Long-term potentiation through calcium-mediated N-Cadherin interaction is tightly controlled by the three-dimensional architecture of the synapse

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

Correlative Light- and Electron Microscopy with chemical tags

AbstractCorrelative 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 25nm 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

Temas y problemas en Antropología Social

El presente texto tiene que ver con el programa de la materia Antropología Cultural y Social dictada en la Facultad de Psicología de nuestra Universidad de La Plata. Sus diversos capítulos cubren varios temas del curso y fueron antecedidos por otros textos temáticos menos formalizados, editados anteriormente por la Cátedra. Nos ha parecido siempre importante adaptar los conocimientos de la Antropología en el marco de las Ciencias Sociales y de la Antropología Social en particular -que constituyen el eje de la materia- con la intención de conformar un eje didáctico de materiales que sean de fácil comprensión y permitan una lectura ulterior de mayor profundidad y continuidad, según el avance en la construcción de los conocimientos por parte de alumnos. Esto es importante por cuanto la disciplina constituye, de acuerdo al nuevo perfil del Plan de Estudios, uno de los cuatro pilares de conocimiento básico de la Psicología. En este sentido, se la considera un ámbito disciplinar académico destacado que aporta a los estudiantes herramientas conceptual-metodológicas básicas para la lectura y comprensión crítica del contexto sociohistórico, cultural y político en el que desarrollan sus prácticas actuales y su futura práctica profesional. Con una perspectiva más amplia se ha pensado, al redactar los capítulos, en el posible interés que puedan tener su lectura en el ámbito general de la Universidad.Facultad de Psicologí

Direct visualization of newly synthesized target proteins in situ

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ

Molecular and functional analyses of the pannexins in the central nervous system

Die Rolle von Pannexin1 und Pannexin2 im zentralen Nervensystem wurde auf molekularer und funktioneller Ebene untersucht. Es wurde gezeigt, dass eine akzessorische Kaliumkanaluntereinheit, Kv$\beta$3, mit Panx1 interagiert und die Wirkung von Pharmaka durch diese Interaktion moduliert wird. Diese beiden Proteine sind in retinalen Ganglienzellen koexprimiert. Des Weiteren wurde die Bedeutung der Cysteine von Pannexin1 für dessen Funktion als Ionenkanal und der Einfluss von Reduktionsmitteln auf die Kanaleigenschaften untersucht. Mutationen einzelner Cysteine führen zu einem Hemikanal mit erhöhter oder erniedrigter Aktivität. Es wurde ein Pannexin2 spezifischer Antikörper hergestellt, mit Hilfe dessen die Expression im zentralen Nervensystem sowie die subzelluläre Lokalisation des Proteins analysiert wurden. Pannexin2 ist in Neuronen in weiten Bereichen des Gehirns vorzufinden. Es liegt vor allem intrazellulär vor, aber geringe Mengen konnten auch in der Plasmamembran nachgewiesen werden

The potassium channel subunit Kv beta 3 interacts with pannexin 1 and attenuates its sensitivity to changes in redox potentials

Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non-junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kv beta 3, previously identified by bacterial two-hybrid strategies. Here, we report on the physical association of Panx1 with Kv beta 3 by immunoprecipitation when co-expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co-expression of Panx1 and Kv beta 3 was shown to occur in murine hippocampus and cerebellum. Kv beta 3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kv beta 3. Apparently, Kv beta 3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kv beta 3 was co-expressed. The influence of Kv beta 3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.Structured digital abstract times MINT-7260843, MINT-7260856: Panx1 (uniprotkb:Q9JIP4) physically interacts ( MI:0915) with Kv beta 3 (uniprotkb:P97382) by anti-tag co-immunoprecipitation ( MI:0007

All mutants exhibit sensitivity to prolonged Ca²⁺-chelation.

<p>(<b>A</b>) Example images for WT and all mutants before and 1 h after BAPTA addition. Scale bar = 10 µm. (<b>B</b>) Quantification of junction disassembly. Mean fluorescence intensity of multiple junctions from the different genotypes was measured over time. The drop in mean fluorescence intensity is specific to the addition of BAPTA (blue), indicated by the arrow, as application of a vehicle (green) did not lead to a decrease in junction intensity. (<b>C</b>) Comparison of junction disassembly between different mutants and WT. Although the time course and degree of disassembly differed for the various mutants, with W2A junctions displaying the highest degree of disassembly; on a longer timescale, none of them were completely Ca²⁺-insensitive. N-cadherin-R14E, which appeared Ca²⁺-insensitive on a short timescale (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081517#pone-0081517-g003" target="_blank">Fig. 3 D</a>) also showed a drop in junction intensity 10 min after BAPTA application. Error bars indicate SEM. n(WT) = 84, n(W2A) = 56, n(R14E) = 84, n(V81D/V174D) = 52 junctions. </p

All mutants differ in cellular spheroid organization on global or cellular levels compared to N-cadherin-WT.

<p>(<b>A</b>) Example images of complete spheroids after fusing the images from 12 different imaging angles. The dramatic difference between WT and W2A is evident, where W2A does not form a cellular spheroid, while WT is an almost circular spheroid. N-cadherin-Venus signal is represented in pseudocolor “Fire” LUT. (<b>B</b>) An XY cross-section through the spheroids. A cross-section through the middle of the spheroid highlights the differences in cellular organization between WT, R14E and V81D/V174D. WT had orderly junctions throughout the XY-plane, while R14E was hollow on the inside as seen by the absence of junctions. Although in V81D/V174D junctions were present, they were not organized. (<b>C</b>) Surface generation of the spheroids after slicing them into half through XY. If junctions are present, they are represented in red. Compared to WT, the empty space inside R14E is apparent here too. Even though the morphologies of the WT, R14E and V81D/V174D spheroids are quite similar on a global scale, there is a dramatic difference between how the cells are organized within a spheroid highlighting the effect of any mutation on junction assembly in 3D.</p

Influence of Ca²⁺-chelation on the individual binding interfaces of N-cadherin.

<p>(<b>A</b>) Disruption of the <i>trans</i> interaction of N-cadherin by BAPTA. Upon disruption of this interaction the fluorophores move further apart, reducing FRET. Examples for ratiometric FRET measurements of the WT (<b>B</b>), W2A (<b>C</b>), R14E (<b>D</b>) and V81D/V174D (<b>E</b>) before and after Ca²⁺-chelation are shown. After baseline recording, the Ca²⁺-chelator BAPTA (20 mM final concentration) was added. The fluorescence of the FRET acceptor Venus (yellow) and the donor Cerulean (cyan) as well as the ratio of the two (blue) are shown. (<b>F</b>) The quantification of the effect of BAPTA on all mutants is shown. While the mutants W2A and V81D/V174D showed a significantly higher decrease of the FRET signal due to Ca²⁺-chelation than the WT, no Ca²⁺-sensitivity was observed for the X-dimer mutant R14E. This confirms the hypothesis that the X-dimer is a Ca²⁺-dependent intermediate step in the formation of the strand-swapped dimer. n(WT) = 8, n(W2A) = 8, n(R14E) = 9, n(V81D/V174D) = 7 junctions. Statistics were conducted using either paired or unpaired t-test.</p