The goal of this research is to determine how both the dynamics and spatial organization of the actin and microtubule cytoskeletons are coordinated by a group of five interacting mammalian proteins: APC, Dia1, EB1, CLIP-170, and capping protein. This work will define the functions and mechanisms of these proteins in microtubule-actin cross-regulation, and will thereby provide a deeper understanding of the molecular activities and interactions that underlie such processes as cell migration, cell adhesion, and cell and tissue morphogenesis. This project uses bulk biochemical experiments combined with novel multi-wavelength single molecule fluorescence methods that we have tailored to directly observe the mechanisms of complex multi- component regulatory systems in vitro. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo to confirm that they are important for specific cellular functions of these proteins. The Specific Aims are: (1) Test the hypothesis that the microtubule plus end-binding protein EB1 directly regulates nucleation of actin filaments by APC-Dia1; (2) Test the hypothesis that the rate and duration of actin filament elongation is controlled by dynamic binding interactions of Dia1, capping protein, microtubules, and/or EB1 at barbed ends; and (3) Define the roles of APC, Dia1, EB1, and CLIP-170 in controlling microtubule plus end dynamics and in triggering ultrafast actin polymerization from microtubule plus ends.