Spatio-temporal structural and functional studies into the novel interaction between LIMD1 and clathrin heavy chain proteins

We have identified the LIMD1 gene that may be damaged or lost in many human diseases and cancers. In order to determine why this protein is so important for human health we performed experiment to determine how this protein functions. We subsequently discovered that LIMD1 interacts with a subset of proteins the function of which is to regulate a critical cellular process called endocytosis. Thus LIMD1's ability to regulate cell growth and proliferation could be through novel interactions with key regulators of endocytosis.

I have recently implicated the putative tumour suppressor gene LIMD1 in the development of lung cancer although the physiological significance/role of LIMD1 function in non-transformed cell is currently unclear. Using in vivo co-immunoprecipitation (co-IP) assays and proteomic analysis I have identified the clathrin heavy chain and Hsc70 proteins as novel binding partners of LIMD1. These interactions implicate LIMD1 in the regulation of clathrin-mediated endocytosis (CME) and/or endosome trafficking. I have further confirmed the specificity of the LIMD1-clathrin interaction via direct in vitro binding assays and in vivo endogenous co-immunoprecipitation experiments. CME and endosome processing/maturation represent critical cellular functions of receptor internalisation and cell signalling control respectively. The objective of this project therefore, is to determine the functional significance of these protein interactions with LIMD1 and thus obtain a greater insight into how loss of LIMD1 function or its de-regulation may affect these fundamental biological processes and therefore contribute to the disease process. This focused application will utilize three established and clearly defined research/experimental approches which represent a logical progression of work and also will complement each other with respect to addressing the hypothesis and project goals. Briefly these are 1) In vitro studies to determine specific protein-protein interactions 2) Spatiotemporal colocalisation analysis using live cell imaging. 3) Functional studies using live cell real-time imaging of fluophor-tagged-proteins