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1.
Bioessays ; 42(12): e2000276, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33145803
2.
Development ; 147(13)2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32541014

RESUMO

The gap junction protein Connexin 43 (Cx43) contributes to cell fate decisions that determine the location of fin ray joints during regeneration. Here, we provide insights into how Cx43, expressed medially, influences changes in gene expression in lateral skeletal precursor cells. Using the Gap27 peptide inhibitor specific to Cx43, we show that Cx43-gap junctional intercellular communication (GJIC) influences Cx43-dependent skeletal phenotypes, including segment length. We also demonstrate that Cx43-GJIC influences the expression of the Smp/ß-catenin pathway in the lateral skeletal precursor cells, and does not influence the Sema3d pathway. Moreover, we show that the cx43lh10 allele, which has increased Cx43 protein levels, exhibits increased regenerate length and segment length. These phenotypes are rescued by Gap27, suggesting that increased Cx43 is responsible for the observed Cx43 phenotypes. Finally, our findings suggest that inhibition of Cx43 hemichannel activity does not influence Cx43-dependent skeletal phenotypes. These data provide evidence that Cx43-GJIC is responsible for regulating cell fate decisions associated with appropriate joint formation in the regenerating fin.

3.
Nature ; 575(7781): 180-184, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695210

RESUMO

Methane is a powerful greenhouse gas and is targeted for emissions mitigation by the US state of California and other jurisdictions worldwide1,2. Unique opportunities for mitigation are presented by point-source emitters-surface features or infrastructure components that are typically less than 10 metres in diameter and emit plumes of highly concentrated methane3. However, data on point-source emissions are sparse and typically lack sufficient spatial and temporal resolution to guide their mitigation and to accurately assess their magnitude4. Here we survey more than 272,000 infrastructure elements in California using an airborne imaging spectrometer that can rapidly map methane plumes5-7. We conduct five campaigns over several months from 2016 to 2018, spanning the oil and gas, manure-management and waste-management sectors, resulting in the detection, geolocation and quantification of emissions from 564 strong methane point sources. Our remote sensing approach enables the rapid and repeated assessment of large areas at high spatial resolution for a poorly characterized population of methane emitters that often appear intermittently and stochastically. We estimate net methane point-source emissions in California to be 0.618 teragrams per year (95 per cent confidence interval 0.523-0.725), equivalent to 34-46 per cent of the state's methane inventory8 for 2016. Methane 'super-emitter' activity occurs in every sector surveyed, with 10 per cent of point sources contributing roughly 60 per cent of point-source emissions-consistent with a study of the US Four Corners region that had a different sectoral mix9. The largest methane emitters in California are a subset of landfills, which exhibit persistent anomalous activity. Methane point-source emissions in California are dominated by landfills (41 per cent), followed by dairies (26 per cent) and the oil and gas sector (26 per cent). Our data have enabled the identification of the 0.2 per cent of California's infrastructure that is responsible for these emissions. Sharing these data with collaborating infrastructure operators has led to the mitigation of anomalous methane-emission activity10.


Assuntos
Monitoramento Ambiental , Metano/análise , Gerenciamento de Resíduos , California , Efeito Estufa , Esterco , Metano/química , Metano/metabolismo , Gás Natural , Indústria de Petróleo e Gás/métodos , Petróleo , Águas Residuárias
4.
Environ Sci Technol ; 53(16): 9636-9645, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31347357

RESUMO

California methane (CH4) emissions are quantified for three years from two tower networks and one aircraft campaign. We used backward trajectory simulations and a mesoscale Bayesian inverse model, initialized by three inventories, to achieve the emission quantification. Results show total statewide CH4 emissions of 2.05 ± 0.26 (at 95% confidence) Tg/yr, which is 1.14 to 1.47 times greater than the anthropogenic emission estimates by California Air Resource Board (CARB). Some of differences could be biogenic emissions, superemitter point sources, and other episodic emissions which may not be completely included in the CARB inventory. San Joaquin Valley (SJV) has the largest CH4 emissions (0.94 ± 0.18 Tg/yr), followed by the South Coast Air Basin, the Sacramento Valley, and the San Francisco Bay Area at 0.39 ± 0.18, 0.21 ± 0.04, and 0.16 ± 0.05 Tg/yr, respectively. The dairy and oil/gas production sources in the SJV contribute 0.44 ± 0.36 and 0.22 ± 0.23 Tg CH4/yr, respectively. This study has important policy implications for regulatory programs, as it provides a thorough multiyear evaluation of the emissions inventory using independent atmospheric measurements and investigates the utility of a complementary multiplatform approach in understanding the spatial and temporal patterns of CH4 emissions in the state and identifies opportunities for the expansion and applications of the monitoring network.


Assuntos
Poluentes Atmosféricos , Metano , Aeronaves , Teorema de Bayes , California , São Francisco
5.
J Biomed Mater Res B Appl Biomater ; 107(4): 886-899, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30267633

RESUMO

For hard tissue regeneration, the bioactivity of a material is measured by its ability to induce the formation of hydroxyapatite (HA) under physiological conditions. It depends on the dissolution behavior of the glass, which itself is determined by the composition and structure of glass. The enhanced HA growth on nanoporous than on nonporous glass has been attributed by some to greater specific surface area (SSA), but to nanopore size distribution by others. To decouple the influence of nanopore size and SSA on HA formation, we have successfully fabricated homogeneous 30CaO-70SiO2 (30C70S) model bioactive glass monoliths with different nanopore sizes, yet similar SSA via a combination of sol-gel, solvent exchange, and sintering processes. After incubation in PBS, HA, and Type-B carbonated HA (HA/B-CHA) form on nanoporous monoliths. The XPS, FTIR, and SEM analyses provide the first unambiguous demonstration of the influence of nanopore size alone on the formation pathway, growth rate, and microstructure of HA/CHA. Due to pore-size limited diffusion of PO4 3- , two HA/CHA formation pathways are observed: HA/CHA surface deposition and/or HA/CHA incorporation into nanopores. HA/CHA growth rate on the surface of a nanoporous glass monolith is dominated by the pore-size limited transport of Ca2+ ions dissolved from nanoporous glass substrates. Furthermore, with increasing nanopore size, HA/CHA microstructures evolve from needle-like, plate-like, to flower-like appearance. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 886-899, 2019.

6.
J Cell Sci ; 131(15)2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30054380

RESUMO

Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Animais , Anticorpos , Arginina/química , Arginina/genética , Membrana Celular/metabolismo , Conexina 43/genética , Cães , Endocitose/genética , Endocitose/fisiologia , Células HeLa , Humanos , Lisina/química , Lisina/genética , Células Madin Darby de Rim Canino , Peso Molecular , Fosforilação/genética , Fosforilação/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologia
7.
Biomed Mater ; 13(2): 025005, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29033393

RESUMO

Tissue regeneration is a significantly improved alternative to tissue replacement by implants. It requires porous bioscaffolds for the restoration of natural tissue rather than relying on bio-inactive, often metallic implants. Recently, we developed technology for fabricating novel, nano-macroporous bioactive 'tailored amorphous multi-porous (TAMP)' hard tissue scaffolds using a 70 mol% SiO2-30 mol% CaO model composition. The TAMP silicate scaffolds, fabricated by a modified sol-gel process, have shown excellent biocompatibility via the rapid formation of hydroxyapatite in biological fluids as well as in early tests with bone forming cells. Here we report an in depth investigation of the response of MC3T3-E1 pre-osteoblast cells and bone marrow derived (BMD) osteoclasts to these TAMP scaffolds. Light and electron microscopic imaging, gene and protein expression, and enzyme activity analyses demonstrate that MC3T3-E1 pre-osteoblasts adhere, proliferate, colonize, and differentiate on and inside the bioactive TAMP scaffolds. Additionally, BMD precursor cells mature into active osteoclasts and remodel the scaffold, highlighting the exceptional qualities of this novel scaffold material for bone tissue regeneration.


Assuntos
Materiais Biocompatíveis , Regeneração Óssea , Vidro , Osteoblastos/citologia , Osteoclastos/citologia , Tecidos Suporte/química , Células 3T3 , Animais , Osso e Ossos/patologia , Adesão Celular , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Durapatita/química , Camundongos , Microscopia Eletrônica de Varredura , Modelos Animais , Porosidade , Ratos , Ratos Sprague-Dawley , Silicatos/química , Dióxido de Silício , Engenharia Tecidual/métodos
8.
Mol Biol Cell ; 28(25): 3595-3608, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29021339

RESUMO

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/fisiologia , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Sítios de Ligação , Conexina 43/genética , Conexina 43/fisiologia , Conexinas/metabolismo , Cães , Endocitose , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Fosforilação/fisiologia , Ligação Proteica , Proteólise , Proteína da Zônula de Oclusão-1/fisiologia
9.
J Mater Sci Mater Med ; 28(10): 161, 2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28905286

RESUMO

We analyzed the biological performance of spinodally and droplet-type phase-separated 45S5 Bioglass® generated by quenching the melt from different equilibrium temperatures. MC3T3-E1 pre-osteoblast cells attached more efficiently to 45S5 Bioglass® with spinodal than to the one with droplet morphology, providing the first demonstration of the role of micro-/nano-scale on the bioactivity of Bioglass®. Upon exposure to biological solutions, phosphate buffered saline (PBS) and cell culture medium (α-MEM), a layer of hydroxyapatite (HA) formed on both glass morphologies. Although both Bioglass® varieties were incubated under identical conditions, and physico-chemical characteristics of the HA layers were similar, the adsorption magnitude of a model protein, bovine serum albumin (BSA, an abundant blood serum component) and its ß-sheet/ß-turn ratio and α-helix content were significantly higher on spinodal than droplet type Bioglass®. These results indicate that: (i) a protein layer quickly adsorbs on the surface of 45S5 Bioglass® varieties (with or without HA layer), (ii) the amount and the conformation of adsorbed proteins are guided by the glass micro-/nano-structure, and (iii) cell attachment and proliferation are influenced by the concentration and the conformation of attached proteins with a significantly better cell adhesion to spinodal type 45S5 Bioglass® substrate. Taken together, our results indicate that the biological performance of 45S5 Bioglass® can be improved further with a relatively simple, inexpensive fabrication procedure that provides a superior glass micro-/nano-structure. A simple modification to the fabrication procedure of classic 45S5 Bioglass® generates spinodal (A(a)) and droplet (A(b)) varieties and has a significant impact on protein adsorption (B) and cell adhesion (C).


Assuntos
Cerâmica/química , Vidro/química , Transição de Fase , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cerâmica/farmacologia , Meios de Cultura/farmacologia , Durapatita/química , Teste de Materiais , Camundongos , Compostos Orgânicos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Propriedades de Superfície
10.
BMC Cell Biol ; 17 Suppl 1: 22, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27230503

RESUMO

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Junções Comunicantes/metabolismo , Animais , Transporte Biológico , Técnica de Fratura por Congelamento , Junções Comunicantes/ultraestrutura , Humanos , Modelos Biológicos , Pontos Quânticos
11.
Cell Biol Int ; 39(11): 1341-7, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26074404

RESUMO

For many years now, researchers have known of a sensory appendage on the surface of most differentiated cell types called primary cilium. Primary cilia are both chemo- and mechano-sensory in function and have an obvious role in cell cycle control. Because of this, it has been thought that primary cilia are not found on rapidly proliferating cells, for example, cancer cells. Here we report using immunofluorescent staining for the ciliary protein Arl13b that primary cilia are frequently found on HeLa (human epithelial adenocarcinoma) and other cancer cell lines such as MG63 (human osteosarcoma) commonly used for cell culture studies and that the ciliated population is significantly higher (ave. 28.6% and 46.5%, respectively in starved and 15.7-18.6% in un-starved cells) than previously anticipated. Our finding impacts the current perception of primary cilia formed in highly proliferative cells.


Assuntos
Cílios/fisiologia , Neoplasias/fisiopatologia , Fatores de Ribosilação do ADP/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Cílios/metabolismo , Imunofluorescência/métodos , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais
12.
Mol Biol Cell ; 26(15): 2755-68, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26063728

RESUMO

Gap junctions (GJs) exhibit a complex modus of assembly and degradation to maintain balanced intercellular communication (GJIC). Several growth factors, including vascular endothelial growth factor (VEGF), have been reported to disrupt cell-cell junctions and abolish GJIC. VEGF directly stimulates VEGF-receptor tyrosine kinases on endothelial cell surfaces. Exposing primary porcine pulmonary artery endothelial cells (PAECs) to VEGF for 15 min resulted in a rapid and almost complete loss of connexin43 (Cx43) GJs at cell-cell appositions and a concomitant increase in cytoplasmic, vesicular Cx43. After prolonged incubation periods (60 min), Cx43 GJs reformed and intracellular Cx43 were restored to levels observed before treatment. GJ internalization correlated with efficient inhibition of GJIC, up to 2.8-fold increased phosphorylation of Cx43 serine residues 255, 262, 279/282, and 368, and appeared to be clathrin driven. Phosphorylation of serines 255, 262, and 279/282 was mediated by MAPK, whereas serine 368 phosphorylation was mediated by PKC. Pharmacological inhibition of both signaling pathways significantly reduced Cx43 phosphorylation and GJ internalization. Together, our results indicate that growth factors such as VEGF activate a hierarchical kinase program--including PKC and MAPK--that induces GJ internalization via phosphorylation of well-known regulatory amino acid residues located in the Cx43 C-terminal tail.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase C/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Linhagem Celular , Junções Comunicantes/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Suínos , Fator A de Crescimento do Endotélio Vascular/farmacologia
13.
FEBS Lett ; 588(8): 1221-9, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24486527

RESUMO

Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Proteólise , Animais , Endossomos/metabolismo , Junções Comunicantes/ultraestrutura , Humanos , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
14.
FEBS Lett ; 588(5): 836-44, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24492000

RESUMO

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies - endogenously expressing the GJ protein connexin43 (Cx43) - with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.


Assuntos
Conexina 43/metabolismo , Células-Tronco Embrionárias/metabolismo , Endocitose , Fator de Crescimento Epidérmico/fisiologia , Processamento de Proteína Pós-Traducional , Animais , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Ativação Enzimática , Junções Comunicantes/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Serina/metabolismo
15.
Mol Biol Cell ; 24(18): 2834-48, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23885125

RESUMO

Gap junction (GJ) channels that electrically and chemically couple neighboring cells are formed when two hemichannels (connexons) of apposed cells dock head-on in the extracellular space. Remarkably, docked connexons are inseparable under physiological conditions, and we and others have shown that GJs are internalized in whole, utilizing the endocytic clathrin machinery. Endocytosis generates double-membrane vesicles (annular GJs or connexosomes) in the cytoplasm of one of the apposed cells that are degraded by autophagosomal and, potentially, endo/lysosomal pathways. In this study, we investigated the structural motifs that mediate Cx43 GJ endocytosis. We identified three canonical tyrosine-based sorting signals of the type "YXXΦ" in the Cx43 C-terminus, two of which function cooperatively as AP-2 binding sites. We generated a set of green fluorescent protein-tagged and untagged Cx43 mutants that targeted these two sites either individually or together. Mutating both sites completely abolished Cx43-AP-2/Dab2/clathrin interaction and resulted in increased GJ plaque size, longer Cx43 protein half-lives, and impaired GJ internalization. Interestingly, Dab2, an accessory clathrin adaptor found earlier to be important for GJ endocytosis, interacts indirectly with Cx43 via AP-2, permitting the recruitment of up to four clathrin complexes per Cx43 protein. Our analyses provide a mechanistic model for clathrin's efficient internalization of large plasma membrane structures, such as GJs.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , Endocitose , Junções Comunicantes/metabolismo , Sinais Direcionadores de Proteínas , Tirosina/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Clatrina/metabolismo , Meia-Vida , Células HeLa , Humanos , Imunoprecipitação , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Ratos , Relação Estrutura-Atividade
16.
Physiology (Bethesda) ; 28(2): 93-116, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23455769

RESUMO

Gap junctions (GJs) are the only known cellular structures that allow a direct cell-to-cell transfer of signaling molecules by forming densely packed arrays or "plaques" of hydrophilic channels that bridge the apposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for all aspects of multicellular life, including coordination of development, tissue function, and cell homeostasis, has been well documented. Assembly and degradation of these membrane channels is a complex process that includes biosynthesis of the connexin (Cx) subunit proteins (innexins in invertebrates) on endoplasmic reticulum (ER) membranes, oligomerization of compatible subunits into hexameric hemichannels (connexons), delivery of the connexons to the plasma membrane (PM), head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic spatially and temporally organized GJ channel plaques, as well as internalization of GJs into the cytoplasm followed by their degradation. Clearly, precise modulation of GJIC, biosynthesis, and degradation are crucial for accurate function, and much research currently addresses how these fundamental processes are regulated. Here, we review posttranslational protein modifications (e.g., phosphorylation and ubiquitination) and the binding of protein partners (e.g., the scaffolding protein ZO-1) known to regulate GJ biosynthesis, internalization, and degradation. We also look closely at the atomic resolution structure of a GJ channel, since the structure harbors vital cues relevant to GJ biosynthesis and turnover.


Assuntos
Junções Comunicantes/metabolismo , Proteínas/metabolismo , Animais , Humanos , Canais Iônicos/metabolismo , Transporte Proteico , Proteólise
17.
Tissue Eng Part A ; 19(13-14): 1632-40, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23427819

RESUMO

Nanoporosity is known to impact the performance of implants and scaffolds such as bioactive glass (BG) scaffolds, either by providing a higher concentration of bioactive chemical species from enhanced surface area, or due to inherent nanoscale topology, or both. To delineate the role of these two characteristics, BG scaffolds have been fabricated with nearly identical surface area (81 and 83±2 m(2)/g) but significantly different pore size (av. 3.7 and 17.7 nm) by varying both the sintering temperature and the ammonia concentration during the solvent exchange phase of the sol-gel fabrication process. In vitro tests performed with MC3T3-E1 preosteoblast cells on such scaffolds show that initial cell attachment is increased on samples with the smaller nanopore size, providing the first direct evidence of the influence of nanopore topography on cell response to a bioactive structure. Furthermore, in vivo animal tests in New Zealand rabbits (subcutaneous implantation) indicate that nanopores promote colonization and cell penetration into these scaffolds, further demonstrating the favorable effects of nanopores in tissue-engineering-relevant BG scaffolds.


Assuntos
Vidro/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Linhagem Celular , Masculino , Camundongos , Porosidade , Coelhos
18.
J Membr Biol ; 245(8): 465-76, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22825714

RESUMO

Gap junctions (GJs) are composed of tens to many thousands of double-membrane spanning GJ channels that cluster together to form densely packed channel arrays (termed GJ plaques) in apposing plasma membranes of neighboring cells. In addition to providing direct intercellular communication (GJIC, their hallmark function), GJs, based on their characteristic double-membrane-spanning configuration, likely also significantly contribute to physical cell-to-cell adhesion. Clearly, modulation (up-/down-regulation) of GJIC and of physical cell-to-cell adhesion is as vitally important as the basic ability of GJ formation itself. Others and we have previously described that GJs can be removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles [termed annular gap junctions (AGJs) or connexosomes] in the cytoplasm of one of the coupled cells. Four recent independent studies, consistent with earlier ultrastructural analyses, demonstrate the degradation of endocytosed AGJ vesicles via autophagy. However, in TPA-treated cells others report degradation of AGJs via the endo-/lysosomal degradation pathway. Here we summarize evidence that supports the concept that autophagy serves as the cellular default pathway for the degradation of internalized GJs. Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway.


Assuntos
Autofagia/fisiologia , Conexinas/metabolismo , Endocitose/fisiologia , Junções Comunicantes/fisiologia , Ativação do Canal Iônico/fisiologia , Lisossomos/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Modelos Biológicos
19.
Autophagy ; 8(5): 794-811, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22635056

RESUMO

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.


Assuntos
Autofagia , Endocitose , Junções Comunicantes/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores/metabolismo , Western Blotting , Membrana Celular/metabolismo , Conexina 43/metabolismo , Fluorescência , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Transporte Proteico , Interferência de RNA , Proteína Sequestossoma-1
20.
Tree Physiol ; 32(2): 200-18, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22323526

RESUMO

The maximum light use efficiency (LUE = gross primary production (GPP)/absorbed photosynthetic photon flux density (aPPFD)) of plant canopies has been reported to vary spatially and some of this variation has previously been attributed to plant species differences. The canopy nitrogen concentration [N] can potentially explain some of this spatial variation. However, the current paradigm of the N-effect on photosynthesis is largely based on the relationship between photosynthetic capacity (A(max)) and [N], i.e., the effects of [N] on photosynthesis rates appear under high PPFD. A maximum LUE-[N] relationship, if it existed, would influence photosynthesis in the whole range of PPFD. We estimated maximum LUE for 14 eddy-covariance forest sites, examined its [N] dependency and investigated how the [N]-maximum LUE dependency could be incorporated into a GPP model. In the model, maximum LUE corresponds to LUE under optimal environmental conditions before light saturation takes place (the slope of GPP vs. PPFD under low PPFD). Maximum LUE was higher in deciduous/mixed than in coniferous sites, and correlated significantly with canopy mean [N]. Correlations between maximum LUE and canopy [N] existed regardless of daily PPFD, although we expected the correlation to disappear under low PPFD when LUE was also highest. Despite these correlations, including [N] in the model of GPP only marginally decreased the root mean squared error. Our results suggest that maximum LUE correlates linearly with canopy [N], but that a larger body of data is required before we can include this relationship into a GPP model. Gross primary production will therefore positively correlate with [N] already at low PPFD, and not only at high PPFD as is suggested by the prevailing paradigm of leaf-level A(max)-[N] relationships. This finding has consequences for modelling GPP driven by temporal changes or spatial variation in canopy [N].


Assuntos
Luz , Nitrogênio/metabolismo , Fotossíntese , Árvores/metabolismo , Nitrogênio/análise , Árvores/efeitos da radiação
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