Effects of ligand and receptor valence on the surface dynamics and nanoscale localization of synaptic membrane proteins

Abstract : Synapses are highly compartmentalized structures packed with interacting protein complexes. The synaptic cleft bridging pre- and post-synaptic compartments is an adhesive zone ~20 nm thick, containing adhesion proteins and neurotransmitter receptors. Progress in super-resolution imaging offers an improved view of the dynamical organization of synaptic proteins. However, a quantitative assessment of the concentration and oligomerization level of those complexes remains difficult. This is in part because traditional ways to label receptors rely on antibodies, whose relatively large size (~15 nm) may lead to steric hindrance and localization bias, while their divalence causes protein cross-linking. Here, we studied the impact of probe and receptor valence on the diffusion and organization of 3 synaptic proteins: neurexin1β, neuroligin1, and the GluK2 kainate receptor subunit. We used single molecule pull-down to characterize the subunit composition of those proteins, by observing immobilized GFP-tagged proteins under TIRF illumination. Neurexin1β shows essentially 1 photo-bleaching step, while neuroligin1 exhibits mostly 2 steps, and GluK2 shows multiple steps, confirming that those proteins assemble into monomers, dimers, and tetramers, respectively. Then, we used FRAP to monitor the surface diffusion of the 3 proteins labeled with probes of different valence. We labeled recombinant proteins carrying a biotinylated N-terminal tag with monomeric, dimeric, or tetrameric streptavidin (or with biotin antibody), all conjugated to the same organic dyes. We also used STORM to characterize the aggregation level of the 3 proteins in response to the different probes. We show drastic effects depending on the nature of the protein used and the probe valence, suggesting that diffusion is slowed down by receptor aggregation, thereby highlighting the crucial issues at stake in labelling strategies, especially in confined spaces such as synapses.
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Camille Saphy. Effects of ligand and receptor valence on the surface dynamics and nanoscale localization of synaptic membrane proteins. Human health and pathology. Université de Bordeaux, 2018. English. ⟨NNT : 2018BORD0243⟩. ⟨tel-02177139⟩

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