Induction of macroautophagy by exogenously introduced calcium.
Macroautophagy can be activated by a broad range of agents and cellular manipulations. In performing cellular transfection using the calcium phosphate method, we noticed that the calcium phosphate precipitates (CPP) could induce LC3 punctation. Because of the wide use of this transfection method in mammalian cells and the potential significance of calcium in autophagy induction, we investigated whether CPP could specifically induce macroautophagy. We found that CPP-induced LC3 punctation was dependent on calcium and could be neutralized by an extracelluar or intracellular calcium chelator. The punctation was not due to nonspecific aggregation of LC3 since it depended on the amino acid residue Glycine120, which is specifically required for LC3 to conjugate to phosphatidylethanolamine (PE). Consistently, there was also a significant increase of the PE-conjugated form of LC3. Electron microscopy confirmed the accumulation of typical autophagosomes following CPP treatment. Flux analysis indicated that CPP induced but did not inhibit autophagic degradation. Finally CPP-induced autophagy depended on the classical macroautophagy machinery including Beclin 1, the class III phosphoinositide-3 kinase and Atg5. Our studies thus indicate that exogenously introduced calcium in the form of CPP could specifically induce macroautophagy, which may have the practical significance in the use of this agent for introducing genes into cells, and for studying the mechanism of autophagy as a model system.
Gao
Wentao
W
Department of Pathology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Ding
Wen-Xing
WX
Stolz
Donna B
DB
Yin
Xiao-Ming
XM
D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D002130:Calcium Phosphates / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010588:Phagosomes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D002130:Calcium Phosphates; D011993:Recombinant Fusion Proteins; C020243:calcium phosphate