title: Artificial induction of autophagy around polystyrene beads in nonphagocytic cells.
abstract: Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.
authors.affiliation: Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Iwaoka-cho, Nishi-ku, Kobe, Japan.
authors.forename: Shouhei
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kojidani
authors.forename: Hiroko
authors.initials: H
authors.lastname: Osakada
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Yoshimori
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002477:Cells / Q000187:drug effects* / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008863:Microspheres; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D010316:Particle Size; D010586:Phagocytes / Q000187:drug effects / Q000502:physiology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D010873:Pinocytosis / Q000502:physiology; D011137:Polystyrenes / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins