Fine tuning the efficiency of synaptic transmission is essential for learning and memory, while its disruption is associated with diverse pathologies including autism spectrum disorder, depression, and anxiety disorders. Thus, identifying mechanisms that regulate synaptic strength is a central goal in neuroscience. Since such plasticity is frequently triggered by activation of NMDA-type glutamate receptors, understanding the regulation of NMDA receptors is particularly critical. Recent studies indicate synaptic strength and NMDA receptor activation can be directly affected by the nanometer-scale organization of proteins within the synapse. Many synaptic proteins, including vesicle release machinery and postsynaptic scaffolds and receptors, display a heterogeneous organization with local regions of high protein density, known as nanodomains (NDs). These NDs can be aligned across the synapse to form a “nanocolumn”, which our lab has demonstrated is the site of action potential-evoked vesicle fusion and maximal receptor activation. New work from our lab has established a novel role for the postsynaptic cell-adhesion molecule (CAM) LRRTM2 in positioning AMPA receptors within the nanocolumn. While CAMs have well-established roles in synapse formation and development, these recent findings highlight the possibility that CAMs may coordinate synaptic nanostructure and function in the mature synapse. However, the mechanism by which presynaptic organization and vesicle fusion sites are communicated to proteins within the postsynaptic density to enable alignment to occur remains unknown. In this proposal I will investigate whether the presynaptic CAM PTPσ coordinates nanocolumn alignment. PTPσ is important for synapse formation, is present in the mature synapse, and forms indirect interactions with both pre- and postsynaptic machinery located within the nanocolumn. Loss of PTPσ impacts both pre- and postsynaptic physiology, most notably NMDA receptor-mediated responses. Previous attempts to study PTPσ have relied on chronic manipulations, such as knockouts and knockdowns. However, interpretations are complicated by its initial role in synapse formation during development. I propose to elucidate the ongoing functions of PTPσ by acutely disrupting its cleft interactions via cleavage by an exogenous protease. This highly specific and acute approach will allow me to manipulate PTPσ’s cleft interactions to isolate their functions, without compromising its earlier role in synapse formation. Throughout this project, I will use super-resolution microscopy, electrophysiology, molecular biology, and live-cell imaging to test the role of PTPσ cleft interactions in maintaining nanocolumn alignment and regulating NMDA receptor-mediated transmission. This work will provide novel insight regarding the roles of a critical family of presynaptic CAMs following development and will test a new candidate mechanism for the coordination of synaptic nanostructure and NMDA receptor...