Project Summary Bidirectional transport is essential for cargo trafficking in cells and is required for proper growth and cell division. Kinesin and dynein are microtubule motors responsible for bidirectional cargo transport in cells. Defects in microtubule motor-based transport are linked to many neurodegenerative diseases including Alzheimer’s, Parkinson’s, spinal muscular atrophy, amyotrophic lateral sclerosis, and Huntington’s disease; thus, understanding the mechanisms underlying bidirectional transport is crucial to understanding transport deficiencies in disease states and developing potential treatments. Despite important advances in understanding the mechanochemical properties of individual motors, many questions remain regarding how motors work as teams, and how kinesins and dyneins coordinate with one another. A widely supported model for bidirectional transport is the ‘tug-of-war’ model in which teams of dynein and kinesin pull in opposite directions and the winning team determines the direction of transport. However, this model cannot account for the motor coordination and other regulatory factors involved. Previous modeling work identified the load-dependent detachment rate as the key parameter that determines whether kinesin or dynein wins in a motor tug-of-war, and recent experimental and theoretical work showed that vertical force inherent to widely used single-bead optical tweezer geometry significantly accelerates motor detachment rates. Consistent with this, when kinesin and dynein were connected through DNA linkages such that forces are only parallel to the microtubule, these two-motor complexes remained attached for much longer times than seen in optical tweezer experiments. The first goal of this project is to establish a novel technique that uses ssDNA as a pN-scale spring, to accurately determine motor stepping characteristics in the absence of vertical forces, mimicking physiological conditions. Aim1 will test the ability of transport kinesins and the dynein-dynactin-BicD2 complex to maintain stepping against a hindering load oriented solely parallel to the microtubule. Initially, motors will be tracked with a fluorescent probe via TIRF microscopy, and later a gold nanoparticle will be used to track in high resolution the load-dependent transitions in the kinesin stepping cycle. Aim 2 will use a DNA origami scaffold to pair gold nanoparticle-labeled kinesin and dynein together and track them via Interferometric Scattering (iSCAT) microscopy. The motor dynamics underlying the bidirectional transport trajectories will be interpreted using a computational model of kinesin-dynein transport. In Aim 3, teams of motors will be tracked to test how assisting and hindering loads inherent to multimotor geometries affect the competition between kinesin and dynein teams. Uncovering the motor dynamics underlying these complex multimotor systems is essential for understanding how intracellular bidirectional transport ensures that speci...