TMOD1 knockdown during human erythroblast differentiation impairs enucleation and increases the amount of cytoplasmic F-actin in polarized erythroblasts. CD34⁺ cells were transduced with either luciferase (control) or TMOD1 (shRNA27 or shRNA28) lentivirus and induced toward erythroid differentiation. Puromycin selection was performed 48 hours posttransduction. (A) Wright Giemsa–stained cytospins of CD34⁺ cells at day 14. Bar, 10 µm. (B) Quantitative reverse transcription polymerase chain reaction of TMOD1 and TMOD3 levels normalized to ACTB in Luciferase control or TMOD1 knockdown. ***P < .001 (N = 3). (C) Representative western blot for Tmod1 and Tmod3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at day 11 and day 14 of differentiation. (D) Late orthochromatic erythroblast populations were flow sorted at day 16 based on the surface expression levels of GPA, α4-integrin, and Band3. SYTO60 was used to measure enucleation within these populations. ***P < .001. (E) Extended-focus projections of confocal Z-stacks of polarized, differentiated CD34⁺ cells (control or TMOD1 knockdown), immunostained for GPA, phalloidin for F-actin, and Hoechst for nuclei. Arrows show increased size of cytoplasmic F-actin spot in TMOD1 knockdown cells. Bar, 4 µm. (F) Average F-actin spot volume in polarized, or enucleating differentiated CD34⁺ cells in panel E. ***P < .001. N = 26 cells for Luciferase control, 29 for shRNA27, and 32 for shRNA28, obtained from 2 separate knockdown experiments. (G) Schematics of GPA sorting (green), F-actin reorganization (red), and nuclear (blue) expulsion during the progression of mouse and human erythroblast enucleation. In both mouse and human erythroblasts, the F-actin network undergoes dramatic reorganization during enucleation. As the erythroblasts polarize, an F-actin spot (the enucleosome) appears at the rear of the nucleus. The enucleosome follows the translocating nucleus and may drive expulsion. Mouse erythroblast nuclei adopt a dumbbell-shaped morphology during expulsion, with prominent F-actin foci associated with the cell surface and nuclear constriction at the Ter119-sorting boundary, whereas human erythroblast nuclei retain a spherical morphology, and only very small F-actin foci are present at the GPA-sorting boundary over the translocating nucleus. Small membrane vesicles are depicted near the rear of the translocating nucleus, based on our (eg, supplemental Figure 3B) and others’ observations.^11,18  For simplicity, microtubules are not depicted.