Molecular mechanism of bidirectional transport

PROJECT SUMMARY In cells, bidirectional transport of vesicles and organelles involves a tug-of-war between kinesin and dynein motors as they carry cargo along microtubule tracks. This transport is particularly important in the elongated axons and dendrites of neurons, and transport defects are linked to neurodegenerative diseases. Microtubules are dynamic polymers that grow and shrink from their ends by the addition and loss of tubulin subunits, and these dynamics are essential for neurogenesis, cell migration, and formation of the mitotic spindle during cell division. The goals of this proposal are to uncover the molecular mechanisms underlying bidirectional transport by kinesin and dynein and the mechanisms by which microtubules grow and shrink from their ends. Many of the molecules involved in bidirectional transport are known, but the working mechanisms of these component parts and how their activities combine to achieve the emergent bidirectional transport are not well understood. The effectiveness of each motor to compete in bidirectional transport is intimately linked to their ability to maintain association with their microtubule track under load. To characterize the load-dependent properties of kinesin and dynein, we will track gold-nanoparticle labeled motors at msec- and nm-scale resolution as they move against an elastic load generated by a DNA ‘spring’. We recently found that all three transport kinesin families act as ‘catch-bonds’ at stall, defined as dissociating more slowly under load. This finding has important implications for how motors compete, and we will vary nucleotide conditions and track motors at high resolution to uncover the underlying mechanism. In parallel, we will construct pairs and teams of motors using DNA origami and measure which motors dominate during these reconstituted ‘tug-of-war’ battles on microtubules built from diverse tubulin isotypes. We will use stochastic computational models of bidirectional transport to understand the emergent behavior in terms of the molecular components. In the second thrust of the proposal, we will build on our ongoing single-molecule investigations into microtubule polymerization dynamics. By linking a 20-nm gold nanoparticles to recombinant tubulin dimers, we are able to visualize the reversible binding kinetics at both stable and growing microtubule ends, and from these measurements extract the on- and off-rate constants and free energies that govern tubulin binding to microtubule ends. We will characterize different tubulin isotypes and mutants to define how subtle modulations of lateral and longitudinal binding free energies lead to observed changes in microtubule dynamics and stability. Finally, we will bring together the transport and dynamics thrusts by investigating how microtubule binding proteins that induce cooperative structural changes in the microtubule lattice alter motor properties and microtubule end dynamics. Understanding polymerization at the level of individual tubulin interactions is vital for understanding the mechanisms by which the diverse protein and small molecule modulators of microtubule dynamics exert their effects on microtubules in cells.