Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL-1b is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R-SNARE Sec22b to recruit cargo to the LC3-II + sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome-lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP-23 and SNAP-29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.
Mammalian autophagosomes mature into autolysosomes through SNARE-driven processes that include syntaxin 17 (Stx17). Kumar et al. show that Stx17 interacts with mammalian Atg8s and with the small guanosine triphosphatase IRGM and that both IRGM and mAtg8s help recruit Stx17 to autophagosomes.
Highlights d Syntaxin 17 functions during autophagy initiation and bulk cargo sequestration d TBK1 phosphorylates syntaxin 17 at Ser202 (Stx17 pS202) d Stx17 pS202 translocates from Golgi to pre-autophagosomal structure upon starvation d Stx17 pS202 controls formation of FIP200-ATG13 preautophagosomal structures
Macroautophagy/autophagy plays a role in unconventional secretion of leaderless cytosolic proteins. Whether and how secretory autophagy diverges from conventional degradative autophagy is unclear. We have shown that the prototypical secretory autophagy cargo IL1B/IL-1b (interleukin 1 b) is recognized by TRIM16, and that this first to be identified secretory autophagy receptor interacts with the R-SNARE SEC22B to jointly deliver cargo to the MAP1LC3B-II-positive sequestration membranes. Cargo secretion is unaffected by knockdowns of STX17, a SNARE catalyzing autophagosome-lysosome fusion as a prelude to cargo degradation. Instead, SEC22B in combination with plasma membrane syntaxins completes cargo secretion. Thus, secretory autophagy diverges from degradative autophagy by using specialized receptors and a dedicated SNARE machinery to bypass fusion with lysosomes. The principal morphological feature of autophagy is the formation of a double-membrane organelle called an autophagosome that encloses cytosolic cargo and typically delivers it to lysosomes for degradation. Unconventionally secreted cytosolic proteins are characterized by the absence of leader peptides; thus, they do not enter the lumen of the ER and do not follow the secretory pathway reserved for conventionally secreted proteins that typically go through the ER and the Golgi apparatus, and are secreted by exocytosis of post-Golgi vesicles. An archetypal example of unconventionally secreted proteins is the proinflammatory cytokine IL1B, which has been reported in 1990 as being secreted from mammalian cells despite the absence of a leader peptide. Recently, autophagy as a process has been implicated in the secretion of IL1B in mammalian cells and of the yeast protein Acb1, a homolog of Dictyostelium discoideum AcbA that stimulates encapsulation of prespore cells in the slime mold.Our previous work has shown that IL1B secretion is dependent on autophagy in primary murine bone marrow-derived macrophages. Others have confirmed utilization of the autophagy apparatus for IL1B secretion. How IL1B is recognized for delivery to autophagic organelles en route for secretion and how it is protected from degradation has hitherto remained unknown. Our present work has identified the first specific receptor for secretory autophagy and defined the SNARE apparatus that bypasses autophagosomal maturation but instead leads to secretion of the cargo at the plasma membrane.Diverse lysosome-damaging agents, such as silica, alum, monosodium urate (MSU), and Leu-Leu-O-Me (LLOMe), activate inflammasome, which in turn processes pro-IL1B into mature IL1B, induce autophagy and trigger IL1B secretion. We first established that autophagy factors, e.g. MAP1LC3B and ATG16L1, are required for the efficient secretion of IL1B upon LLOMe treatment. Next, we searched for an IL1B receptor for selective secretory autophagy. The TRIM family proteins (with over 80 members in humans) have been shown to mediate autophagy, act as selective autophagic cargo receptors, and act as organizer...
Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.
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