PROJECT SUMMARY (See instructions): Intricate cellular morphology is essential for neuron function. Axons in particular form thin and extremely long cables with synaptic contacts along their length to communicate with their target neurons. This cable property of the axons allows rapid conduction of electrical signals, or action potentials, to activate synaptic communication. Traditionally, axon diameter is thought to be relatively uniform. However, recent studies suggest that axon diameter is highly dynamic and can be controlled by neuronal firing. Our preliminary data further the view of dynamic axon morphology by revealing that axons are not simple cylindrical tubes, but rather exhibit peals-on-a-string morphology between synaptic varicosities. This structure is reminiscent of lipid bilayer pearling, generated through a tension-driven instability. In support of this, in silico modeling suggests that axon pearling depends on membrane mechanics such as bending modulus and tension. In fact, axon pearling is lost when hyperosmotic solution is applied, while it is exacerbated in hypoosmotic conditions. Thus, pearled axon morphology is tightly coupled to the biophysical properties of membranes. Two of the major determinants of membrane mechanics are lipid composition and cytoskeletal structure. Importantly, lipid composition and cytoskeletal structure also control the localization of transmembrane proteins such as voltage-gated sodium and potassium channels, essential for action potential firing. Therefore, membrane properties likely regulate both axon morphology and function. In this proposal, we will test this hypothesis by 1) determining the contribution of lipid composition and cytoskeletal structure to the axon morphology, channel localization, and action potential firing, and 2) determining how these factors change with plasticity induced by repetitive neuronal firing. Since many factors controlling membrane mechanics are implicated in neurological disorders such as epilepsy and depression, this study will potentially reveal the underling mechanisms by which misregulation of biophysical factors leads to pathophysiological conditions. We will achieve these goals with the expertise in theoretical modeling by Pl Rangamani and the expertise in ultrastructural analysis of neurons by co-Pl Watanabe. Together, we will elucidate the fundamental biophysical principles governing axon morphology and function.