Developmental regulation of lipid -droplet transport in <i>Drosophila</i>
The timing and directionality of microtubule-based transport in cells are regulated in a highly dynamic and cargo-specific fashion. My thesis research aims to gain mechanistic insights into these regulation processes, by studying two genes regulating lipid-droplet transport in early Drosophila embryos. After briefly over-viewing the general question and the experimental system we study (Chapter I), I focus the first part of my thesis (chapter III and IV) on the gene klarsicht (klar), it encodes a component of the motor regulatory machinery. I found the klar gene encodes distinct isoforms undergoing complex gene regulations and the Klar protein regulates distinct microtubule-based transport processes. In chapter III, I discuss the mechanism of how this single gene regulates distinct transport process in different cells. After demonstrating that localization of Klar to its cargoes is crucial for Klar function, I identify functionally important regions of Klar that confer distinct cargo specificity. Since the variation in cargo targeting domains is likely a result of complex gene regulation, I characterize (in chapter IV) the complexity of Klar gene expression and show preliminary evidence relating different Klar isoforms to distinct transport processes. To study cargo-targeting domains of Klar, I developed a novel cell-culture system to study lipid-droplet associated proteins. Chapter V gives an overview of this system and two examples of its applications. In the last part of my thesis (Chapter VI), I summarize my contribution to the cloning and identification of a novel directionality signal, Halo. Halo appears to be the first member of a new class of transport regulators, and I found that the timing of switches in net droplet transport is correlated with changes in expression of the halo gene. These studies further our understandings of how microtubule-based cargo transport processes are regulated, and open up new areas of investigation. I discuss remaining challenges and unsolved questions in the final chapter.
0307: Molecular biology