step of polarized transport and the molecules involved there. Usually, epithelial cells and neurons are two well-known examples of polarized cells. Thus, I next describe the similarity and difference in polarized transport between these two cell types. Though closely connected, the relationship between cell-cell adhesion and polarized transport remain poorly understood. I will take a few examples indicating the relationship between them. Finally, I will present the future directions in this field.