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Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2

Abstract

Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane1,2,3. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments4,5,6, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts.

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Figure 1: Enucleation of mammalian erythroid cells in vivo and during in vitro culture.
Figure 2: Deregulation of Rac GTPases blocks enucleation.
Figure 3: Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner.
Figure 4: Depletion of mDia2 inhibits enucleation.
Figure 5: Constitutively active mutant of mDia2 rescues the defects of enucleation induced by dominant-negative mutants of Rac GTPases.

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Acknowledgements

We thank: A. Alberts for critical reading of the manuscript; S. Lux for helpful comments; J. Zhang for help with the in vitro culture system; G. Pardis for help with flow cytometry. This study was supported by National Institutes of Health (NIH) grant (P01 HL 32262) and a research grant from Amgen, Inc. to H.F.L.

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Authors

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P. J. and S. R. J. performed the experiments. P. J. and H. F. L. contributed to the experimental design, data analysis and writing the paper.

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Correspondence to Harvey F. Lodish.

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Supplementary figures S1, S2, S3, S4, S5 and S6 (PDF 1241 kb)

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Ji, P., Jayapal, S. & Lodish, H. Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2. Nat Cell Biol 10, 314–321 (2008). https://doi.org/10.1038/ncb1693

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