A Facility for the Analysis of Radio-labelled Molecules

细胞器是真核细胞的“器官”。有许多细胞器类型，每一种都具有不同的生物物理，生物化学和功能特性，或细胞器身份。例如，内质网是蛋白质合成发生的小管迷宫，而溶酶体是圆形的，充满了消化酶。 我研究的长期目标是了解建立，维持和改变细胞器身份的分子机制。磷脂酰肌醇（PtdInsP）信号脂质是细胞器身份的关键建筑师，其中有七种。每个PtdInsP具有特定的细胞器分布，并结合到一组不同的同源效应蛋白。 以这种方式，一个特定的PtdInsP的存在“油漆”的主机细胞器的分子身份。 因此，PtdInsPs的过度、不足或错误定位的合成或周转对细胞有害。 因此，我的实验室试图了解调节PtdInsPs的分子机制，以了解细胞器的身份过程。为了剖析PtdInsP的调节，我们必须能够在各种遗传和生化条件下量化细胞中每种PtdInsP的水平，而我目前还不能。 这项资助的目标是建立一个“放射性标记分子分析设施”，这将支持我的研究以及Antonescu，Coe，Foster，Karshafian和马歇尔博士的研究，他们有兴趣了解PtdInsPs在胞吞作用，膜运输，感染，声孔作用和受体信号传导等过程中的作用。 有了这个设施，我们将处理和定量放射性标记的PtdInsPs的高效液相色谱法（HPLC）。 为了建立该设施，我们需要：i）专门用于处理放射性标记样品的设备; ii）用于处理样品的离心真空装置; iii）用于实时定量HPLC洗脱液中放射性信号的流动闪烁器。 该设施利用瑞尔森大学的新HPLC。 这项研究可能有助于制药和生物技术行业开发针对PtdInsP故障引起的疾病的疗法，并减少其对受影响的加拿大人和加拿大经济的负面影响。

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.