Development of biophysical principles of vehicle coating

University Of Alabama At Birmingham, Birmingham AL

Investigators

Abstract

All mammalian cells transport proteins via the secretory pathway to sustain indispensable cell activities such as growth, division and differentiation. Transport is mediated by small vesicles that bud from one compartment, move towards the next compartment, and then fuse in order to deliver their cargo. Vesicular traffic involves a hierarchy of molecular subprocesses linked to one another in time and space. The goal of this project is to model vesicle traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process. In addition, this project will support interdisciplinary synergy by providing cross-discipline training opportunities and developing workshops and courses to effectively bridge the gap between biological and physical fields. Importantly, a focus is to advance scientific equity by actively recruiting and mentoring under-represented groups and by participating in programs aimed at increasing representation of minorities and promoting and developing community outreach programs to increase science awareness and literacy. This project focuses on the formation of vesicles that transport proteins at the ER-Golgi interface. Such vesicles form through the recruitment of the coatomer complex (also called COPI complex) to the nascent bud. The recruitment is mediated by a small GTPase of the ARF (ADP-ribosylation factor) family. Like all GTPases, ARF acts as a molecular switch by cycling between an inactive state when it is bound to a GDP, and an activated state when it is bound to GTP. Only the active form of ARF can recruit coatomer and initiate vesicle formation. The activation of ARF is mediated by a nucleotide exchange factor (GEF) called GBF1. Hence, GBF1 activates ARF, which then recruits coatomer to make COPI vesicle. In addition to coat recruitment, ARF also regulates the sorting of cargo proteins into the nascent bud, and this process requires that ARF continuously cycle from between the GDP and the GTP-bound state. To facilitate the GTP to GDP conversion, a GTPase activating protein (GAP) called ArfGAP1 is required. Hence, the process of vesicle formation requires, at the very minimum, 4 key components: GBF1, ARF, coatomer and ArfGAP1. While we have descriptive knowledge of each molecular subprocess mediated by each component, we lack the knowledge of how these processes are connected in time and space to result in vesicle formation. We also lack even basic understanding of the governing biophysical principles. Thus, the aim of this project is to elucidate this enigma. We propose four Specific Aims: (1) Determine diffusion type and parameters of the 4 key components in cytosol; (2) Determine membrane association processes for the 4 key components; (3) Determine volume and surface densities and define binding constants and reaction rates for the 4 key components; and (4) Develop mathematical models describing the formation of the ternary coating complex. We will measure the dynamic properties of the 4 key components by fluorescence recovery after photobleaching (FRAP) under various experimentally induced conditions to mathematically describe their behavior. The resulting models will be refined through targeted perturbations of the system. Our models will provide the framework for future integration of other participants such as cargo proteins, lipids and a multitude of known regulatory factors that together ensure specificity to vesicular traffic. We stress that the machineries and the mechanisms we will model are highly conserved, and that analogous processes mediate coating of other types of vesicles for transport between different cellular compartments. Thus, our models will be generally applicable and will form an obligatory foundation for understanding the biophysical processes that govern vesicle traffic. A systems understanding of vesicle coating based on the laws of physics is crucial for future manipulation in vivo and for building synthetic organelles and cells. The funding for this project comes from both the Division of Molecular and Cell Biology and the Division of Mathematical Sciences