A Facility for the Analysis of Radio-labelled Molecules

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Organelles are the "organs" of eukaryotic cells. There are many organelle types, each with distinct biophysical, biochemical and functional properties, or organelle identity. For example, the endoplasmic reticulum is a labyrinth of tubules in which protein synthesis occurs, while lysosomes are round and full of digestive enzymes. The long-term goal of my research is to understand the molecular mechanisms that establish, maintain and change organelle identity. The phosphoinositide (PtdInsP) signaling lipids are key architects of organelle identity, of which there are seven species. Each PtdInsP has a particular organelle distribution and binds to a distinct set of cognate effector proteins. In this way, the presence of a specific PtdInsP "paints" the host organelle with molecular identity. Consequently, excessive, inadequate or mis-localized synthesis or turnover of PtdInsPs is detrimental to cells. Therefore, my laboratory seeks to understand the molecular mechanisms that regulate PtdInsPs in order to understand organelle identity processes. To dissect PtdInsP regulation, we must be able to quantify the levels of each PtdInsP in cells under various genetic and biochemical conditions, which I currently cannot. The goal of this grant is to establish a "Facility for the Analysis of Radio-labelled Molecules", which will support my research and that of Drs. Antonescu, Coe, Foster, Karshafian and Marshall, who are interested in understanding the role of PtdInsPs in processes like endocytosis, membrane transport, infection, sonoporation and receptor signaling. With this Facility, we will process and quantify radio-labelled PtdInsPs by High-Performance Liquid Chromatrography (HPLC). To establish the Facility, we need: i) equipment dedicated to process radio-labelled samples; ii) a centrifuge-vacuum to process samples and iii) a flow scintillator to quantify radioactive signal from the HPLC eluant in real-time. The Facility makes use of a new HPLC at Ryerson University. This research may help the the pharmaceutical and biotechnology industries to develop therapies against ailments caused by PtdInsP malfunction, and decrease their negative impact on affected Canadians and the Canadian economy.