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M. Moras et al.
monitored by MGG coloration. VDAC1 downregulation did not alter erythroid differentiation before 3 days post- transduction (D7) (Figure 1B). However, by day 10 of dif- ferentiation, shVDAC1 transduced cells exhibited an accelerated terminal differentiation as compared to con- trol cells, shown by a significantly higher percentage of orthochromatic erythroblasts as compared to earlier basophilic and polychromatic erythroblasts (Figure 1C). These data were confirmed by evaluating a4- integrin/Band3 profile by flow cytometry allowing the terminal stage of differentiation to be distinguished as previously described28 (Figure 1D). Consistent with the data above, shVDAC1-transduced progenitors showed no difference in Band3 expression (Figure 1E) but a signif- icantly decreased levels of a4 integrin, a marker which is lost during terminal differentiation, as compared to shSCR-transduced cells (Figure 1F). Furthermore, we observed a decreased proliferation rate in shVDAC1- transduced cells starting at day 10 (Figure 1G). Altogether, these data point to the importance of VDAC1 in regulat- ing terminal erythroid differentiation.
VDAC1 downregulation impairs erythroblast enucleation
The main morphological feature of terminal mammalian erythroid differentiation is the enucleation, leading to the production of a reticulocyte and a pyrenocyte from an orthochromatic erythroblast. Despite the accelerated dif- ferentiation observed in shVDAC1-transduced cells,
orthochromatic erythroblast percentage was increased whereas reticulocyte level was significantly decreased from day 14 until day 17 as evaluated by MGG coloration (Figure 2A and B) as well as Hoechst staining (Figure 2C).
Enucleation starts with the polarization of the nucleus towards the plasma membrane, allowing the separation of the nucleus from the yet-to-be formed reticulocyte by a mechanism similar to cytokinesis.30 In order to assess whether the decrease in enucleation in shVDAC1-trans- duced cells was due to an impairment in the polarization of the nucleus, we sorted orthochromatic erythroblasts at day 12 of differentiation and analyzed these cells by imaging flow cytometry 24 hours (h) later. We measured the distance between the center of the cell and the center of the nucleus (delta centroid XY from the IDEAS soft- ware). No significant differences in delta centroid XY were observed between shVDAC1- and shSCR-trans- duced cells, suggesting that VDAC1 downregulation does not block the polarization of the nucleus (Online Supplementary Figure S2).
In order to assess whether the block in enucleation affects cell viability, we evaluated the level of apoptosis as a function of annexin V staining of exposed phos- phatidylserine. While annexin V staining was not altered in shVDAC1-transduced cells as compared to shSCR- transduced cells at days 7 and 10 of differentiation, levels were significantly higher at days 14 and 17, strongly sug- gesting that an attenuated enucleation was associated with apoptosis (Figure 2D).
AB
CD
Figure 2. Enucleation is impaired in short hairpin VDAC1-transduced erythroid progenitors. (A) The progression of terminal erythroid progenitors to the reticulocyte stage of differentiation was evaluated following scramble shRNA (shSCR) and VDAC1 shRNA (shVDAC1) transduction. Representative May-Grünwald-Giemsa (MGG) images (left) and quantification of the different progenitor stages (right) at day 14 and (A) day 17 (B) are presented and means ± standard error (SE) are shown. Scale bar =20 mm. (C) The percentages of enucleated cells (glycophorin A postive [GPA+], Hoechst- cells) were quantified by flow cytometry at day 10, 14 and 17 of differentiation (n=4) and means ± SE are shown. (D) Apoptosis was evaluated by annexin V staining and the quantification of annexin V+ cells is presented at day 7, 10, 14 and 17 (n=3). *P<0.05, **P<0.01, ****P<0.0001. BasoE: basophilic erythroblasts; PolyE: polychromatic erythroblasts; OrthoE: orthochromatic erythrob- lasts; Retic: reticulocytes.
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