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Poster presentation: Twenty Second Annual Computational Neuroscience Meeting: CNS*2013. Paris, France. 13-18 July 2013.
The synaptic cleft is an extracellular domain that is capable of relaying a presynaptically received electrical signal by diffusive neurotransmitters to the postsynaptic membrane. The cleft is trans-synaptically bridged by ring-like shaped clusters of pre- and postsynaptically localized calcium-dependent adhesion proteins of the N-Cadherin type and is possibly the smallest intercircuit in nervous systems [1]. The strength of association between the pre- and postsynaptic membranes can account for synaptic plasticity such as long-term potentiation [2]. Through neuronal activity the intra- and extracellular calcium levels are modulated through calcium exchangers embedded in the pre- and postsynaptic membrane. Variations of the concentration of cleft calcium induces changes in the N-Cadherin-zipper, that in synaptic resting states is rigid and tightly connects the pre- and postsynaptic domain. During synaptic activity calcium concentrations are hypothesized to drop below critical thresholds which leads to loosening of the N-Cadherin connections and subsequently "unzips" the Cadherin-mediated connection. These processes may result in changes in synaptic strength [2]. In order to investigate the calcium-mediated N-Cadherin dynamics at the synaptic cleft, we developed a three-dimensional model including the cleft morphology and all prominent calcium exchangers and corresponding density distributions [3-6]. The necessity for a fully three-dimensional model becomes apparent, when investigating the effects of the spatial architecture of the synapse [7], [8]. Our data show, that the localization of calcium channels with respect to the N-Cadherin ring has substantial effects on the time-scales on which the Cadherin-zipper switches between states, ranging from seconds to minutes. This will have significant effects on synaptic signaling. Furthermore we see, that high-frequency action potential firing can only be relayed to the Calcium/N-Cadherin-system at a synapse under precise spatial synaptic reorganization.
Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells
(2013)
Cadherins, Ca2+-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca2+-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca2+-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca2+-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.