Molecular mechanism of bidirectional transport

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PROJECT SUMMARY Bidirectional transport of vesicles and organelles in cells involves a tug-of-war between the microtubule motors kinesin and dynein. This transport is particularly important in the axons and dendrites of neurons and in cilia and flagella, and transport defects are linked to neurodegenerative diseases such as Alzheimer’s and ALS, as well as ciliopathies. Although many of the molecular players are known, the working mechanisms of these component parts and how their activities combine to achieve the emergent property of bidirectional transport are not sufficiently understood. The goal of this proposal is to bridge the gulf in understanding between the mechanochemistry of single kinesin and dynein motors and the bidirectional transport dynamics of vesicles and organelles observed in cells. Unresolved questions include: How does load affect the mechanochemistry and detachment kinetics of different kinesins and dynein? How do opposing motors coordinate and compete to achieve bidirectional transport? How do regulatory proteins, microtubule associated proteins and tubulin post- translational modifications alter the balance of plus- and minus-end directed motility to achieve proper vectorial transport? To address these questions, Interferometric Scattering (iSCAT) microscopy with nanometer spatial precision and millisecond temporal resolution will be used to track individual motor domains, single motor proteins, and multi-motor assemblies as they step along their microtubule tracks. These microscopy studies will be complemented by stopped-flow kinetics investigations, in vitro reconstitution experiments, and computational modeling to understand assemblies of increasing complexity. Specific motor mechanisms to be investigated include the origin of the fast speed and superprocessivity of kinesin-3, the polymerase mechanism of kinesin-5, and the molecular basis of dynein activation by its adapter proteins. A DNA tensiometer will be developed to understand the influence of mechanical load on kinesin and dynein mechanochemistry, and statistical tools will be developed to extract load-dependent detachment kinetics from these experiments. Finally, multi-motor assemblies will be built using DNA origami, which allows for precise control of motor number and positioning, and reconstituted lipid vesicles, which mimic the mechanical and diffusional properties of intracellular cargo. This work will advance our understanding of how organelles are correctly positioned in cells and how specific intracellular cargo are reliably targeted to their proper cellular locations.