Abstract
N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.
Highlights
Cadherins are Ca2+ dependent homophilic adhesion molecules
Structured illumination microscopy can resolve different synaptic domains Conventional light microscopy has a resolution limit of ~200-250 nm, which is insufficient to resolve details within synapses which generally range in diameter from ~200-500 nm
To visualize how N-cadherin localizes within synapses, we turned to structured illumination microscopy (SIM), a super-resolution microscopy technique with a two-fold greater resolution along the lateral and axial directions, resulting in an x-y resolution of ~100 nm [27,28,29]
Summary
Cadherins are Ca2+ dependent homophilic adhesion molecules. In the nervous system they are involved in neural tube formation, stratification of cells into layers, tissue coherence and laminar targeting [1,2]. N-cadherin endocytosis is regulated by NMDAR activation and reciprocally, stabilization of surface N-cadherin blocks short term NMDAR-dependent synaptic plasticity [21]. N-cadherin endoyctosis is regulated by the protocadherin arcadlin after synaptic stimulation [22] These activity dependent changes in Ncadherin endocytosis may be involved in linking synaptic activity with N-cadherin localization and spine morphology [21,22]. The functional and structural roles of cadherin are likely to be interlinked, and we hypothesized that synaptic activity could change the localization of N-cadherin within synapses, and this could be one mechanism via which N-cadherin regulates synaptic strength and stability. We found that in contrast to a defined ring structure or simple cluster, N-cadherin localizes in a spectrum of patterns within the synapse, from clusters adjacent to the active zone to an even distribution along the active zone, with variations in between these extremes. Synaptic stimulation converts N-cadherin localization from a peripheral to a more central distribution within the synapse
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