Throughout a lifetime of an organism synapse addition and elimination is on-going to ensure proper function of neuronal circuits. Growing evidence have revealed complex interactions involving intrinsic and extrinsic factors in synapse refinement with temporal and neuronal-type specificity. Studies using C. elegans have continued to expand the understanding of molecular and genetic pathways with single- synapse resolution. The locomotor circuit consists of several classes of excitatory cholinergic motor neurons and two classes of GABAergic motor neurons, and is a highly tractable system to discover mechanisms underlying synapse formation and refinement. Each neuron forms stereotyped pattern and number of synapses, providing an accurate readout to examine how synapses are dynamically regulated. Moreover, the development of the mature locomotor circuit involves a precisely timed remodeling of the embryonically born GABAergic neurons, known as “DD synapse remodeling”, in the absence of axonal morphological changes. We developed the first in vivo visualization approach to examine DD synapse remodeling. In our recent studies, we have defined critical roles of microtubule dynamics in promoting cargo and motor interaction in the formation of new synapses in DD remodeling. Our findings underscore the concept that microtubules are not passive tracks but play an active role in cellular signaling. In the specific Aim 1 of this renewal application we will leverage our expertise in genetic pathway dissection with in vivo imaging of microtubule components to dissect the roles of a novel kinase in DD synapse remodeling. In parallel, we have investigated the mechanisms regulating the cholinergic neuron synapses, and have uncovered roles of inter-tissue interaction mediated by a IgSF transmembrane domain protein ZIG-10. Our studies show that ZIG-10 regulates phagocytotic pathway via a SRC kinase in the adjacent non-neuronal tissues. In specific Aim 2, we will tackle the cellular action and the physiological impact of this pathway using innovative technologies. We will further examine how neuronal activity regulates this pathway. In Aim 3, we will investigate the role of a conserved MAGUK protein that may link the ZIG-10 pathway to phospholipid biosynthesis in synapse maintenance. Genetic mutations of homologous molecules in human have been linked to various neurological diseases. Together our findings will provide important insights to the underlying signaling network and advance our knowledge in the understanding of human diseases.