title: Autophagy facilitates organelle clearance during differentiation of human erythroblasts: evidence for a role for ATG4 paralogs during autophagosome maturation.
abstract: Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B (C74A) ; ATG4D (C144A) ) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B (C74A) -expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, UK.
authors.forename: Virginie M S
authors.initials: VM
authors.lastname: Betin
authors.forename: Belinda K
authors.initials: BK
authors.lastname: Singleton
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Parsons
authors.forename: David J
authors.initials: DJ
authors.lastname: Anstee
authors.forename: Jon D
authors.initials: JD
authors.lastname: Lane
mesh_terms: D001343:Autophagy; D000071183:Autophagy-Related Proteins; D002451:Cell Compartmentation; D002454:Cell Differentiation; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D050505:Mutant Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation
chemical_list: D000071183:Autophagy-Related Proteins; D050505:Mutant Proteins; C474025:ATG4A protein, human; C507524:ATG4B protein, human; C543490:ATG4D protein, human; D003546:Cysteine Endopeptidases; D003545:Cysteine
keywords: ATG4B; ATG4D; amphisome; autophagosome; electron microscopy; erythropoiesis; mitochondrion