Skip to Content
Merck
  • Autophagy induced by calcium phosphate precipitates targets damaged endosomes.

Autophagy induced by calcium phosphate precipitates targets damaged endosomes.

The Journal of biological chemistry (2014-03-13)
Xi Chen, Bilon Khambu, Hao Zhang, Wentao Gao, Min Li, Xiaoyun Chen, Tamotsu Yoshimori, Xiao-Ming Yin
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydroxyapatite, nanoparticles, dispersion, 10 wt. % in H2O, <200 nm particle size (BET)
Sigma-Aldrich
Hydroxyapatite, nanopowder, <200 nm particle size (BET), contains 5 wt. % silica as dopant, synthetic
Sigma-Aldrich
Hydroxyapatite, synthetic, 99.8% trace metals basis (excludes Mg)
Sigma-Aldrich
Hydroxyapatite, nanopowder, <200 nm particle size (BET), ≥97%, synthetic
Sigma-Aldrich
Hydroxyapatite, reagent grade, powder, synthetic
Sigma-Aldrich
Calcium phosphate tribasic, suitable for plant cell culture, BioReagent, powder
Sigma-Aldrich
Calcium phosphate tribasic, 34.0-40.0% Ca basis