![]() 2 Nuclear polarization and movement also require degradation of dynein microtubule motors by Trim58, a ubiquitin ligase upregulated during terminal erythroid differentiation. ![]() Local activation of phosphoinositide-3-kinase near the microtubule-organizing center regulates cell polarity and nuclear movement to the opposing side of the erythroblast. 3 - 7 Many genes have been linked directly or indirectly to enucleation, 8, 9 from transcription factors (eg, KLF1 10) to signaling molecules (eg, Rac GTPases 11 - 14), structural proteins (eg, mDia2, 14 dematin 15), molecular motors (eg, dynein 16, 17 and nonmuscle myosin IIB 2, 11, 12), and endocytic pathway components (eg, EPS15, clathrin 18, 19), converging on a model in which enucleation is proposed to be analogous to asymmetric cell division, with microtubules important for cell polarization and nuclear movement, and F-actin assembly into an actomyosin contractile ring driving nuclear expulsion. Mammalian erythroblast enucleation is a rapid cellular process (<10 minutes in cultured erythroblasts 1, 2) involving nuclear polarization to one side of the cell, membrane protein sorting to remove unwanted membrane proteins from the nascent reticulocyte, nuclear translocation and expulsion, followed finally by separation of the pyrenocyte (expelled nucleus) from the nascent reticulocyte. This novel structure, the “enucleosome,” may mediate common cytoskeletal mechanisms underlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34 + hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. ![]() We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. ![]() Here, we employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained.
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