Deciperhing inter-organelle cross talk in epithelial architecture

The liver is composed mostly of specialised cells known as hepatocytes. Hepatocytes perform vital functions including bile secretion, uptake of nutrients such as glucose and lipids from the blood, as well as the removal of toxins from the blood. To perform these distinct tasks, cell-cell junctions form between hepatocytes to maintain the bile-blood barrier. Defects in junctions between hepatocytes are a hallmark of liver disease including fatty liver disease and liver cancer. It is therefore crucial to understand how cell-cell junctions are established and maintained in hepatocytes.

Recently, using electron microscopy, we have observed that junctional complexes in the cell membrane (termed the plasma membrane, PM) are often found in close proximity to an intracellular membrane compartment termed the endoplasmic reticulum (ER). Thus, intracellular ER membrane compartments are extensively localised at regions in the PM that form junctions between hepatocytes. Contacts between the ER and PM (ER-PM contacts) are important sites for the transfer of newly synthesised membrane lipids from the ER to the PM. Our ongoing studies are revealing how ER-PM contacts maintain the precise supply of lipids to the PM needed for the stability of junctions between hepatocytes.

Hepatocytes are susceptible to damage upon chronic overload of lipids from the blood, termed lipotoxicity. Non-alcohol related fatty liver disease (NAFLD), a condition caused by the accumulation of fat in the liver, is a growing health concern in the UK and the western world. According to the British Liver Trust, it is estimated that one in three people have early-stage NAFLD and that 20% of patients with NAFLD may develop more severe liver disorders including liver cancer. A hallmark of metastatic cancer cells is the loss of cell-cell junctions that allows subsequent cancer cell migration. Consequently, our studies to reveal how the intracellular ER maintains normal hepatocyte architecture and function may lead to new insight into the progression of liver diseases caused by alterations in membrane lipids and cell-cell junctions.

Our lab has discovered that contacts between the endoplasmic reticulum and the plasma membrane (intracellular ER-PM contacts) are enriched at the lateral domain that forms intercellular junctions between hepatocytes. We postulate that membrane lipid exchange taking place at ER-PM contacts stabilises cell-cell junctions in part by facilitating the nanoscale clustering of junctional proteins. To test this, we will 1) determine how ER-PM contacts direct membrane lipid composition and organisation at cell-cell junctions, 2) determine how alterations in ER-PM contacts impact the stability and function of junctional complexes including adherens junctions and tight junctions, 3) elucidate how ER-PM contacts are formed in proximity to cell-cell junctions, and 4) establish conserved organising principles of ER-PM contact distribution and function in epithelial architecture. The experimental plans will use 2D and 3D model cell culture systems, combined with cutting-edge imaging methods including correlative super-resolution microscopy and 3D scanning electron microscopy array tomography to study the dynamics of ER-PM contact and cell-cell junction assembly. We will use quantitative lipidomics to measure PM lipid composition upon modulation of ER-PM contacts, including CRISPR/Cas9 knockout cell lines already available in the lab, as well as validated biosensors to monitor membrane lipid composition in specific PM domains (apical, basal, and the lateral domains that form cell-cell junctions). Advanced biophysical and spectroscopy techniques will be employed along with atomistic modelling to study interactions between junctional proteins and lipids, as well as to interrogate nanoscale membrane lipid organisation in the lateral domain. As a result, we expect to gain new mechanistic understanding of how ER-PM contacts regulate PM lipid organisation and cell-cell adhesion in hepatocytes as well as additional epithelial cells and tissues.