High content analysis of apical versus basolateral trafficking

点击翻译按钮获取中文摘要

Proteins are required to be sent to various cellular compartments in order for those distinct compartments to function efficiently. Small structures called vesicles mediate the sorting and trafficking of proteins. This current research program aims to elucidate how vesicles mediate the sorting and intracellular trafficking to the plasma membrane of polarized cells. Aquaporins are water permeable membrane channels found on the outer membrane of cells. Mammalian cells express 13 aquaporins, with aquaporins-0, -1, -2, -4, -5, -6 and -8 being permeable only to water, while aquaporins-3, -7, -9 and -10 are also permeable to small molecules such as urea and glycerol. Within cells, aquaporins can localize to specific membrane domains. For example, aquaporin-4 (AQP4) traffics and is localized to the basolateral membrane (the side and bottom of cells), while AQP5 is found on the apical plasma membrane (top of the cell). Although AQP expression is restricted, the molecular mechanisms that regulate this trafficking have not been fully elucidated. We have recently shown AQP4 vesicles are actively transported in cells, but to our surprise, found that vesicles are not directed towards their destination. Instead, we found that, vesicle dynamics in non-polarized (cells that do not have specific sides, bottom or top) and polarized cells was similar. This suggested that trafficking vesicles "sample" membrane targets and fuse to the appropriate target membrane to localize proteins, a significant shift away from current thinking.***In this proposal, we will elucidate the machinery required to package AQP4 into trafficking vesicles, study the mechanism of interaction of AQP carrying vesicles with target membranes and determine whether or not AQP vesicle dynamics differs in cells in the process of polarizing (acquiring sides, bottom and top). In order to accomplish our research goals, we will use our tracking algorithms to determine whether or not vesicle dynamics are altered in cell lacking various known trafficking determinants and we will use biochemical tools to study properties of AQP interactions with membranes. Since our tracking algorithm enables us to study hundreds of cells and thousands of vesicles, our methods provide data with statistical significance, which differs greatly from the majority of data obtained in previous studies on AQP trafficking which relied on protein localization or single images.***This proposal will provide excellent training in both cell biology and biophysics. Trainees will learn a variety of methods in these interdisciplinary projects such as Western blotting, cell culture, transfection, microscopy, programming in Matlab and image analysis. ***The successful completion of this proposal will lead to a greater understanding of the mechanisms cells use to sort and traffic proteins to various membrane domains in polarized cells (apical versus basolateral). *** **