In pancreatic islet β cells, insulin granules (IG) are formed at the Golgi complex deep inside the cytoplasm. They need to be actively transported from the site of production to underneath the plasma membrane for regulated secretion. It was previously assumed that MT-dependent transport directionally delivers IGs to the cell periphery along the straight microtubule (MT) tracks, predicting a positive role of MTs in insulin secretion. However, our data over the previous grant cycle show that MT-dependent transport restricts secretion of pre-existing IGs, which are normally present in excessive numbers. We have also found that this negative regulatory function was ascribed to the unique configuration of β-cell MTs. In many other secretory cells, MTs are assembled from the centrosome and organized as radial tracks that allow directional cargo movement. In contrast, most β-cell MTs are nucleated at the Golgi (Golgi-derived MTs, GDMTs) and are organized as a dense, non-directional meshwork in the cell interior, with addition of stable sub-membrane MT bundles at the cell periphery. Experimental tests and mathematical modeling indicate that this configuration leads to trapping of IGs within the cytoplasm, storing them for sustainable insulin release during long-term β-cell function to avoid insulin-insufficiency-induced diabetes. This function also prevents insulin over-secretion at each stimulus to avoid hyperinsulinemia-induced hypoglycemia. Importantly, we show that glucose stimulus triggers reconfiguration of the MT networks: it induces new GDMT nucleation via the cAMP/EPAC2-mediated signals, which is essential for new IG biosynthesis. It also induces MT disassembly in β-cell periphery to enhance insulin secretion by phosphorylating tau, a well-established microtubule associated protein (MAP). However, a big part of intracellular mechanisms that are responsible for β-cell MT organization and its action downstream of glucose remains elusive. Intriguingly, our preliminary data suggest that glucose-induced MT remodeling depends on the islet microenvironment. In this proposal, we will test a hypothesis that optimal dynamic architecture of the β-cell MT networks, modulated by intracellular molecular machinery and intercellular paracrine signals, is essential for β-cell function and glucose homeostasis. By addressing our Specific Aims, we will: (1) investigate building and regulating the unique MT networks in β cells, (2) determine MT-dependent regulation of IG transport and positioning in β cells, and (3) determine roles of MTs in paracrine α-β cell communication in islets.