Fig 2: Pleckstrin-2 activation and cofilin binding prevents mitochondrial translocation of cofilin in ß-thalassemic erythroblasts.a Western blot analysis of cofilin protein concentration in bone marrow erythroblasts from WT, ß-thalassemic, and apoTf ß-thalassemic mice (n = 3 mice/group; MW = cofilin 19 kDa, actin 42 kDa). b Statistical analysis of (a) demonstrates no differences in cofilin protein concentration between erythroblasts from WT, ß-thalassemic, and apoTf ß-thalassemic erythroblasts. c Confocal immunofluorescence microscopy of cofilin expression in sorted (2–3 mice pooled per data point, n = 3–4 data points per category) Pro-Es from WT, ß-thalassemic, and apoTf-treated ß-thalassemic mice. d Statistical analysis of cofilin MFI in (c) demonstrates no differences between Pro-Es from WT, ß-thalassemic, and apoTf-treated ß-thalassemic mice. e Confocal immunofluorescence microscopy of plek2 and cofilin co-localization in sorted (2–3 mice pooled per data point, n = 3–4 data points per category) Pro-E from WT, ß-thalassemic, and apoTf-treated ß-thalassemic mice. The confocal images are representative optical sections of at least 50 cells in each condition in 2 different experiments. Scale bar 5 µm. f MFI of plek2:cofilin co-localizations in Pro-Es from WT, ß-thalassemic, and apoTf-treated ß-thalassemic mice demonstrates higher co-localization predominantly in ß-thalassemic relative to WT, normalized after apoTf treatment. All data are reported as mean ± s.e.m. and p < 0.05 (one-way ANOVA) is considered statistically significant. g Western blot analysis and h quantification of mitochondrial cofilin expression demonstrates no differences in cofilin translocation to the mitochondria in bone marrow erythroblasts from ß-thalassemic relative to WT mice. (MW = cofilin 19 kDa, cytochrome c 14 kDa, VDAC1 35 kDa). i Confocal immunofluorescence microscopy of cofilin (green) and mitochondria (magenta) co-localization in Pro-E from WT and ß-thalassemic mice. Scale bar 30 µm. The confocal images are representative of at least 20 cells analyzed in each condition. j Statistical analysis of (i) demonstrates no difference in cofilin/mitochondria co-localization in ß-thalassemic erythroblasts compared to WT. k Confocal immunofluorescence microscopy of cofilin (green) and mitochondria (magenta) co-localization in fetal liver cells from WT and ß-thalassemic mice transfected with either nt-shRNA or plek2-shRNA. Scale bar 50 µm. The confocal images are representative of at least 20 cells analyzed in each condition. l Statistical analysis of (k) demonstrates borderline decreased cofilin/mitochondria co-localization in nt-shRNA ß-thalassemic erythroblasts compared to nt-shRNA WT (p = 0.06) as well as increased cofilin/mitochondria co-localization in both WT and borderline increased cofilin/mitochondria co-localization ß-thalassemic (p = 0.07) erythroblasts transfected with plek2-shRNA compared to their respective controls (nt-shRNA). All data are reported as mean ± s.e.m. and p < 0.05 was considered statistically significant (Student’s unpaired t-test) (*p = 0.05, **p = 0.005, ***p = 0.0005). WT = wild type; apoTf = apo-transferrin; plek2 = pleckstrin-2; Pro-E = pro-erythroblasts; Baso-E = basophilic erythroblasts; Poly-E = polychromatophilic erythroblasts; Ortho-E = orthochromatophilic erythroblasts; MFI = mean fluorescent index.

Fig 3: Mechanism by which pleckstrin-2 supports proliferation, decreases apoptosis, and enhances enucleation in ineffective erythropoiesis.a The normal balance of differentiation and proliferation enables effective erythropoiesis in WT mouse bone marrow. In ß-thalassemic bone marrows, ineffective erythropoiesis results from an imbalance, with relatively more proliferating erythroblasts that differentiate and enucleate to form mature RBCs. When ROS are increased in ß-thalassemic erythroblasts, elevated Epo enables multiple downstream mechanisms. Plek2 serves as a mechanism downstream of Epo by which more ß-thalassemic erythroblasts evade apoptosis and increase erythroblast enucleation (green line). In thal plek2+/- bone marrow, more erythroblasts undergo apoptosis (red X) and fewer enucleate (green line), resulting in worsening anemia compared with ß-thalassemic mice. b When membrane-associated pleckstrin-2 is increased, it’s binding to cofilin and consequent prevention of cofilin translocation to the mitochondria results in decreased apoptosis despite elevation of ROS in ß-thalassemic erythroblasts. In addition, interaction with and activation of Rac1 results in enhanced enucleation in high-pleckstrin-2 states. Thus, when membrane-associated pleckstrin-2 is decreased in the setting of increased erythroblast ROS, relatively more cofilin translocation to the mitochondria leads to apoptosis and decreased Rac1 activation results in relatively decreased enucleation, leading to worsening ineffective erythropoiesis in ß-thalassemia. WT = wild type; thal = ß-thalassemia; plek2 = pleckstrin-2; ROS = reactive oxygen species.

Fig 4: Pleckstrin-2 haplo-insufficiency in ß-thalassemic mice results in exacerbation of ineffective erythropoiesis.a Splenomegaly observed in ß-thalassemic relative to WT mice is not reversed in plek2 haplo-insufficient ß-thalassemic (thal plek2+/-) mice (n = 5–7 mice/group). b Flow cytometry analysis reveals an increased fraction of bone marrow erythroblasts in ß-thalassemic relative to WT mice, borderline further increased in thal plek2+/- relative to ß-thalassemic mice (n = 4–5 mice/group). c Serum erythropoietin concentration reveals an increase in ß-thalassemic relative to WT mice, further increased in thal plek2+/- relative to ß-thalassemic mice (n = 4–5 mice/group). d Circulating RBC count and e hemoglobin were also unaltered in thal plek2+/- relative to ß-thalassemic mice (n = 10–11 mice/group). f Fam132b and g FasL mRNA expression in bone marrow CD45- erythroblasts further increased in thal plek2+/- relative to ß-thalassemic mice (n = 5–6 mice/group). h Flow cytometry analysis reveals unchanged caspase 3 activation in erythroblasts from ß-thalassemic mice compared to WT, but increased caspase3 activation in thal plek2+/- relative to ß-thalassemic mice despite increased serum Epo (n = 5 mice/group). i Flow cytometry analysis reveals an expected decrease in the fraction of enucleated:nucleated bone marrow erythroblasts (n = 3 mice/group). All data are reported as mean ± s.e.m. and p < 0.05 was considered statistically significant (one-way ANOVA) (*p = 0.05, **p = 0.005, ***p = 0.0005, ****p = 0.00005). WT = wild type; apoTf = apo-transferrin; RBC = red blood cell; Hb = hemoglobin; Fam132b = erythroferrone.

Fig 5: Early pleckstrin-2 activation and membrane localization during ß-thalassemic erythroid precursor differentiation.a Confocal immunofluorescence microscopy of plek2 (green) localization in sorted (2–3 mice pooled per data point, n = 3 data points per category) WT, ß-thalassemic, and apoTf-treated ß-thalassemic erythroblasts demonstrate an increased percentage of membrane plek2 localization relative to total plek2 in ß-thalassemic relative to: b WT Pro-E and c Baso-E from mice, normalized in apoTf-treated ß-thalassemic Pro-E (b) and Baso-E (c) with no differences in: d Poly-E and e Ortho-E. The confocal images were representative optical sections of the cell from at least 50 cells in each condition in 2 different experiments. Scale bar 5 µm. All data are reported as mean ± s.e.m. and p < 0.05 was considered statistically significant (one-way ANOVA) (*p = 0.05, **p = 0.005, ***p = 0.0005). WT = wild type; apoTf = apo-transferrin; plek2 = pleckstrin-2; Pro-E = pro-erythroblasts; Baso-E = basophilic erythroblasts; Poly-E = polychromatophilic erythroblasts; Ortho-E = orthochromatophilic erythroblasts.