Characterization of Polarised Trafficking Machinery in Epithelial to Mesenchymal Transition

Exocytosis of cellular cargo, such as proteins, enables communication between cells and is essential for maintaining proper cell structure and function. To ensure the accurate release of cargo at the right time and location, all cells contain a core set of eight proteins, collectively known as the exocyst. This protein complex interprets signals from specialized lipids and other molecular machinery to precisely regulate exocytosis in both space and time.
Different cell types have distinct needs for cargo transport to specific locations at various times. For instance, epithelial (stationary) cells require cargo delivery to the junctions between cells, which is essential for forming and maintaining a monolayer. In contrast, mesenchymal (migratory) cells need constant delivery of cell adhesion components to the basal membrane, supporting their ability to migrate.
The exact mechanism by which the exocyst, a single protein complex, regulates material delivery across different cell types remains unknown. Disruptions in material delivery can impair cell behaviour, potentially causing disease, depending on the specific cell type affected. Therefore, understanding of how the exocyst functions in various cell types is crucial.
The overarching aim of this project is to investigate how the exocyst regulates cargo delivery in a spatiotemporal manner. Specifically, we aim to:
1. Characterize the exocyst in epithelial and mesenchymal cells.
2. Explore how the exocyst works alongside other proteins and determine how cargo delivery is co-ordinated.
To address the above aims, this project will utilise a Exoc4-sfGFP endogenous CRISPR knock in NMuMG cell line to study the exocyst in epithelial and mesenchymal cell states.
1. Visualise the exocyst using live/fixed microscopy.
2. Characterise the exocyst and identify other proteins which assist in delivery using organelle immunoprecipitation (organelle-IP) and proteomics.
3. Validate identified proteins in cargo trafficking assays.