Regulation of Dynein-Mediated Transport

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PROJECT SUMMARY Organization is a fundamental and defining feature of life at all degrees of scale, from the subcellular level all the way up to the level of the organism. On the cellular level, molecules and organelles must be situated with appropriate temporal and spatial precision such that processes may proceed according to the needs of the cell. Similarly, cells are arranged into appropriate layers to define underlying tissue organization, which provides the basis for organ function, and to support health of the organism. Major determinants of subcellular and cellular organization are molecular motors that transport diverse cargoes throughout the cellular environment. One such motor is cytoplasmic dynein, which transports numerous types of cargoes along microtubule tracks during all cell cycle stages, and within many cell types. For instance, dynein is the major retrograde microtubule motor that transports many vesicular and protein cargoes toward the cell body of neurons. In addition to orchestrating appropriate subcellular organization, dynein plays a major role in the establishment and maintenance of tissue architecture. For instance, a major determinant of cell fate and consequent tissue organization is the orientation and position of the mitotic spindle with respect to the boundaries of the cell. In addition to localizing to the membrane of small vesicular cargoes – from where it effects their transport – dynein motors are anchored at the plasma membrane from where they orient and position the spindle through precisely tuned interactions with microtubules. During such processes as organismal development and tissue homeostasis, spindle orientation and position dictate the plane of cell division, and thus whether a cell divides symmetrically or asymmetrically. Symmetric stem cell divisions result in two identical stem cells, whereas a switch to asymmetric division results in one stem cell and a differentiated cell, which promotes tissue stratification. Thus, dynein is a critically important molecule that dictates biological organization on many levels of scale. The precise mechanisms by which dynein performs all these disparate functions with appropriate spatial and temporal control are unclear. The lack of such information presents an impediment towards the development of effective therapies that may prevent or reverse defects in cellular and tissue organization that can lead to various devastating disorders (e.g., malformations of cortical development, motor neuron diseases). In the proposed studies, we will use a combination of in vitro and cell biological approaches to determine the mechanisms by which dynein is regulated to perform its cargo transport functions. Specifically, we will: (1) resolve the mechanism by which the lissencephaly-related protein LIS1 initiates dynein-mediated cargo transport; (2) determine how dynein effects spindle movements with precise directional precision; (3) determine how, and the molecular basis by which various critical regulators affect dynein activity; and, (4) investigate the coordination and interplay between cell cycle state and dynein activity. Our studies will provide critical insight into fundamental mechanisms that dictate transport of numerous cargoes with spatial and temporal precision such that cellular and ultimately organismal health is established and maintained.