The identification and interrogation of novel intracellular proteins that underlie the distinct nanoscopic architecture of Neurexin-1 and Neurexin-3 - Project Summary/Abstract Recent developments in super-resolution imaging have revealed that proteins involved in neurotransmitter release and detection are non-uniformly distributed and assemble into regions of high density called nanoclusters (NCs). Pre- and post-synaptic NCs often align across the synapse to form transsynaptic nanocolumns, which are thought to be required for efficient synaptic communication. Transsynaptic cell-adhesion molecules, which span the synaptic cleft and are capable of participating in bidirectional signaling via intracellular and extracellular sequences, are essential for the nanoarchitecture of synapses. Neurexins (Nrxns) are a family of evolutionarily conserved presynaptic adhesion molecules. We recently performed the first 3D dSTORM super-resolution imaging of endogenous neurexins using our novel double epitope-tag mouse line that permits the immunolabeling of Nrxn1 and Nrxn3. We found that Nrxn1 and Nrxn3 each form spatially discrete and non- overlapping NCs. Additionally, Nrxn1 NCs and Nrxn3 NCs preferentially align transsynaptically with GluD1 and LRRTM2, respectively. We proposed that this spatially discrete nano-organization of neurexins and their ligands results in parallel signaling, which explains how neurexin-1 and neurexin-3, despite sharing high sequence homology, control distinct and non-overlapping properties of synapse function. A fundamentally important question is: how are the intrinsically monomeric neurexins organized into discrete and homogeneous NCs? We hypothesize that differential interactions with cytoplasmic proteins establish the discrete nano-organization of Nrxn1 and Nrxn3 NCs. To test our hypothesis, we used purified hippocampal synaptosomes from our double epitope-tag mouse line to perform the first co-immunoprecipitation mass spectrometry of endogenous neurexins and identified cytoplasmic proteins that exclusively co-immunoprecipitated with Nrxn1 or Nrxn3. Importantly, due to limitations circumvented by our novel mouse line, no new neurexin-interacting cytoplasmic proteins have been identified in over twenty years. The top candidate is p130Cas, which exhibits exclusive co-immunoprecipitation with Nrxn1. This proposal builds on our published results and compelling preliminary data that indicate that p130Cas controls the surface expression of neurexin-1, the volume of Nrxn1 NCs, and presynaptic release probability at excitatory synapses. We will use orthogonal 3D STED and 3D dSTORM imaging to dissect how p130Cas regulates neurexin-1 and the nanoarchitecture excitatory synapses in Aim 1. In Aim 2, we will use classic structure-function approaches to define the sequences necessary and sufficient for p130Cas and neurexin-1 binding, and systematically assess the functional implications of Nrxn1 – p130Cas interactions using electrophysiology. Here, we will provide the first critical insight into how neurexin NCs are established, start to define how individual Nrxns differentially control intracellular signaling and, importantly, create a methodological framework to interrogate our other candidates.
