RESUMO
Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.
Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Ratos , Eletricidade EstáticaRESUMO
SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q(a)-, Q(b)-, Q(c)- and R-SNARE-motif form a complex. The core complex represents a Q(a)Q(b)Q(c)R-SNARE-motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)-studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q(a)), SNAP25 (Q(b)/Q(c)) and synaptobrevin 2 (R)]. We find that the Q(b)-SNARE-motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q(b)-SNARE-motif and depends on its position within the molecule. We conclude that a zippered Q(a)Q(b)-SNARE complex represents a short-lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.
Assuntos
Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos , Animais , Recuperação de Fluorescência Após Fotodegradação , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Estrutura Secundária de Proteína , Transporte Proteico , Ratos , Proteínas SNARE/química , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The spatial distribution of the target (t-)SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two- and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.
Assuntos
Membrana Celular/química , Lipídeos de Membrana/química , Proteínas Qa-SNARE/química , Proteína 25 Associada a Sinaptossoma/química , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Células PC12 , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.
Assuntos
Células do Tecido Conjuntivo/citologia , Endotélio Vascular/citologia , Placenta/irrigação sanguínea , Trofoblastos/citologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Neurônios/citologia , Placenta/citologia , GravidezRESUMO
Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.
Assuntos
Encéfalo , Meios de Cultivo Condicionados/efeitos adversos , Hipóxia , Neurônios/efeitos dos fármacos , Oxigênio/farmacologia , Placenta/química , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Hipóxia Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/química , Dendritos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Feto , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/patologia , Hipóxia/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/fisiologia , Placenta/citologia , Gravidez , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Cultura de TecidosRESUMO
For membrane fusion to occur, opposed lipid bilayers initially establish a fusion pore, often followed by complete mixing of the fusing membranes. Contemporary views suggest that during fusion lipid bilayers are continuous passive platforms that are disrupted and remodeled by catalytic proteins. Some models propose that even the architecture and composition of the fusion pore might be dominated by proteins rather than lipids. Hence, lipids have no regulatory contribution to this process; they simply adapt their shape passively for filling space between otherwise autonomous protein machineries. However, an increasing number of experimental findings indicate that membrane fusion critically depends on a variety of lipids and lipid derivatives. Therefore, a purely proteocentric view describes fusion mechanisms insufficiently. Instead, lipids have functions probably at different levels, as (i) a general influence on the propensity of lipid bilayers to fuse, (ii) a role in recruiting exocytotic proteins to the plasma membrane, (iii) a role in organizing membrane domains for fusion and (iv) direct regulatory effects on fusion protein complexes. In this review we have made an attempt to bring together the large body of evidence supporting a major role for lipids in membrane fusion either directly or indirectly.