The synaptic cleft is a conserved and integral component of central synapses. It is comprised of protein complexes that span across it, and their adhesive interactions and signaling roles guide synapse development. Their relevance is underscored by the mutations in synapse-organizing proteins that are linked to complex brain disorders including autism-spectrum disorders and schizophrenia. Yet, the molecular patterning and dynamics of synaptic cleft components remain largely unknown. This contrasts with our understanding of the nanoscale organization of pre- and post-synaptic compartments, which is elucidating our understanding of synaptic structure and function. Moreover, acute synapse-organizing roles of cleft proteins at mature synapses and the functional interplay of trans-synaptic interaction systems remain to be defined. Our central hypothesis is that the properties and plasticity of mature synapses are dynamically instructed by select cleft components. This proposal builds on preliminary and previously published results by the collaborating groups that synaptic adhesion complexes are differentially localized within the cleft, shape this compartment, can undergo rapid changes in synaptic abundance upon plasticity induction, and alter long-term synaptic plasticity. Three specific aims will be pursued to test our hypothesis. First, we will test roles of trans-synaptic interactions in acutely instructing pre- and postsynaptic function. Second, it is our aim to determine the molecular and organizational dynamics of the cleft during long-term plasticity. Third, we will identify cleft proteins that guide changes during plasticity. Our approaches include tools to acutely perturb trans-synaptic interactions, proximity labeling to identify cleft-resident molecules and monitor them during plasticity, superresolution imaging to map their cleft locations, and cell biological and physiological functional assays. We anticipate to determine the molecular patterning and dynamics of the synaptic cleft and to identify how trans-synaptic interactions actively shape synaptic function. This expected progress is significant because it will define the cleft as a molecularly organized and acutely controlled cellular compartment that instructs synaptic properties. Moreover, this research can determine how a dynamic remodeling of the cleft architecture underlies the activity-dependent plastic changes at synapses. Synaptic organization and function are disrupted in neurodevelopmental and neurological disorders, and this program will provide information for defining how these diseases impact the cleft and may even originate in it to alter synaptic properties.