decoupling internalization, acidification and phagosomal-endosomallysosomal fusion during phagocytosis of inla coated beads in epithelial cells解耦内化、酸化和phagosomal-endosomallysosomal融合在上皮细胞吞噬作用u201d涂层的珠子.pdfVIP

Decoupling Internalization, Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells ¤ Craig D. Blanchette, Youn-Hi Woo, Cynthia Thomas, Nan Shen, Todd A. Sulchek , Amy L. Hiddessen* Physical Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, United States of America Abstract Background: Phagocytosis has been extensively examined in ‘professional’ phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in ‘non-professional’ phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal- endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Methodology/Principal Findings: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor- mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales f