Project Summary Nonmuscle Myosin IIB (NMIIB) is an ATPase motor complex that generates force on actin filaments. This force generation is an essential driver of the actin re-organization that occurs in dendritic spines, actin dense post- synaptic structures, that allows spines to enlarge when stimulated. When a neuron is stimulated, actin mobilization in spines, spinal enlargement, and then actin stabilization of the enlarged structures occurs, and this dynamic process results in plasticity. Spine plasticity in regions of the brain such as the hippocampus (HPC) and basolateral amygdala (BLA) contributes to the molecular basis of learning and memory storage. While NMIIB is known to be a critical contributor to the structural plasticity underlying learning and memory, surprisingly little is known about its action and regulation in mature excitatory neurons. Previous work from our group established that NMIIB is a driver of actin polymerization in rodent hippocampal neurons and that it is regulated as a part of the NMDA receptor pathway upon synaptic stimulation. Inhibiting NMIIB in the HPC results in disruption of memory. Our group has also discovered a regionally specific role of NMIIB in the BLA. Methamphetamine (METH) exposure induces the actin cytoskeleton of a subset of spines to remain constitutively active in an NMIIB-dependent manner. Upon NMIIB inhibition, this overactive population returns to normal motility. Accordingly, NMIIB inhibition after METH exposure disrupts METH-associated memories and drug seeking, establishing NMIIB as a therapeutic target. With new advances in molecular level imaging, a comprehensive cellular biological study of NMIIB is now possible to elucidate its regulation of synaptic actin dynamics. To support this, I have generated and validated a novel endogenously tagged NMIIB knock-in mouse line containing both 3x FLAG and Halo tags as a tool compatible with super resolution imaging and biochemical analysis. Super resolution imaging is necessary to address our questions about NMIIB localization and dynamics because at 300nm, myosin filaments are just at the diffraction limit and any non- filamentous myosin structures will be even smaller. Preliminary data shows NMIIB interacts with proteins in the shaft and at tips of spines, suggesting that dynamic changes in subcellular localization are occurring on a scale < 1 micron and therefore super resolution, and even more specifically, single molecule localization microscopy (SMLM) is most suitable to investigate. In Aim1 we will determine the subcellular distribution on NMIIB in neurons from our tagged NMIIB line using stochastic optical reconstruction microscopy (STORM) in fixed samples. We will also treat neurons to simulate synaptic plasticity to determine if that changes NMIIB distribution. In Aim 2 we use live neurons from NMIIB mice to track the subcellular location of NMIIB and measure its trafficking dynamics within the spine using single particle tracking photoac...