Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2020 Nov 23:8:592035.
doi: 10.3389/fcell.2020.592035. eCollection 2020.

Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics

Alvaro M Gonzalez-Ibanez  1   2 , Lina M Ruiz  3 , Erik Jensen  1 , Cesar A Echeverria  4 , Valentina Romero  5 , Linsey Stiles  6   7 , Orian S Shirihai  6   7 , Alvaro A Elorza  1   2
Affiliations

"VSports手机版" Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics

"V体育2025版" Alvaro M Gonzalez-Ibanez et al. Front Cell Dev Biol. .

Abstract

Erythropoiesis is the most robust cellular differentiation and proliferation system, with a production of ∼2 × 1011 cells per day. In this fine-tuned process, the hematopoietic stem cells (HSCs) generate erythroid progenitors, which proliferate and mature into erythrocytes. During erythropoiesis, mitochondria are reprogrammed to drive the differentiation process before finally being eliminated by mitophagy. In erythropoiesis, mitochondrial dynamics (MtDy) are expected to be a key regulatory point that has not been described previously. We described that a specific MtDy pattern occurs in human erythropoiesis from EPO-induced human CD34+ cells, characterized predominantly by mitochondrial fusion at early stages followed by fission at late stages. The fusion protein MFN1 and the fission protein FIS1 are shown to play a key role in the progression of erythropoiesis. Fragmentation of the mitochondrial web by the overexpression of FIS1 (gain of fission) resulted in both the inhibition of hemoglobin biosynthesis and the arrest of erythroid differentiation, keeping cells in immature differentiation stages VSports手机版. These cells showed specific mitochondrial features as compared with control cells, such as an increase in round and large mitochondrial morphology, low mitochondrial membrane potential, a drop in the expression of the respiratory complexes II and IV and increased ROS. Interestingly, treatment with the mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporin A, rescued mitochondrial morphology, hemoglobin biosynthesis and erythropoiesis. Studies presented in this work reveal MtDy as a hot spot in the control of erythroid differentiation, which might signal downstream for metabolic reprogramming through regulation of the mPTP. .

Keywords: cell differentiation; erythropoiesis; fission and fusion; mitochondria; permeability transition pore; stem cell V体育安卓版. .

PubMed Disclaimer

"V体育官网入口" Figures

FIGURE 1
FIGURE 1
Mitochondrial features throughout erythroid progression. Human hematopoietic CD34+ stem cells isolated from umbilical cord were cultured and EPO-induced to erythropoiesis for 3, 5, 8, 10, 13, and 16 days and pooled together in just one pre-sort sample. (A) Cell separation of different erythroid populations. Pre-sort samples were labeled with CD71 and GPA surface markers to follow up erythroid progression. Five erythroid populations are distinguished: R1 progenitors, R2 proerythroblast, R3 basophilic erythroblast (these cells start making hemoglobin), R4 polychromatophilic erythroblast and R5 orthochromatophilic erythroblast and reticulocytes. After labeling, samples were sorted to obtain four highly pure cell populations as seen as in the post-sort panel for R1, R2, R3, and R4 + R5. (B) Mitochondrial biomass and morphology analysis. Each sorted cell population was stained with DAPI (nucleus, blue) and MitoTracker Red FM (mitochondria, red), and immunolabeled with anti CD71 antibody, green. Confocal microcopy was done to quantify mitochondrial mass and circularity, a form factor where 1 is a perfect circle and 0, an elongated or branched mitochondrion. From R1 to R4 + R5, mitochondrial biomass decreases, and circularity goes from round mitochondria in R1 to elongated in R2 and then to come back to intermediate values in R3 and R4 + R5. Mitochondrial biomass was calculated as the percentage of total cell volume. Statistical analysis was ANOVA followed by Bonferroni post hoc test. Mitochondrial Circularity was plotted as a frequency distribution and statistically analyzed through contingency tables and a Chi-square test (p < 0.05). (C) Mitochondrial membrane potential analysis. Sorted cell populations were stained with TMRE dye in a non-quenching mode and visualized under confocal microscope. Membrane potential goes from average in R1 to high in R2. Then, it starts to decrease toward R4 + R5 population. (D) Mitochondrial Dynamics gene expression analysis. Total RNA, cDNA synthesis and RT-PCR were performed in each sorted population for the mitochondrial fission genes fis1, mff, mief1, and mief2; and for the mitochondrial fusion genes mfn1, mfn2, and opa1. 18s was used as a loading control. In general, fusion is predominant at early stages and the fission, at later stages of erythroid differentiation. All plotted values in panels (C,D) are ±SEMs of n = 3. Statistical analysis was ANOVA followed by Bonferroni post hoc test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).
FIGURE 2
FIGURE 2
Functional characterization of FIS1 in erythropoiesis. (A) Western blot to detect the knock-down or over-expression of FIS1 protein. Quantification was performed by band densitometry. Values are ±SEM, n = 3. (B) Visual inspection of cellular pellet. FIS1 OX cells do not differentiate under EPO-induced erythroid differentiation. Cell cultures at D16 were spun down and the pellet’s color, visualized. Red pellets were observed in all conditions, but not in FIS1 OX cells whose pellet was white. (C) Quantification of hemoglobin carrying cells. Cells from D10, D13, and D16 of differentiation were staining with benzidine to detect hemoglobin. Blue cells (benzidine+) were counted. FIS1 OX cells do not make hemoglobin. Plotted values are ±SEM, n = 3. (D) Analysis of erythroid progression. FIS OX cells are mainly arrested at the level of progenitors and proerythroblasts. This was seen by flow cytometry analysis of control and FIS OX cells labeled with the surface markers anti-CD71-APC and anti-GPA-PE at D5, D8, D10, D12, and D16 of EPO-induced differentiation (n = 3). Statistical analysis, ANOVA followed by Bonferroni post hoc test (∗ p < 0.05, ∗∗ p < 0.01, **** p < 0.001).
FIGURE 3
FIGURE 3
Effects of FIS1 OX on mitochondrial morphology. (A) Mitochondria visualization by confocal microscopy. Immunofluorescence microscopy at D5, D10, and D16 of erythroid differentiation for pWPI control and FIS1 OX cells. Anti-VDAC1 antibody labeled mitochondria (red) and the DAPI dye, the nucleus (blue). (B) Mitochondrial morphometric analysis in term of Area at D5, D10, and D16 of differentiation for control pWPI and FIS1 OX cells was performed using the Z-slides from GFP+ cells. Each dot represents a mitochondrial unit in terms of Area (Y axes) and Circularity (X axes). Also, a frequency histogram of mitochondrial circularity and area were added. Linear regression analysis was performed to compare slopes, which were significantly different (p < 0.05). R2 and MSE were also calculated (C) similar to panel (B). Mitochondrial morphometric analysis in term of Perimeter. Each dot represents a mitochondrial unit in terms of Perimeter (Y axes) and circularity (X axes).
FIGURE 4
FIGURE 4
Effects of FIS1 OX on mitochondrial bioenergetics. (A) OXPHOS protein expression levels. Total protein lysate from FIS1 OX and pWPI control cells at D10, D13, and D16 of erythroid differentiation were assayed for immunoblot to detect the OXPHOS protein expression level. Band densitometry analysis was performed for Complex II and Complex IV. The plotted values are ±SEM. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (* p < 0.05, **p < 0.01). (B) Measurement of mitochondrial membrane potential. FIS1 OX and pWPI control cells at D10, D13, and D16 erythroid differentiation were stained with 10 nM TMRE in non-quenching mode and analyzed by flow cytometry. 10 uM FCCP was used as negative control. TMRE mean intensity was quantified. The values plotted are ±SEMs. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (* p < 0.05, *** p < 0.001). (C) Measurement of mitochondrial superoxide radical. FIS1 OX and pWPI control cells at D10, D13, and D16 erythroid differentiation were stained with 5 μM MitoSOX and analyzed by flow cytometry. MitoSOX mean intensity was quantified and the ratio MitoSOX/TMRE was calculated to obtain a normalized value of the amount of ROS between the cell groups, because MitoSOX uptake is dependent on mitochondrial membrane potential. The values plotted are ±SEMs. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01).
FIGURE 5
FIGURE 5
Assessment of apoptosis in FIS1 OX cells. Total protein lysates from FIS1 OX and pWPI control cells at D8, D10, D13, and D16 of erythroid differentiation were examined by Western blot. (A) Protein levels of Procaspase 9 and cleaved active forms P37 and P35. This is for intrinsic apoptosis. HSP70 were used as housekeeping control. Band densitometry for p37, as a percentage of total caspase 9, was plotted. Values are ±SEM. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test. (B) Protein levels of Procaspase 8 and cleaved active forms P42/P43. This is for extrinsic apoptosis. HSP70 were used as housekeeping control. (C) Protein levels of Procaspase 3 and cleaved active form P17. This is a general apoptotic effector. HSP70 were used as housekeeping control. Band densitometry for p17, as a percentage of total caspase 3, was plotted. Values are ±SEM. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (*** p < 0.001).
FIGURE 6
FIGURE 6
Effect of MFN1 KD on mitochondrial morphology and erythroid differentiation. (A) Western blot to detect the knock-down of MFN1. Total protein lysates from pLVCTH control and MFN1 KD cells at D3 of erythroid differentiation were examined by Western blot to check MFN1 expression. Quantification was performed by band densitometry. Plotted values are ±SEM. n = 3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (* p < 0.05). (B) Mitochondria visualization by confocal microscopy. Mitochondria were stained with 10 nM MitoTracker Red CMXRos and visualized at D5, D8, D10, D13, and D16 of erythroid differentiation for pLVCTH control and MFN1 KD cells. The nucleus was stained with DAPI (blue). (C) Mitochondrial morphometric analysis in term of Area at D5, D10, and D16 of erythroid differentiation for pLVCTH control and MFN1 KD cells. It was performed using the Z-slides from GFP+ cells. Each dot represents a mitochondrial unit in terms of area (Y axes) and circularity (X axes). Also, a frequency histogram of mitochondrial circularity and area were added. Linear regression analysis was performed to compare slopes, which were significantly different (p < 0.05). R2 and MSE were also calculated. (D) Similar to panel (C). Mitochondrial morphometric analysis in term of Perimeter. Each dot represents a mitochondrial unit in terms of Perimeter (Y axes) and circularity (X axes). (E) Erythroid progression in MFN1 KD cells. Flow cytometry analysis of surface markers anti-CD71-APC and anti-GPA-PE in pLVCTH control and MFN1 KD cells at D5, D10, and D16 days of EPO-induced differentiation. All analysis (n = 3) correspond GFP+ cells. MFN1 KD cells have a bigger R2 and smaller R3 population at D13 and D16 than control cells, meaning a delay in erythroid progression as compared with control cells.
FIGURE 7
FIGURE 7
Assessment of the mPTP in FIS1 OX cells. (A) Ultrastructure visualization of mitochondria. Transmission electron microscopy (TEM) was performed for pWPI control and FIS1 OX cells at D13 and D16 of erythroid differentiation. FIS1 OX displayed heterogeneous mitochondrial morphologies. Some mitochondria look like immature mitochondria with short and few cristae. It was also found swollen-like mitochondria as compared with control cells. Magnification 6000×. (B) Ultrastructure visualization of mitochondria in CsA-treated cells. TEM analysis showed normal mitochondrial structure and electron density in CsA-treated FIS1 OX cells as compared with CsA-treated pWPI control cells. (C) Mitochondrial circularity analysis. It was calculated from TEM images of pWPI control and FIS1 OX cells treated or not treated with CsA at D13 of erythroid differentiation. Frequency distributions showed that the treatment with CsA fully rescued the mitochondrial morphology. Data was analyzed using contingency tables and a Chi-square test (p < 0.05). (D) Erythroid progression analysis of CsA-treated FIS1 OX cells. Cells were treated with CsA on D9 and collected at D10, D13, and D16. Right after collection, cells were immunolabeled with anti-CD71-APC and anti-GPA-PE and analyzed by flow cytometry. FIS1 OX cells with no CsA treatment are also shown. All analysis (n = 3) correspond to GFP+ cells. In addition, cells were spun down to observe the cell pellet’s color. Treatment with CsA rescued erythroid differentiation and hemoglobin biosynthesis in FIS1 OX cells.
See this image and copyright information in PMC

References

    1. Ahlqvist K. J., Suomalainen A., Hämäläinen R. H. (2015). Stem cells, mitochondria and aging. Biochim. Biophys. Acta Bioenerget. 1847 1380–1386. 10.1016/j.bbabio.2015.05.014 - DOI - PubMed
    1. Ansó E., Weinberg S. E., Diebold L. P., Thompson B. J., Malinge S., Schumacker P. T., et al. (2017). The mitochondrial respiratory chain is essential for haematopoietic stem cell function. Nat. Cell Biol. 19 614–625. 10.1038/ncb3529 - DOI - PMC - PubMed
    1. Arciuch V. G. A., Elguero M. E., Poderoso J. J., Carreras M. C. (2012). Mitochondrial regulation of cell cycle and proliferation. Antioxid. Redox Signal. 16 1150–1180. 10.1089/ars.2011.4085 - DOI - PMC - PubMed
    1. Bernardi P., Di Lisa F. (2015). The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J. Mol. Cell Cardiol. 78 100–106. 10.1016/j.yjmcc.2014.09.023 - DOI - PMC - PubMed
    1. Betin V. M. S., Singleton B. K., Parsons S. F., Anstee D. J., Lane J. D. (2013). Autophagy facilitates organelle clearance during differentiation of human erythroblasts: evidence for a role for ATG4 paralogs during autophagosome maturation. Autophagy 9 881–893. 10.4161/auto.24172 - DOI - PMC - PubMed