Direct lysosome-based autophagy of lipid droplets in hepatocytes Ryan J. Schulzea,b,1, Eugene W. Kruegera,b,1, Shaun G. Wellera,b, Katherine M. Johnsona,b, Carol A. Caseyc, Micah B. Schotta,b, and Mark A. McNivena,b,2 aDepartment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905; bDivision of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905; and cDepartment of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198 Edited by Tobias C. Walther, Harvard School of Public Health, Boston, MA, and accepted by Editorial Board Member Joseph L. Goldstein October 13, 2020 (received for review June 4, 2020) Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) process that appears to play an especially important role in the in the lysosome-directed process of autophagy. An organelle- degradation of hepatocellular LDs (15). A selective form of LD- selective form of this process (macrolipophagy) results in the engulf- centric autophagy known as lipophagy is thought to involve the ment of LDs within double-membrane delimited structures (auto- recognition of as-of-yet unidentified LD-specific receptors to phagosomes) before lysosomal fusion. Whether this is an promote the localized assembly and extension of a sequestering exclusive autophagic mechanism used by hepatocytes to catabolize phagophore around the perimeter of the LD (16, 17). How this LDs is unclear. It is also unknown whether lysosomes alone might be phagophore is targeted to (and extended around) the LD surface to sufficient to mediate LD turnover in the absence of an autophago- facilitate lipophagy remains unclear. Once fully enclosed, the somal intermediate. We performed live-cell microscopy of hepato- double-membrane lipoautophagosome undergoes fusion with the cytes to monitor the dynamic interactions between lysosomes and lysosome to form a degradative organelle known as an autolyso- LDs in real-time. We additionally used a fluorescent variant of the some. Lysosomal lipases deposited within the autolysosome are LD-specific protein (PLIN2) that exhibits altered fluorescence in re- then ultimately responsible for the acid hydrolysis of the LD-stored sponse to LD interactions with the lysosome. We find that mamma- neutral lipids and subsequent release of free fatty acids (18–20). lian lysosomes and LDs undergo interactions during which proteins The two pathways of lipolysis and lipophagy likely work in tandem and lipids can be transferred from LDs directly into lysosomes. Elec- as coordinated processes (21). Indeed, we recently reported that tron microscopy (EM) of primary hepatocytes or hepatocyte-derived lipolysis can act to rapidly reduce the size of large LDs to diameters CELL BIOLOGY cell lines supports the existence of these interactions. It reveals a more appropriate in size for engulfment by lipophagic vesicles (22). dramatic process whereby the lipid contents of the LD can be “ex- To better evaluate the role and consequences of these LD– truded” directly into the lysosomal lumen under nutrient-limited lysosomal interactions in the context of mammalian lipophagy, conditions. Significantly, these interactions are not affected by per- we used a combination of live-cell laser-scanning confocal and turbations to crucial components of the canonical macroautophagy electron microscopy (EM) to study these contacts further. We machinery and can occur in the absence of double-membrane lip- identified a subpopulation of these associations that appear to oautophagosomes. These findings implicate the existence of an promote the direct exchange of both protein and lipid from LDs autophagic mechanism used by mammalian cells for the direct trans- fer of LD components into the lysosome for breakdown. This pro- Significance cess further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying Lipid droplets (LDs) are specialized fat-storage organelles that lipid homeostasis in the liver. can be used as a fuel source by many types of cells when nu- trients are scarce. One mechanism used by hepatocytes (the lipid droplet | microautophagy | lipolysis | lysosome | hepatocyte functional cells of the liver) for the catabolism of these energy reserves is the lysosome-directed process of autophagy. Tra- hallmark of fatty liver disease and related metabolic disorders ditionally, autophagy necessitates the enclosure of cargo Ais the intracellular accumulation of triacylglycerols and other within a double-membrane autophagosome before delivery to neutral lipids within organelles called lipid droplets (LDs) (1–3). As the lysosome for degradation. Here, we use live-cell and elec- a central site of lipid metabolism, the liver usually can tolerate tron microscopy to demonstrate that stable contacts between significant fluctuations in the LD content of its parenchymal cells, LDs and lysosomes in hepatocytes can result in the transfer of the hepatocytes (4). Chronic dysregulation in levels of LDs is as- both proteins and lipids from LDs directly into the lysosome in sociated with liver disease and necessitates a better understanding the absence of an autophagosomal intermediate. These find- of the mechanics underlying hepatic LD metabolism (5). Delimited ings reveal a mechanism used for lipid homeostasis in the liver. by a phospholipid monolayer and decorated with a dynamically changing proteome, a primary function of the LD is to sequester Author contributions: R.J.S., E.W.K., S.G.W., M.B.S., and M.A.M. designed research; R.J.S., E.W.K., S.G.W., K.M.J., and M.B.S. performed research; C.A.C. contributed new reagents/ esterified fatty acids away from the aqueous cytoplasm as neutral analytic tools; R.J.S., E.W.K., S.G.W., K.M.J., C.A.C., M.B.S., and M.A.M. analyzed data; and lipids, thus averting the lipotoxicity associated with high cytoplasmic R.J.S., E.W.K., S.G.W., M.B.S., and M.A.M. wrote the paper. – concentrations of free fatty acids (6 9).Inadditiontoaprotective The authors declare no competing interest. function, LDs also provide a supply of lipids for cellular membrane This article is a PNAS Direct Submission. T.C.W. is a guest editor invited by the production and readily oxidizable substrates used to support mito- Editorial Board. chondrial adenosine triphosphate (ATP) generation (10, 11). While This open access article is distributed under Creative Commons Attribution-NonCommercial- complex, the mechanisms underlying the regulated breakdown of NoDerivatives License 4.0 (CC BY-NC-ND). hepatic LDs are primarily thought to require the involvement of two See online for related content such as Commentaries. central catabolic pathways: cytosolic lipolysis and autophagy (12). 1R.J.S. and E.W.K. contributed equally to this work. Hepatic lipolysis is generally regarded as a rapid process pro- 2To whom correspondence may be addressed. Email: [email protected]. moting the release of free fatty acids from parent triacylglycerols This article contains supporting information online at https://www.pnas.org/lookup/suppl/ stored within the LD by a cascade of cytoplasmic lipases (13, 14). doi:10.1073/pnas.2011442117/-/DCSupplemental. Autophagy, on the other hand, represents a lysosome-driven First published December 7, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2011442117 PNAS | December 22, 2020 | vol. 117 | no. 51 | 32443–32452 Downloaded by guest on September 29, 2021 that are actively “sampled” by the lysosome. Importantly, this variant of PLIN2 in AML12 hepatocytes that exhibits differen- process seems to result in the accumulation of LD-derived pro- tial fluorescence dependent upon the pH of its surrounding en- tein and lipids in the lysosome in the absence of canonical vironment. We have previously shown that this construct, like autophagy proteins. Together, these findings provide evidence wild-type PLIN2, localizes to the surface of LDs and leaves be- for a lysosome-centric, LD catabolic process that is independent hind small RFP+-only puncta upon interaction with acidic lyso- of traditional autophagy/lipophagy and plays a meaningful role somes (Fig. 1G and SI Appendix, Fig. S1F) (27). Long-term live- in hepatocellular lipid homeostasis. cell imaging showed instances of prolonged interactions between lysosomes and reporter-coated LDs that resulted in the eventual Results formation of RFP+-only puncta that then dissociated from the Stable Interactions between Lysosomes and LDs Support LD Protein source LD together with the lysosome, suggestive of a piecemeal Transfer in Hepatocytes. To study the dynamics of LD–lysosome removal of protein from the surface of the LD (Fig. 1H and interplay within the hepatocyte, we used AML12 mouse hepa- Movie S2). Stable expression of this mRFP1-EGFP-PLIN2 re- tocytes as an initial model imaging system. These cells maintain porter in AML12 hepatocytes revealed significant numbers of many characteristics of normal primary hepatocytes (23) and flat- RFP+-only puncta spread throughout the cytoplasm, indicating ten well on collagen-coated glass imaging dishes to support time- that a considerable quantity of the PLIN2 reporter is continually lapse imaging of individual organelles in live cells. Labeling of LDs being sampled by lysosomes (SI Appendix, Fig. S1G), even when and lysosomes with BODIPY-FL-C12 and LysoTracker Deep Red, cells are cultured in nutrient-rich conditions. respectively, allowed monitoring of dynamic interactions between these two compartments. As shown in Fig. 1A and Movie S1,dis- Persistent Interactions between Lysosomes and LDs Support Direct crete contacts between the two organelles were observed that were Lipid Transfer. Aside from proteins residing on the LD surface, often highly transient. Under basal cell culture conditions another cargo that could be exchanged as a result of direct (i.e., growth in full-serum medium, 37 °C, 5% CO2), the persistence contacts between the two organelles is lipid itself. Live-cell im- of thousands of individual interactions were measured and found aging of AML12 hepatocytes cultured under nutrient-replete to average 30.26 s (Fig. 1B, control), a timescale consistent with conditions was therefore performed to determine whether direct previously published data (24).