Optogenetic control of intracellular membrane trafficking

Abstract

Intracellular membrane trafficking is a complex conserved aspect of eukaryotic cells. Trafficking events (e.g., transport, tethering, docking and fusion) mediate and participate in diverse intracellular processes. An impairment in any trafficking step can alter cell metabolism, cell polarization, and/or specific signaling pathways and may be associated with severe diseases, including cancer, immunodeficiency, and neurological disorders. Because of the high dynamics and multi-step characteristics, intracellular membrane trafficking is difficult to study in a spatiotemporal manner. This study is divided into two parts and focuses on the study of intracellular membrane trafficking using a new optogenetic approach. Part I is the development of the system for spatiotemporal targeting and control of various intracellular membranes, using blue-light-induced hetero-interaction between Arabidopsis’s Cryptochrome 2 and CIB1. To target specific intracellular membranes, I utilize several membrane targeting proteins, especially Rab GTPases-the small GTPase family that are well-known as membrane identities. In this system, illumination induces either a rapid recruitment of protein to the targeted membrane, or an aggregation that disrupts the dynamics and functions of the targeted membrane. I apply the tool to specifically perturb several trafficking processes, including lipid modification on the early endosomes, multi-step transportation of receptors, proteins trafficking, secretory pathway and signals initiated from endosomes. In Part II, I demonstrate the examples of studies that can exploit the advantages of the developed optogenetic system in Part I. In neuronal growth study, I utilize this tool to reveal different functions of local Rab5- and Rab11-mediated membrane trafficking in the growth cones and somas of young hippocampal neurons. The result reveals an important role of Rab5-mediated trafficking in maintaining protrusion rate during the rapid growing stage. In cancer signaling study, I showed that several Rab GTPases localize to the protrusion area in invasive breast cancer cell line, and the potential of using this new optogenetic tool to study distinct roles of different Rab GTPases during tumor cell migration. In summary, in this study I develop a new optogenetic system to control diverse intracellular membrane trafficking processes. My results prove that the developed system is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in many models, especially in polarized complex such as neurons, and in vivo studies in tissues or whole organisms.