Project Summary/Abstract: Microtubules are critical for nearly every function of eukaryotic cells, from their ability to divide and move to their ability to adopt specific morphologies and withstand mechanical forces. Microtubule assembly, dyamics, and functions are dictated and regulated by a large number of cellular factors including microtubule associated proteins (MAPs) and molecular motors in the kinesin and dynein superfamilies. Our overall goal is to define the mechanisms by which microtubules and kinesin motor proteins drive intracellular trafficking in mammalian cells. To do this, we combine powerful biophysical and biochemical methods that provide mechanistic detail on motor mechanics and motility with cellular assays that report on regulation and function within the complex cellular environment. We will continue to utilize these multi-disciplinary approaches to investigate critical gaps in our knowledge of mechanisms and regulation of intracellular trafficking. We will define mechanisms for targeting of proteins to the primary cilium, a microtubule-based organelle that protrudes from the surface of the cell and drives cell motility and signaling. We will utilize a novel chemical-genetic approach that we developed for engineering inhibitable motors to probe the functions of kinesins critical for the assembly and function of primary cilia. We will determine the motility and force-generating properties of kinesins using both in vitro and cellular assays and use this knowledge to understand how these properties were selected through evolution for specific motor functions in cells. Finally, we will test models of motor regulation by signaling pathways such as Hedgehog ligand. As defects in microtubules and kinesin motors are linked to developmental disorders, neurodegenerative diseases, and cancer, these studies will advance our understanding of their functions in cell biology and disease.