title: Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells.
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.
  
  
  RESULTS
  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 for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification.
  
  
  CONCLUSIONS
  Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems.
authors: Physical & Life Sciences, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors: Craig D
authors: CD
authors: Blanchette
authors: Youn-Hi
authors: YH
authors: Woo
authors: Cynthia
authors: C
authors: Thomas
authors: Nan
authors: N
authors: Shen
authors: Todd A
authors: TA
authors: Sulchek
authors: Amy L
authors: AL
authors: Hiddessen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018938:Caco-2 Cells; D004285:Dogs; D011992:Endosomes / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008089:Listeria monocytogenes / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D041681:NIH 3T3 Cells; D010587:Phagocytosis
chemical_list: D001426:Bacterial Proteins; C069761:internalin protein, Bacteria