De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy

Abstract

2020) Denovo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy, ABSTRACT Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13 C-labeled choline and 13 C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/ alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagyinduced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues.

Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets

“…The present result clarified the physiological function of CCTβ3. This result differs from that of a recent study that concluded CCTα as the critical CCT isoform in autophagosome formation 38 . We argue that the prolonged loading of propargylcholine used for PC labeling in the study 38 (i.e., 6 vs. 1 h pulse loading in the present study) is not appropriate for visualizing de novo-synthesized PC because all cellular membranes including cargos engulfed by autophagosomes are intensely labeled by the protocol (see Fig.…”

The influence of choline treatment on behavioral and neurochemical autistic-like phenotype in Mthfr-deficient mice

“…1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), an entity of phosphatidylcholine, is concentrated at the presynaptic area, playing a role in neuronal synapses in the brain 89 . On another level, by promoting de novo phosphatidylcholine synthesis, choline supplementation may affect the formation of autophagosomes 90 . Indeed, choline treatment normalized the levels of the autophagy markers towards those in the control group (Wt-Wt).…”

Lysosomal dysfunction and autophagy blockade contribute to autophagy-related cancer suppressing peptide-induced cytotoxic death of cervical cancer cells through the AMPK/mTOR pathway

“…Autophagy is a lysosomal-mediated form of degradation in cells. The process of autophagy includes the formation of autophagosomes, autophagosome-lysosomal fusion, and autophagolysosomal degradation [12]. During degradation, an acidic pH and the enzymatic action of lysosomal hydrolase leads to destruction of the inner membrane of autophagosomes and the elimination of autophagolysosomes, thereby maintaining cell homeostasis [13].…”