Abstract

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca²⁺-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5–50 μM), sorafenib (1–40 μM), staurosporine (20–500 nM), or thapsigargin (20–500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca²⁺ chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca²⁺/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10–30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1–10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca²⁺ mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α₁/α₂-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca²⁺-dependent signaling, including CaMKI independent of AMPKα₁/α₂. Our data also suggest that AMPKα₁/α₂ might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.