Project description:LC3-associated phagocytosis (LAP) represents a non-canonical function of autophagy proteins in which ATG8 family proteins (LC3 and GABARAP proteins) are lipidated onto single-membrane phagosomes as particles are engulfed by phagocytic cells. LAP plays roles in innate immunity, inflammation and anti-cancer responses and is initiated upon phagocytosis of particles that stimulate Toll-like receptors (TLR), Fc-receptors, and upon engulfment of dying cells. However, how this molecular process is initiated remains elusive. Here we report that receptors that engage LAP enrich phosphatidylserine (PS) in the phagosome membrane via membrane-proximal domains that are necessary and sufficient for LAP to proceed. Subsequently, PS recruits the Rubicon-containing PI3-kinase complex to initiate the enzymatic cascade leading to LAP. Manipulation of plasma membrane PS content, PS-binding by Rubicon, or the PS-clustering domains of receptors prevents LAP and phagosome maturation. Therefore, the initiation of LAP represents a novel mechanism of PS-mediated signal transduction upon ligation of surface receptors.
Project description:Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an anti-inflammatory adipokine, and we observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-/- AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing and decreased activation of LC3-associated phagocytosis (LAP). Furthermore, AM treatment with the AdipoR agonist AdipoRon partially rescued deficient killing in APN-/- AMs that was dependent on both receptors. Our study identifies a novel role for APN in LC3-mediated killing of A.fumigatus.
Project description:FAM134B, also known as RETREG1 or JK-1, was first identified in esophageal cancer and later identified as an ER autophagy receptor. The protein contains a Reticulon-homology domain(RHD) and an LC3 interaction region (LIR), where RHD is responsible for sensing and inducing ER membrane curvature, while LIR binds to LC3 on the phagocytic membrane, and the ER membrane and its contents are subsequently encapsulated into autophagosomes and transported to lysosomes for degradation. Now FAM134B, as one of the receptors involved in ER autophagy, has received more and more attention.We knocked down FAM134B in HK-2cells and researched which genes and pathways were been influenced.
Project description:Organelle fragmentation is crucial for the onset of autophagic programs that control lysosomal clearance of portions of organelles to be removed from cells. It is driven by membrane-bound organello-phagy receptors that display cytoplasmic intrinsically disordered modules (IDRs) containing short LC3-interacting regions (LIRs). Studies on ER-phagy receptors of the FAM134 family revealed the importance of transcriptional induction, and receptors phosphorylation, ubiquitylation and clustering for execution of the ER-phagy programs. In this model, ER fragmentation is promoted by the membrane-remodeling function of FAM134 reticulon homology domains (RHDs)18,19. However, RHDs are not conserved in ER-phagy receptors, nor in receptors for autophagy of other organelles that also require fragmentation such as the mitochondria. Thus, membrane remodeling by RHDs is unlikely to be a conserved mechanism to regulate organelle turnover. Here, we show that the membrane-tethering modules of ER-phagy receptors (RHDs for FAM134B, single/multi spanning transmembrane domains for TEX264 and SEC62) determine the sub-compartmental distribution of the receptors but are dispensable for ER fragmentation, regardless of their propensity to remodel the ER membrane. Our experiments reveal that the information encoding for ER fragmentation is contained in the cytoplasmic IDR modules of the ER-phagy receptors, which also control delivery of the ER fragments within degradative acidic compartments upon engagements of lipidated LC3/GABARAP proteins via their LIRs. Notably, the transplantation of ER-phagy receptors IDRs at the mitochondrial membrane induces DRP1-driven mitochondrial fragmentation and mitophagy, and the transplantation of mitophagy receptors IDRs at the ER membrane induces ER fragmentation and ER-phagy. Our work reveals the functional conservation of membrane-exposed IDRs in promoting organelle fragmentation and offers a method to control integrity and activity of intracellular organelles by surface activation of IDR modules with net negative charges.
Project description:Mycobacterium tuberculosis impairs host lysosomal trafficking pathways. Xenophagy and LC3-associated phagocytosis (LAP) are lysosomal trafficking pathways that normally clear intracellular microbes. The xenophagy and LAP pathways depend upon ATG5 and ATG7, which result in the lipidation of LC3-I to LC3-II. How Mtb undermines these innate immune defenses of the host in not well understood. We used a transposon sequencing (Tn-seq) screen to identify bacterial factors that are required for the bacilli to resist ATG5 and ATG7-dependent processes. We found that that mutants defective in production of PDIM are attenuated in murine macrophages, and they are able to survive in macrophages lacking ATG5 or ATG7.
Project description:Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP).
