retrieval of the vacuolar h+-atpase from phagosomes revealed by live cell imaging检索的空泡的时间显示的h + atp酶活细胞成像.pdfVIP

+ Retrieval of the Vacuolar H -ATPase from Phagosomes Revealed by Live Cell Imaging 1 1 2 ¨ 3 Margaret Clarke *, Lucinda Maddera , Ulrike Engel , Gunther Gerisch 1 Program in Genetic Models of Disease, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America, 2 Nikon Imaging Center at the ¨ University of Heidelberg, Bioquant, Heidelberg, Germany, 3 Max-Planck-Institut fur Biochemie, Martinsried, Germany Abstract Background: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. Methodology: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V- ATPase, in parallel with stage-specific endosomal markers or in combination with