The long-term goals of this proposal are to understand the regulatory mechanisms that allow organelle transport to be integrated with development. Motor-driven transport along microtubules plays many essential cellular roles, yet the mechanisms with which cells control its specificity, timing and destination remain a mystery. The proposal focuses on the rules of regulation for bidirectional transport, a kind of intracellular transport in which motor cooperation results in novel emergent phenomena not easily understood by studying the individual motors in isolation. A unique model system, lipid-droplet motion in Drosophila embryos, provides the opportunity to attack this long-standing problem in cell biology with an interdisciplinary approach that combines experimental techniques from genetics, biochemistry, molecular biology, and biochemistry. The proposal focuses on Phase II of droplet transport in which the Halo protein acts as a directionality determinant: in the absence of Halo, transport is minus-end directed; in the presence of Halo, it is plus-end directed. The questions asked are designed to unravel how the Halo signal is ultimately transmitted into changed activity of the plus-end motor kinesin-1 and the minus-end motor cytoplasmic dynein. Two complementary approaches are proposed, focusing on the one hand on the signal Halo and on the other hand on Sfo, a component of the motor machinery physically connected to the motors. 1) To understand how Halo acts as a signal, protein partners of Halo will be identified and a structure-function analysis of Halo will be performed. For selected Halo mutants, physical motion parameters will be quantified, using nanometer-scale tracking and stall-force measurements with optical tweezers. 2) Sfo has been proposed to be a """"""""conductor"""""""", a molecule that fine-tunes how far cargoes travel in a single uninterrupted motion. Experiments are proposed to characterize the developmental expression and localization of Sfo, to determine if Sfo is required to transduce any aspects of the Halo signal, and to identify Sfo domains important in vivo. These experiments will in particular critically test whether Sfo indeed acts as a conductor. If successful, these studies will provide a framework for how a transacting signal is transduced by the transport machinery to affect properties of motors on a specific cargo. Because Halo is the founding member of a whole protein family in Drosophila, because Sfo is conserved from fungi to mammals and because both molecules are expressed throughout Drosophila development, these studies are expected to shed light on many different transport processes, both in flies and other eukaryotes. ? ?
The proposed work has broad significance for human health since aberrant motor function is linked to diseases ranging from Alzheimer's to cancer. These studies will also illuminate the basic biology of lipid droplets, organelles important in obesity and cardiovascular disease. ? ? ?
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