Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

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. RAW264.7 cells were plated on 6-well plates in complete media and harvested in cold PBS the next day. Total RNA was extracted using the RNeasy Kit (Qiagen), quantified by RiboGreen (ThermoFisher), and quality-checked on a 4200 TapeStation HS RNA ScreenTape (Agilent). Libraries were prepared from 500 ng total RNA with the TruSeq Stranded mRNA Kit (Illumina), with insert sizes verified on TapeStation D1000 and concentrations measured by PicoGreen (ThermoFisher). Paired-end 100 bp sequencing was performed on a NovaSeq 6000 (Illumina) at the St. Jude Hartwell Center.