Efferocytosis meets endocytosis

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Abstract Apoptosis happens continuously along with the active clearance of apoptotic cells (ACs) by efferocytes, termed “efferocytosis”, to maintain tissue homeostasis. Our recent studies revealed a novel role of efferocytosis in accelerating tissue repair as it promotes inflammation resolution by inducing the biosynthesis of specialized pro- resolving mediators (SPMs) that stop inflammatory responses. Therefore, understanding how efferocytosis is successfully carried out is of paramount importance. Much has been learned about the mechanisms of AC recognition and uptake, but how efferocytes degrade ACs and process the metabolic cargo, e.g., cholesterol released from AC digestion, is incompletely understood. Moreover, although efferocytosis and endocytosis share common features, such as involving cytoskeleton rearrangement and intracellular transport of vesicular membrane-bound cargoes, whether efferocytes hijack the endocytic machinery to process AC- derived cargo remains uncertain. In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes) and LC3-II lipidation, key features in LC3-associated phagocytosis (LAP)-mediated corpse degradation. As our recent study showed that the activation of MerTK, the efferocytosis receptor, was required for RvD1 biosynthesis, these results indicate a novel role of MerTK-RvD1 signaling in LAP-mediated AC degradation. To study whether the key endocytic regulators—the C-terminal Eps15 Homology Domain (EHD) proteins comprising EHD1, EHD2, EHD3, and EHD4—are involved in efferocytosis-related events, we analyzed a single-cell RNA-sequencing (scRNA-seq) dataset from atherosclerotic lesions where a lot of cells undergo apoptosis and found that EHD proteins had heterogeneous expression with high expression of EHD1 and EHD4 in macrophages, the professional efferocytes. We further found that EHD1 enhanced the cell surface levels of the cholesterol efflux transport protein ABCA1 in macrophages during efferocytosis, which indicates that EHD1- mediated endocytic trafficking of ABCA1 may play a role in removing the excess free cholesterol released from digested ACs. Here, we propose to combine approaches in cell biology, biochemistry, mouse genetics, and functional genomics to determine the function and mechanisms of MerTK-RvD1 signaling in LAP (Project 1) and EHD proteins in efferocytosis-related events including maintaining cellular cholesterol homeostasis (Project 2) and controlling endocytic trafficking of MerTK (Project 3). All the three projects require confocal microscopy to image AC- containing compartments including efferosomes, LAPosomes, endosomes and lysosomes in both fixed and live cells. Given our intensive usage needs for confocal microscopy, this Administrative Supplement requests the acquisition of the STELLARIS 5 confocal microscope from Leica microsystems, which will allow us to access to complementary layers of information for dynamic, structural, and mechanistic insights into vesicle trafficking. In summary, the STELLARIS 5 will significantly accelerate our research described in the funded parent R35 award.