Removal of hypersignaling endosomes by simaphagy

Autophagy. 2024 Apr;20(4):769-791. doi: 10.1080/15548627.2023.2267958. Epub 2023 Oct 25.

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.Abbreviations: AKT: AKT serine/threonine kinase; APEX2: apurinic/apyrimidinic endodoexyribonuclease 2; ctrl: control; EEA1: early endosome antigen 1; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HGS/HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; IF: immunofluorescence; ILV: intralumenal vesicle; KO: knockout; LIR: LC3-interacting region; LLOMe: L-leucyl-L-leucine methyl ester (hydrochloride); MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NBR1: NBR1 autophagy cargo receptor; PAG10: Protein A-conjugated 10-nm gold; RB1CC1/FIP200: RB1 inducible coiled-coil 1; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TUB: Tubulin; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VCL: Vinculin; VPS37: VPS37 subunit of ESCRT-I; WB: western blot; WT: wild-type.

Keywords: Autophagy; ESCRT; endosome; receptor degradation; signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagosomes / metabolism
  • Autophagy* / physiology
  • Cell Movement
  • Endocytosis / physiology
  • Endosomal Sorting Complexes Required for Transport* / metabolism
  • Endosomes* / metabolism
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Sequestosome-1 Protein / metabolism
  • Signal Transduction*

Substances

  • Endosomal Sorting Complexes Required for Transport
  • NBR1 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Sequestosome-1 Protein
  • SQSTM1 protein, human

Grants and funding

HS was supported by grants from the Norwegian Cancer Society (grant no. 182698), the Research Council of Norway (grant no. 302994), the South-Eastern Norway Regional Health Authority (grant no. 2018081) and the European Research Council (advanced grant no. 788954). CR and EMW were supported by the Norwegian Cancer Society (grant numbers 198140 and 246670). Mass spectrometry-based proteomic analyses were performed by the Proteomics Core Facility, Department of Immunology, University of Oslo/Oslo University Hospital, which is supported by the Core Facilities program of the South-Eastern Norway Regional Health Authority. This core facility is also a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910). This work was partly supported by the Research Council of Norway through its Centers of Excellence funding scheme, project number 262652 (CanCell).