Dissecting pre- vs postsynaptic actin dynamics in synapse structure and strength

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Bidirectional, spatially restricted actin filament dynamics modify synapses by controlling the structure and function of each cell creating the synaptic contact. Dynamic actin filaments are disrupted in psychiatric disease, so understanding their role in regulating pre- vs postsynaptic structure and function will be key for elucidating molecular mechanisms of memory formation and developing precise disease treatment strategies. Unfortunately, most existing tools to experimentally manipulate actin lack control of one or more of these biological factors, making it difficult to parse specific contributions of pre- vs. postsynaptic actin dynamics to synapse strength. The overarching goal of this proposal is to clarify the particular roles of actin at each side of the synapse. To facilitate this goal, I propose to develop new tools to spatiotemporally, bidirectionally, and synapse-specifically manipulate actin dynamics. The tools will be valuable additions to the arsenal of reagents in diverse fields of neuroscience and in other areas of cell biology. In my project, I will use these tools to answer two critical questions of how pre- vs postsynaptic actin dynamics regulate synapse strength. My first aim is to validate tools for precise, bidirectional control of actin dynamics. To drive actin depolymerization, I will develop photoactivateable (PA) DeActs by caging these published, genetically encodable actin depolymerizing proteins with photo-dimerizable pdDronpa. To drive actin polymerization, I will optimize an existing PA-Rac1 probe, which drives actin branching via Arp2/3. This set of tools will be highly useful to broad areas of science, and I will make new transfectable and recombinant AAV versions publicly available. My second aim is to elucidate how pre- vs postsynaptic actin regulates subsynaptic nanoorganization. Pre- and postsynaptic proteins form subsynaptic, nanoscale clusters that align across the synapse, a newly discovered organization expected to influence synaptic strength. However, we do not understand the biology that forms and maintains synaptic nanoorganization. My preliminary data suggest alignment requires actin dynamics, consistent with actin being a key regulator of nanostructure. I will combine my tools and 2-color 3D dSTORM to determine how actin in each synaptic compartment controls nanoorganization. My third aim is to interrogate the role of acute actin dynamics in synapse strength. The precise and independent roles of pre- and postsynaptic actin in synapse strength have been clouded by non-specific manipulations. I will use the tools developed in Aim 1 in conjunction with in vivo electrophysiology and in vitro imaging techniques to answer how bidirectional perturbation of presynaptic actin dynamics changes aspects of vesicle release and whether postsynaptic actin polymerization is sufficient to drive receptor plasticity. These aims synthesize my biochemical background, my sponsor’s synaptic optical imaging expertise, and the experience of the diverse UMB faculty to improve our understanding of how actin controls the basic cellular and molecular physiology of synapses, and provide a strong platform to launch my independent career in science.