title: A resealed-cell system for analyzing pathogenic intracellular events: perturbation of endocytic pathways under diabetic conditions.
abstract: Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan.
authors.forename: Fumi
authors.initials: F
authors.lastname: Kano
authors.forename: Daiki
authors.initials: D
authors.lastname: Nakatsu
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Noguchi
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Masayuki
authors.initials: M
authors.lastname: Murata
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002772:Cholera Toxin / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D003920:Diabetes Mellitus / Q000378:metabolism / Q000473:pathology*; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D066246:ErbB Receptors / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008954:Models, Biological; D008970:Molecular Weight; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D013301:Streptolysins / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003911:Dextrans; D005452:Fluoresceins; D018129:Phosphatidylinositol Phosphates; D013301:Streptolysins; D014168:Transferrin; C052526:fluorescein-dextran; C055525:phosphatidylinositol 3-phosphate; C025964:streptolysin O; D002772:Cholera Toxin; D066246:ErbB Receptors; D048051:p38 Mitogen-Activated Protein Kinases; D002122:Calcium Chloride