Developing quantitative understanding of adaptor-clathrin coating at the trans-Golgi network

All cells contain organelles that are specialized to perform a specific function. The function of these organelles depends on the correct transport of proteins into and out of each organelle. This transport is mediated by small vesicles that carry various cargos around the cell in a highly specific orchestration of movement.

Thus, the formation of vesicles that will specifically deliver select proteins, to a specific organelle, is critically important to the normal function of cells. The project seeks to understand how specific vesicles containing specific proteins form in the cells. The aim is to unite molecular/biochemical experiments with computational/arithmetic modeling to understand vesicular traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process.

Broader Impact activities include the intrinsic merit of the research itself, e.g., the lack of delivery of enzymes to various organelles causes severe diseases. The project will provide cross-discipline training opportunities and develop workshops and courses to effectively bridge the gap between biological and physical fields. Another goal of the project 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.

Protein transport between the compartments of the secretory and endosomal pathways is essential for activities such as growth, division and differentiation. Transport is mediated by vesicles that select cargo from one compartment, and then deliver the cargo to the next compartment. Cargo selection is mediated by coating lattices that assemble on the cytoplasmic aspect of nascent buds in a process involving a hierarchy of subprocesses systematically linked to one another causally or functionally in time and space.

The goal is to identify underlying principles that govern coating and to develop a predictive understanding of the emergent properties of the regulatory networks that facilitate coating. The project will focus on the subprocesses that assemble coating lattices for sorting cargo at the trans-Golgi network (TGN). Lattice formation at the TGN proceeds via a complex mechanism, in which an inner core of adaptors assembles first, followed by an outer layer of clathrin.

The project seeks to understand the dynamics and the biophysical parameters regulating the formation of adaptor-clathrin (AC) coating modules composed of the tetrameric AP1 complex or three monomeric Golgi-localized -adaptin ARF-binding (GGA1-3) adaptors and clathrin. The adaptors assemble on the membrane by interacting with activated Arf GTPases and the activation of such Arfs at the TGN is mediated by the BIG1 and the BIG2 guanine nucleotide exchange factors.

The project will use CRISPR/Cas9-modified knock-out (KO) cell lines to identify the specific Arfs and BIGs that mediate the recruitment of each AC coating module. Adaptor-clathrin (AC) coating will be modeled by mathematically describing the behavior of key components: BIG1, BIG2, Arf1-3, AP1, GGA1-3 and clathrin. The overall coating process is multi-step, multi-component, and variable.

Experiments alone cannot deal with this complexity, and computational modeling is needed to illuminate the spatio-temporal parameters of the process. Furthermore, the complexity of AC vesicle formation requires the development of sophisticated mathematical methods to model the behavior of the system. This project combines diverse expertise and ultimately seeks to decipher the general principles of coating module assembly which will define one of the Rules of Life.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.