title: Autophagy induced by calcium phosphate precipitates targets damaged endosomes.
abstract: Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62.
authors.affiliation: Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310000, China,; Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Bilon
authors.initials: B
authors.lastname: Khambu
authors.affiliation: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Wentao
authors.initials: W
authors.lastname: Gao
authors.affiliation: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202,; Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China, and.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Genetics, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Yoshimori
authors.affiliation: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202,. Electronic address: xmyin@iupui.edu.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D001798:Blood Proteins; D002130:Calcium Phosphates / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects* / Q000235:genetics; D011992:Endosomes / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism; D037161:Galectins; D057809:HEK293 Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D002130:Calcium Phosphates; D037502:Galectin 3; D037161:Galectins; D051907:Lysosomal Membrane Proteins; D000071456:Sequestosome-1 Protein; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C428445:LGALS3 protein, human; C099718:SQSTM1 protein, human; C000716463:rab7 GTP-binding proteins, human; C020243:calcium phosphate
keywords: Autophagosome; Autophagy; Calcium Phosphate Precipitate; Cell Signaling; Endosomal Damage; Endosomes; Lysosomes; Signaling