An autophagy-independent role for ATG16L1: promoting lysosome-mediated plasma membrane repair

ABSTRACT

There is growing evidence in the literature for unconventional roles of autophagy-related (ATG) proteins, outside of their function in canonical autophagy. Here we discuss our recent study that revealed a novel ATG16L1-dependent pathway that promotes plasma membrane repair upon bacterial pore-forming toxin damage. Disruption of the ATG16L1-dependent pathway leads to an accumulation of cholesterol in lysosomes, which affects lysosomal exocytosis required for efficient membrane repair. Our study provides insights into the role of ATG16L1 in cholesterol homeostasis and plasma membrane integrity.

Punctum

Macroautophagy (hereafter autophagy) is a bulk degradative process that is crucial for cellular homeostasis. During nutrient deprivation, pathogen invasion, or organelle damage, autophagy sequesters and delivers cargoes to the lysosome for degradation and recycling. Autophagy is regulated by more than 30 ATG proteins that are highly conserved across eukaryotes. Increasingly in the past decade, ATG proteins have been found to regulate pathways other than autophagy such as phagocytosis, apoptosis and protein secretion. Recently we discovered a surprising new function of the core autophagy component ATG16L1 aside from its canonical role in LC3 lipidation and autophagosome formation. We demonstrated that an ATG16L1-dependent pathway is capable of promoting membrane repair and restricting bacteria spread [1].

To assess ATG proteins in the context of plasma membrane repair, we performed a repair assay using the pore-forming toxin pneumolysin and propidium iodide to measure damaged membrane. We found that cells deficient in ATG16L1, as well as its binding partners ATG5 and ATG12, are defective in membrane repair. Other key regulators of autophagy such as ATG3, ATG9, ATG14 and RUBCN/RUBICON do not affect membrane repair, confirming a bona fide pathway separate from canonical autophagy. In addition to the ATG16L1 complex, we found that ATG16L1 cells harboring the T300A mutation alone, which is associated with inflammatory bowel disease (IBD) and Crohn disease, also present with decreased membrane repair when challenged with pneumolysin. The defect in membrane repair appears to correct itself given a long enough recovery time, which suggests other repair pathways may eventually compensate for the lack of a repair pathway in ATG16L1-deficient cells.

In additional to toxin-induced membrane repair, we also looked at the effects of ATG16L1 on bacteria infection. During Listeria monocytogenes infection, membrane repair plays a key role in preventing efficient cell-to-cell spread by reducing phosphatidylserine exposure that occurs after listeriolysin O toxin damages the plasma membrane. We speculated that ATG16L1 may play a similar function by limiting membrane repair during L. monocytogenes infection. Indeed, cells deficient in ATG16L1, as well as ATG5 or ATG12, and ATG16L1 cells with the T300A mutation, display a large increase in bacteria cell-to-cell spread, as well as an increase in membrane damage.

Diverse cellular repair mechanisms are employed to maintain plasma membrane integrity in a Ca²⁺-dependent manner. Rapid lysosomal exocytosis upon calcium influx promotes plasma membrane repair likely by providing membrane and releasing tension. The exocytosis of lysosomal enzymes such as acid sphingomyelinases is thought to promote endocytosis of the bacteria toxin-induced pore, and subsequent degradation within endolysosomes. Alternatively, pores can be released in the extracellular medium within microvesicles/ectosomes in an endosomal sorting complex required for transport (ESCRT)-dependent manner. Additionally, influx of extracellular calcium can induce blebbing, which consists in the detachment of the plasma membrane from the actin cortex driven by actomyosin forces, as well as triggering the recruitment of annexins to the neck of the bleb for sealing, thereby creating a Ca²⁺-enriched microenvironment to favor plasma membrane repair.

To gain further insight into the role of ATG16L1 in plasma membrane repair, we assessed the impact of ATG16L1 deficiency on known repair pathways. Both annexins and ESCRT components appear to be properly recruited to the damaged plasma membrane upon toxin damage, indicating that ATG16L1 likely acts independently of those pathways. Conversely, by staining exofacial LAMP1 we showed that ATG16L1 deficiency impairs Ca²⁺-dependent lysosomal exocytosis at the plasma membrane damage sites (marked by exofacial phosphatidylserine). We further showed that ATG16L1 deficiency also affects blebbing responses, causing the formation of more blebs that are smaller but cannot retract. Although it remains to be tested, it is plausible that the defect in plasma membrane blebbing is a consequence of impaired lysosomal exocytosis.

Cholesterol regulates intracellular trafficking of late endosomes and lysosomes, notably by affecting their RABs and SNAREs composition and their interaction with molecular motors. We hypothesized that ATG16L1 deficiency might disturb cholesterol homeostasis leading to lysosomal exocytosis dysfunction. Using biochemical and immunofluorescence assays, we showed that cholesterol accumulates particularly within LAMP1-positive compartments in cells lacking ATG16L1, ATG5 and ATG12, but not ATG3. Using a conditional atg16l1 knockout mouse model, we also observed cholesterol accumulation in intestinal epithelial cells in vivo.

In order to link cholesterol accumulation to dysfunctional lysosome exocytosis and plasma membrane repair, we used chemical drugs that either induce or block intracellular cholesterol accumulation. Importantly, chemically increasing cholesterol in control cells basically recapitulates the ATG16L1-deficient phenotypes, namely impaired lysosomal exocytosis, defect in plasma membrane repair upon pore-forming toxin damage, and increased L. monocytogenes cell-to-cell spread. Conversely, chemically decreasing cholesterol accumulation in ATG16L1-deficient cells was sufficient to rescue those phenotypes.

ATG proteins playing a non-canonical role is a rapidly emerging field. Here we showed a novel ATG16L1-dependent pathway, independent of canonical autophagy, that regulates cholesterol homeostasis, thereby contributing to efficient lysosomal exocytosis to promote plasma membrane repair. In addition to microbial pathogen clearance by autophagy, we further showed that ATG16L1 limits L. monocytogenes infection by promoting membrane repair during cell-to-cell spread. Bacteria often use effectors and toxins to avoid being targeted for autolysosomal degradation, and escape the autophagosome. It is intriguing how the cell has evolved another checkpoint involving ATG16L1 to further prevent bacterial spread and toxin-induced membrane damage by regulating lysosomal cholesterol. While ATG16L1 does not affect membrane cholesterol and toxin binding, we speculate that ATG16L1 may be controlling cholesterol trafficking to and from the lysosomes, as well as cholesterol efflux through exosome release. Other ATG proteins have been previously suggested to play a role in cholesterol homeostasis, such as in macrophage foam cells by regulating lipid metabolism and delivery to the lysosomes by ATG5. Our data support the view that an ATG16L1-dependent pathway affects lysosomal accumulation of cholesterol in a similar manner, independently of autophagy. It is still unclear how ATG16L1, and its binding partners, serves as a checkpoint in regulating cholesterol in the endocytic pathway, a loss of which leads to lysosomal dysfunction. Future studies on this topic will prove to be invaluable in the treatment of bacterial infections and inflammatory disorders.

Funding Statement

This work was supported by the Canadian Institutes of Health Research (MOP97756, PJT148668 and FDN154329).

Disclosure statement

No potential conflict of interest was reported by the authors.