Mechanisms of Clathrin-Independent Endocytosis

我们一直在研究网格蛋白独立形式的内吞作用（CIE），并已确定了一些内源性PM蛋白，通过这种机制进入细胞。 我们已经开始详细研究这些蛋白质，试图了解它们如何在细胞中旅行，以及它们是否与细胞机制特异性相互作用。 我们已经确定了CD44，CD98和CD147的细胞质尾部中的信号，这些信号负责改变它们的运输，并正在寻找负责识别和分选这些信号的细胞机制。 了解这些蛋白质如何进出细胞是很重要的，因为这些蛋白质参与与细胞外基质（CD44）的相互作用，参与营养转运（CD98），并与整合素和基质金属蛋白酶（CD147）相互作用。 为了方便这些研究，我们已经开发了一种方法来共价标记PM蛋白，使我们可以定量跟踪他们进入细胞的CIE和他们随后的行程。 使用SNAP标签技术，我们创建了在蛋白质的细胞外部分上含有SNAP标签的融合货物蛋白，然后对荧光标记的SNAP配体进行修饰，使得我们可以用细胞不可渗透的还原剂释放荧光标记（科尔和唐纳森，2012）。 这使我们能够在去除细胞表面池的同时追踪内化的SNAP蛋白。 我们正在使用这种技术来研究SNAP标记的货物蛋白的运输和周转以及相互作用蛋白的鉴定。

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.