We have been studying clathrin-independent forms of endocytosis (CIE) and have identified a number of endogenous PM proteins that enter cells through this mechanism. We have begun to study these proteins in detail in an attempt to understand how they travel in cells and whether they specifically interact with cellular machinery. We have identified signals in the cytoplasmic tails of CD44, CD98 and CD147 that are responsible for their altered trafficking and are looking for cellular machinery that is responsible for recognition and sorting of these signals. Understanding how these proteins move into and out of cells is important because these proteins are involved in interaction with the extracellular matrix (CD44), are involved in nutrient transport (CD98) and interact with integrins and matrix metalloproteinases (CD147). To facilitate these studies we have developed a method to covalently label PM proteins so that we can follow quantitatively their entry into cells by CIE and their subsequent itinerary. Using SNAP tag technology, we created fusion cargo proteins that contain the SNAP tag on the extracellular portion of the protein and then made a modification to the fluorescently labeled SNAP ligand so that we could release the fluorescent label with cell impermeable reducing agent (Cole and Donaldson, 2012). This allows us to track the internalized SNAP protein while removing the cell surface pool. We are using this technology to study the trafficking and turnover and identification of interacting proteins of the SNAP tagged cargo proteins.
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