Autophagy induction in the quiescent state: In vitro and in vivo characterization of cellular self-cannibalism in quiescent cells

Quiescence, or reversible exit from the cell cycle in early G1, is a conserved response to cues including nutrient deprivation and high cell density. We investigated functional characteristics important for quiescent cells to maintain homeostasis and cellular integrity over prolonged periods of non-division. All cells require timely degradation of unwanted or damaged proteins, with proliferating cells primarily relying on the ubiquitin-proteasome pathway. We show that contact-inhibited, quiescent dermal fibroblasts induce autophagy even though they are not starved. Quiescent fibroblasts deficient for the essential autophagy protein Beclin1 accumulate oxidized proteins, underscoring the importance of autophagy in non-dividing cells. Autophagy in quiescent fibroblasts is associated with increased expression of the ATG protein conjugation systems, but proceeds despite active signaling through the mTOR axis. Mass spectrometry-based identification of phosphorylated residues of the autophagy kinase ULK1 has uncovered that important cell cycle regulators, cyclin dependent kinases (CDKs), may phosphorylate ULK1 to provide a novel layer of regulation of the autophagy pathway. Quiescent fibroblasts decrease autophagy as they transition into proliferative, wound-healing fibroblasts in vitro and in mice. Extending our analysis to another cell type, we found that B lymphocyte quiescence is also associated with autophagy, although the classic pathway of mTOR inhibition characterizes autophagy in these cells. Finally, we show that contact-inhibited quiescent fibroblasts maintain low levels of free radicals, which are sustained by enzymatic detoxification. We found that selective mitophagy is not a significant contributor to handling ROS in quiescent fibroblasts. In summary, autophagy is activated in multiple quiescent models through cell type-specific signaling pathways. Quiescence-associated autophagy facilitates the clearance of damaged proteins, but does not appear to affect detoxification of ROS or mitochondrial degradation. In conclusion, we have identified autophagy as a functional mechanism utilized by reversibly cell cycle arrested cells for maintenance of protein quality control during non-division.