The transport of organelles and macromolecular complexes through the cytoplasm is essential for the function of every eukaryotic cell. This process is performed by motor proteins that move their cargo along microtubules and actin filaments. The spatial and temporal control of motor-dependent transport is critical for cell division, organelle transport and positioning, and the movement of RNA and protein complexes within the cell. The goal of this proposal is to understand the molecular mechanisms, which regulate organelle transport and coordinate the work of molecular motors. Our previous work established a permanent cell line of pigment cells (melanophores) from the frog Xenopus laevis as a model system to study the regulation of organelle movement. Movement of pigment organelles in these cells is initiated by changes in the concentration of cAMP and is performed by two microtubule motors of opposite polarity, cytoplasmic dynein and kinesin-II, and an actin motor, myosin-V. Organelle transport by these three motors is regulated by intracellular cAMP concentration. Furthermore, the activity of two microtubule motors is coordinated. This proposal has three specific aims: (i) find what regulates organelle transport along microtubules in the melanophore system: we will identify differentially regulated protein kinase(s) bound to organelles and determine how they regulate organelle transport; (ii) find the mechanism of coordination between plus and minus-end movement of pigment organelles along microtubules: we will determine if coordination is governed by cooperative differential binding of plus and minus-end directed motors to receptor proteins on the surface of organelles or it is determined by a stochastic mechanism; (iii) find how myosin-V is regulated in melanophores and other cell types: we will explore mechanisms of regulation of myosin-V in interphase cells using Xenopus melanophores and mouse macrophages and determine the role of p21-activated kinase (PAK1) in its regulation.