Projects
Communication between and within cells are important cellular processes. In our lab we use both cell and molecular approaches to investigate mechanisms of membrane trafficking. We use shRNA and CRISPR-Cas9 gene knockout to investigate the molecular and morphological impact of loss of a protein and what the function impacts are. Biochemical techniques are used to investigate protein-protein interactions and explore the impact on cell signalling and other cellular functions. We use a variety of imaging techniques to explore the membrane trafficking mechanisms and sub-cellular localisation, such as confocal imaging, live-cell imaging and transmission electron microscopy. This gives us a unique opportunity to identify the location of individual proteins and the morphological impact on membrane structures and organelles upon loss of function of a specific protein or protein interaction. Together these approaches provides us with a unique set of tools to give new molecular insights into membrane trafficking and how these mechanisms may become dysfunctional during pathogenesis such as cancer.
Investigating the role of CaMKK2 in membrane trafficking
A collaborative project with Prof. Ian Mills (University of Oxford & QUB) has uncovered a new role for calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) in membrane trafficking. We demonstrate that CaMKK2 associates with a number of proteins involved in COPI-mediated vesicle trafficking, in particular Gemin4 and delta-COP. Functionally, this was important as either knockdown or inhibition of CaMKK2 activity resulted in reduced levels of COPI coatomer, increased Golgi area, reduced lysosomal acidification and impaired autophagy. More broadly the dynamic regulation of vesicle trafficking by phosphorylation is a vitally important and nascent research are in attempting to define how signalling affects organelle biogenesis and function. Our data indicate that the contribution that CAMKK2 plays to the function of the secretory pathway and to Golgi-ER homeostasis is critical for the metabolic health of prostate cancer cells and for tumorigenesis. This is particularly relevant in prostate cancer where CaMKK2 is overexpressed in later stages of the disease.
Regulatory roles of clathrin-mediated receptor endocytosis
Clathrin-mediated endocytosis occurs by an orchestrated set of weak protein-protein interactions that together recruits cargo, invaginates the membrane and polymerises the coat protein clathrin resulting in clathrin-coated pit formation. Key regulatory mechanisms for this to occur in a spatially and temporally regulated manner are phospholipids and cargo-binding. Regulation of clathrin-mediated endocytosis by post-translational modifications have not been explored in great detail, but are emerging as important regulators of cell-surface expression of receptors in cancer. To follow on from my previous publications I have chosen to investigate the regulatory role of phosphorylations on FCHo2 protein interactions and have identified a kinase with a significant role. This is a significant contribution to the field of endocytosis and membrane trafficking as EGFR signalling is frequently deregulated in cancer biology and the kinase that I have identified also has an already established role in cancer biology. This research project aims to demonstrate that post-translational protein modifications can act as a rheostat for rewiring key molecular interactions and therefore the speed of receptor endocytosis and cell signalling.
I am also investigating the role of ubiquitin E3 ligases in the endocytosis and trafficking of death receptors in colorectal cancer (collaboration with Prof. Dan Longley). This research is important for understanding the mechanisms for how cancer cells become pro-survival and become resistant to TRAIL therapy. We are focussing on proteins involved in trafficking and ubiquitin ligases that are known to ubiquitinate or interact with endocytic proteins with the aim to identifying strategies to amplify cell death signals and promote apoptosis by regulating the sub-cellular localisation of death receptors. This research has potential impact on death receptor signalling, drug delivery and biomarker development for patient stratification.
The role of BAR proteins and lipid metabolising enzymes in inter-organelle contact formation.
Membrane-contact sites between mitochondria and the ER are functionally important for regulating lipid synthesis, stress and energy homeostasis by facilitating a local transfer of lipids and calcium to the mitochondria. These are dynamic membrane structures and the molecular composition of these membrane contact sites and the adaptations that they undergo in response to stress and changes in cell metabolism are still being characterised. During my research on FCHo2 in clathrin-mediated endocytosis I stumbled on an uncharacterised isoform of FCHo2 that associates with the ER and promotes ER membrane contact sites. These membrane contact sites are relevant in the disease biology of cancer, obesity and neurodegenerative diseases. Therefore, to include a translational research aspect of this theme I have used my expertise in imaging and have generated pilot data from prostate tissue by using electron microscopy in collaboration with Prof. Lisa Butler (University of Adelaide). The data shows a significant increase in mitochondria-ER membrane contact sites in prostate cancer tissue compared to a normal control. This is the first observation of this type of organelle contact sites in prostate cancer, a disease that is characterised by a prominent increase in lipid synthesis.
Another strand to this research theme in prostate cancer is investigation of ER stress mechanisms and in particular lipid droplet formation, which is conducted in collaboration with Dr. Emma Allott (QUB), a cancer epidemiologist. Her research has identified an upregulation of ER stress pathways in patients that were taking cholesterol lowering drugs (statins). The role of the unfolded protein response and ER stress is emerging as an important pathway in prostate cancer progression. Lipid droplet formation by the ER is an important cellular mechanism for alleviating ER stress which is a trigger for inflammation. These droplets act as a sink for excess lipids (triglycerides, cholesterol esters) to prevent lipotoxicity and serve as an important energy source and signalling hub. This is potentially a very significant biology as increased lipid synthesis and storage is one of the key hallmarks of prostate cancer. As such, this critical pathway represents an unexplored biology in prostate cancer that could further our understanding of the molecular mechanisms that contribute to the observed statin-induced effects in prostate cancer.