Autophagy facilitates organelle clearance during differentiation of human erythroblasts

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.

Keywords: :

• ATG4B
• ATG4D
• erythropoiesis
• electron microscopy
• autophagosome
• amphisome
• mitochondrion

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We acknowledge the support of the Medical Research Council in providing an Infrastructure Award to establish the School of Medical Sciences Cell Imaging Facility at Bristol University, and the Wolfson Foundation for recent funds to modernize and expand the facility. We thank Dr. Paul Verkade for invaluable advice on the identification and interpretation of membrane compartments by electron microscopy, and for training and advice on the technique of high-pressure freezing. The authors also wish to acknowledge the assistance of Dr. Andrew Herman for cell sorting, and the University of Bristol Faculty of Medical and Veterinary Sciences Flow Cytometry Facility. This work is funded by a project grant from the NHS Blood and Transplant (Ref: PG07/1).

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/autophagy/article/24172