Essential role for Nix in autophagic maturation of erythroid cells - PubMed (original) (raw)

Essential role for Nix in autophagic maturation of erythroid cells

Hector Sandoval et al. Nature. 2008.

Abstract

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), in the regulation of erythroid maturation through mitochondrial autophagy. Nix(-/-) mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix(-/-) mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(m) is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.

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Figures

Figure 1. Reticulocytosis and retention of mitochondria in _Nix_−/− RBCs

a, Polychromasia (Wright–Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or _Nix_−/− mice (scale bar, 20 µm). b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P <0.05; **P <0.01; ***P<0.001. d, e, Analyses of RBCs as in b by immunocytochemistry (d; scale bar, 10 µm) or transmission electron microscopy (e; scale bar, 0.5 µm).

Figure 2. Decreased survival of RBCs in _Nix_−/− mice

a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3). b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length. c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in _Nix_−/− RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3). d, Mitotracker versus annexin V staining of RBCs after in vitro culture. e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in _Nix_−/− RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol. f, Haematoxylin and eosin (H&E) staining of spleen sections of 9-week-old wild-type and _Nix_−/− mice. Arrows denote iron deposits within macrophage cytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm. g, Real-time RT–PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P<0.05; **P<0.01; ***P<0.001.

Figure 3. Defective clearance of mitochondria by autophagy in _Nix_−/− reticulocytes

a, Ter119+ CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining. b, CD71+ Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm. c, Cells as in b were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; _Nix_−/−, 0.44 ± 0.036 (n = 35, P = 0.0006). d, Cells sorted as in b were analysed by electron microscopy. Scale bar, 0.5 µm.

Figure 4. Removal of mitochondria by autophagy in FCCP-treated or ABT-737-treated _Nix_−/− reticulocytes

a, Ter119+ CD71+ reticulocytes were sorted from the peripheral blood of wild-type and _Nix_−/− mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119. b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.

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Essential role for Nix in autophagic maturation of erythroid cells - PubMed (original) (raw)