RETRACTED ARTICLE: Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins

Abstract

LC3-associated phagocytosis (LAP) is a process wherein elements of canonical autophagy conjugate LC3 to the membranes of phagosomes, facilitating maturation upon fusion to lysosomes. Here, we characterize the molecular requirements for LAP, and identify a protein, Rubicon, required for LAP but not canonical autophagy. During LAP, Rubicon is recruited to the LAP-engaged phagosome (“LAPosome”) and is required for the activity of a Class III PI3K complex containing Beclin-1, UVRAG, and VPS34, but lacking the canonical autophagy components ATG14 and Ambra1. This allows for the sustained localization of PI(3)P, which is critical for the recruitment of downstream autophagic proteins and the stabilization of the NOX2 complex to produce ROS. Both PI(3)P and ROS, as well as ATG7, ATG3, ATG5, ATG12, and ATG16L, are required for the conjugation of LC3 to the LAPosome and subsequent association with LAMP1+ lysosomes. LAP is also induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in the response to Aspergillus fumigatus infection.

Autophagy proteins are not universally required for phagosome maturation

“…4,6 LC3 conjugation to a phagosome is observed in hematopoietic cell types, including macrophages, dendritic cells, and neutrophils. [4][5][6][7][8][9][10][11][12] It has also been observed in retinal pigment epithelial cells, 13,14 which are macrophage-like and expresses some hematopoietic markers. 15 The role of LAP in nonhematopoietic cell types is not yet clear.…”

“…4 These receptors initiate reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase, an event that is required for LAP. 5,6 In fact, intraphagosomal ROS in the absence of other signals is sufficient to recruit LC3 to phagosomes. 6 Other receptors implicated in LAP, such as FCGR2A/FcgR2A and CLEC7A/dectin-1, also result in CYBB/NOX2-dependent ROS production.…”

“…5,6 In fact, intraphagosomal ROS in the absence of other signals is sufficient to recruit LC3 to phagosomes. 6 Other receptors implicated in LAP, such as FCGR2A/FcgR2A and CLEC7A/dectin-1, also result in CYBB/NOX2-dependent ROS production. 5,7 LAP is triggered in response to particles that stimulate TLRs, FCGR2A/FcgR2A, CLEC7A/dectin-1, and other cargo such as apoptotic and entotic cells.…”

“…5,7 LAP is triggered in response to particles that stimulate TLRs, FCGR2A/FcgR2A, CLEC7A/dectin-1, and other cargo such as apoptotic and entotic cells. [4][5][6][7][8][9][10] In addition to ROS, LAP requires a subset of autophagy components, such as ATG5, ATG7, ATG12, ATG16L1, BECN1/Beclin 1, and PIK3C3/VPS34, whereas others, such as ULK1 (unc51-like kinase 1), ATG13, and RB1CC1/FIP200 are dispensable. 4,6 LC3 conjugation to a phagosome is observed in hematopoietic cell types, including macrophages, dendritic cells, and neutrophils.…”

“…[4][5][6][7][8][9][10] In addition to ROS, LAP requires a subset of autophagy components, such as ATG5, ATG7, ATG12, ATG16L1, BECN1/Beclin 1, and PIK3C3/VPS34, whereas others, such as ULK1 (unc51-like kinase 1), ATG13, and RB1CC1/FIP200 are dispensable. 4,6 LC3 conjugation to a phagosome is observed in hematopoietic cell types, including macrophages, dendritic cells, and neutrophils. [4][5][6][7][8][9][10][11][12] It has also been observed in retinal pigment epithelial cells, 13,14 which are macrophage-like and expresses some hematopoietic markers.…”