RESUMO
Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.
Assuntos
Actomiosina , Miotonina Proteína Quinase , Fagocitose , Actinas/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Miotonina Proteína Quinase/metabolismo , Fagocitose/fisiologia , Proteínas Tirosina Quinases , Receptores Fc , c-Mer Tirosina Quinase/metabolismoRESUMO
Evidence is emerging that the tumour necrosis factor (TNF-alpha) is a potent signal that induces neural stem cell proliferation and migration. We show that NSC self-renewal is controlled by bi-directional cross-talk between the endocannabinoid system and the TNF signalling pathway. By blocking endogenous TNF-alpha activity, we demonstrate that the TNF system is critical for the proliferation of NSC. Furthermore, we show that pharmacological blockade of the CB1/CB2 cannabinoid receptors dramatically suppresses TNF-alpha-induced NSC proliferation. Interestingly, we found that CB1 or CB2 agonists induce NSC proliferation coupled to a significant increase in both TACE/ADAM 17 and TNF-alpha levels. Overall these data suggest a novel mode of action for the endocannabinoid system in NSC proliferation that is coupled to TNF signalling and that may be of therapeutic interest in the emerging field of brain repair.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Proliferação de Células , Endocanabinoides , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Agonistas de Receptores de Canabinoides , Antagonistas de Receptores de Canabinoides , Diferenciação Celular/fisiologia , Células Cultivadas , Camundongos , Neurônios/citologia , Receptores de Canabinoides/fisiologia , Células-Tronco/citologiaRESUMO
Tight junctions are required for the formation of tissue barriers and function as suppressors of signalling mechanisms that control gene expression and cell behaviour; however, little is known about the physiological and developmental importance of such signalling functions. Here, we demonstrate that depletion of MarvelD3, a transmembrane protein of tight junctions, disrupts neural crest formation and, consequently, development of neural crest-derived tissues during Xenopus embryogenesis. Using embryos and explant cultures combined with a small molecule inhibitor or mutant mRNAs, we show that MarvelD3 is required to attenuate JNK signalling during neural crest induction and that inhibition of JNK pathway activation is sufficient to rescue the phenotype induced by MarvelD3 depletion. Direct JNK stimulation disrupts neural crest development, supporting the importance of negative regulation of JNK. Our data identify the junctional protein MarvelD3 as an essential regulator of early vertebrate development and neural crest induction and, thereby, link tight junctions to the control and timing of JNK signalling during early development.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases , Proteínas com Domínio MARVEL/genética , Crista Neural/embriologia , Crista Neural/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas com Domínio MARVEL/metabolismo , Fenótipo , XenopusRESUMO
BACKGROUND: Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. RESULTS: We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. CONCLUSION: Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP110/metabolismo , Junções Íntimas/metabolismo , Junções Aderentes/metabolismo , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Linhagem Celular , Polaridade Celular/fisiologia , Impedância Elétrica , Epitélio/crescimento & desenvolvimento , Inativação Gênica , Proteínas de Choque Térmico HSP110/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismoRESUMO
Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca(2+)-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.
Assuntos
Anexina A1/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Transporte Biológico/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Células HeLa , Humanos , Lipoproteínas LDL/farmacologia , Corpos Multivesiculares/efeitos dos fármacos , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptores de Esteroides/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis.
RESUMO
Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO)-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5'-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Western Blotting , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular , Polaridade Celular/genética , Proliferação de Células , Forma Celular/genética , Forma Celular/fisiologia , Cães , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Síndrome Oculocerebrorrenal/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Proteínas da Zônula de Oclusão , Proteína da Zônula de Oclusão-1 , Proteína da Zônula de Oclusão-2 , alfa Catenina/metabolismoRESUMO
Interactions between the neural cell adhesion molecules NCAM and N-cadherin with the fibroblast growth factor receptor (FGFR) are important for a number of developmental events and have also been implicated in tumor progression. The factors regulating these interactions are not known. We have used co-immunoprecipitation and co-clustering paradigms to show that both adhesion molecules can interact with the 3Ig IIIC isoform of the FGFR1 in a number of cell types. Interestingly, whereas the interaction can be seen over most of the cell surface, it is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. We also demonstrate for the first time that all of the major isoforms of NCAM can interact with the FGFR. Using deletion mutagenesis we have found that the adhesion molecule/FGFR interaction can withstand the removal of most of any one of the FGFR immunoglobulin-like domains (D1-D3). In contrast, the FGFR interaction with N-cadherin and NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif that can be found in the linker region between D1 and D2. As this motif can be spliced out of all four FGFRs, it suggests that this is one mechanism that can regulate the interaction of the receptor with different ligand classes.