Equipment supplement - Refeyn TwoMP iSCAT microscope

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Project Summary/Abstract Intracellular cargo transport, such as lipid-bound insulin granules and presynaptic vesicles that are destined for secretion at the plasma membrane, rely on the concerted effort of kinesin-1 (kin1) and myosin Va (myoVa) molecular motors. These double-headed molecular motors carry their common cargo by stepping processively for considerable distances along their respective cytoskeletal tracks, i.e. microtubules (MTs) for kin1 and actin filaments for myoVa. To successfully deliver cargo, teams of kin1 and myoVa motors on the cargo surface, must overcome the physical challenges presented by the 3-dimensional (3D) complex network of MTs and actin filaments that comprise the cell's cytoskeleton, which also serves as these motors' highways. To determine how efficient intracellular cargo transport and delivery are accomplished despite the physical challenges presented by the cell's complex cytoskeletal highway, we have developed a near-physiological in vitro model system of kin1 and myoVa transport that is composed of a complex, but well-defined, 3- dimensional (3D), MT and actin filament network. Physiologically-relevant lipid-bound liposomes will be formed having Rab receptor proteins embedded in the liposome membranes so that molecular motors can be linked to the Rab receptors through their respective adapter proteins as in vivo. Once formed, motor-coated liposomes are introduced into the 3D networks and their transport trajectories defined using state-of-the-art single molecule biophysical techniques with high spatial and temporal resolution. Track-binding proteins to MTs (MAP7, Tau) and actin filaments (tropomyosins) will be added to dictate the direction of liposome transport. Key questions to be addressed using this well-defined model system are: 1) How are motors loaded onto the cargo surface? 2) Are motors on the cargo surface that are not engaged in transport, passive hitchhikers or are they cargo tethers that electrostatically interact with the heterologous track to enhance cargo transport by the actively engaged motors? 3) Is cargo hand-off from MT- to actin-based transport a coordinated event or the result of a tug of war? 4) Do track-binding proteins help to sort cargo by enhancing or inhibiting transport on specific MT and actin filaments? To interpret the results of these experiments, we will develop a mechanistic in silico transport model that incorporates the mechanical interactions between motor teams on the liposome surface. We propose that a functional interplay exists between the properties of the cytoskeletal tracks, motors, and cargos, which determines how teams of molecular motors meet the cellular demands placed on them by 3D cytoskeletal highways. The data obtained will provide a rich, mechano-spatial knowledgebase for the field and serve as a foundation for understanding molecular motor transport in the complex cytoarchitectural environment of the cell and how efficient motor transport systems are designed for delivery and retention of cargo at its destination.