| 1. |
- Knævelsrud, Helene, et al.
(author)
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Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation
- 2013
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In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 202:2, s. 331-349
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Journal article (peer-reviewed)abstract
- The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
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| 2. |
- Knævelsrud, Helene, et al.
(author)
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The membrane-remodeling PX-BAR protein SNX18 is required for autophagy
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Other publication (other academic/artistic)abstract
- Autophagy is a catabolic pathway targeting cytoplasmic material for lysosomal degradation,thereby protecting cells from accumulation of toxic components and enabling cells to survivescarce nutrient supplies. Macroautophagy is characterized by the sequestration of cytoplasmicmaterial into double-membrane vesicles, but the membrane remodeling events required forformation of autophagic vesicles are still not completely understood. However, the class IIIphosphatidylinositol 3-kinase (PI3K)/Vps34 complex and phosphatidylinositol-3-phosphate(PI3P) are of core importance to induction of autophagy. Since PX domain containingproteins are known to bind PI3P and other phosphoinositides and mediate membraneremodeling and trafficking events, we performed an imaging-based siRNA screen targetingPX domain proteins using formation of GFP-LC3 positive autophagosomes as a read-out.The PX-BAR protein SNX18 was found to strongly inhibit autophagosome formation. In linewith this, overexpression of SNX18 increased LC3 lipidation and GFP-LC3 spot formationand we demonstrate that membrane binding of SNX18 is required for efficientautophagosome formation. Moreover, SNX18 colocalizes and interacts with the autophagyassociatedproteins LC3 and TBK1. Our study identified the PX-BAR protein SNX18 to beinvolved in membrane events required for autophagosome formation.
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| 3. |
- Migliano, Simona M., et al.
(author)
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Removal of hypersignaling endosomes by simaphagy
- 2024
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 20:4, s. 769-791
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Journal article (peer-reviewed)abstract
- Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination.
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| 4. |
- Nahse, Viola, et al.
(author)
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ATPase activity of DFCP1 controls selective autophagy
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- The endoplasmic reticulum protein DFCP1 is found on omegasomes implicated in autophagosome biogenesis, but its function has remained unknown. Here, Nahse et al. show that DFCP1 is an ATPase that mediates selective autophagy by promoting constriction of large omegasomes. Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.
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