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Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2

Nature Cell Biology volume 10, pages 314321 (2008) | Download Citation

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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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References

  1. 1.

    , & Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol. 15, 146–155 (2005).

  2. 2.

    , & New insights into erythropoiesis. Curr. Opin. Hematol. 9, 93–100 (2002).

  3. 3.

    & Jak2: normal function and role in hematopoietic disorders. Curr. Opin. Genet. Dev. 17, 8–14 (2007).

  4. 4.

    & Staying in shape with formins. Dev. Cell 10, 693–706 (2006).

  5. 5.

    & Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76, 593–627 (2007).

  6. 6.

    Formin-induced nucleation of actin filaments. Curr. Opin. Cell Biol. 16, 99–105 (2004).

  7. 7.

    & Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells. J. Cell Physiol. 137, 65–74 (1988).

  8. 8.

    , , , & Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development. J. Cell Biol. 177, 871–880 (2007).

  9. 9.

    & The mechanism of denucleation in circulating erythroblasts. J. Cell Biol. 35, 237–245 (1967).

  10. 10.

    & An electron microscopic study of nuclear elimination from the late erythroblast. J. Cell Biol. 33, 625–635 (1967).

  11. 11.

    & A reevaluation of the process of enucleation in mammalian erythroid cells. Prog. Clin. Biol. Res. 55, 679–692 (1981).

  12. 12.

    , , & Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system. Blood 102, 3938–3946 (2003).

  13. 13.

    , & Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts. J. Cell Biol. 109, 3005–3013 (1989).

  14. 14.

    , , & Membrane assembly and remodeling during reticulocyte maturation. Blood 74, 1112–1120 (1989).

  15. 15.

    Rho GTPases in neuronal morphogenesis. Nature Rev. Neurosci. 1, 173–180 (2000).

  16. 16.

    , , , & Structure-function based design of small molecule inhibitors targeting Rho family GTPases. Curr. Top. Med. Chem. 6, 1109–1116 (2006).

  17. 17.

    , , , & Rational design and applications of a Rac GTPase-specific small molecule inhibitor. Methods Enzymol. 406, 554–565 (2006).

  18. 18.

    Formin proteins: a domain-based approach. Trends Biochem. Sci. 30, 342–353 (2005).

  19. 19.

    & The Rho family GTPase Rif induces filopodia through mDia2. Curr. Biol. 15, 129–133 (2005).

  20. 20.

    et al. A novel Rho–mDia2–HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts. J. Cell Sci. 118, 2901–2911 (2005).

  21. 21.

    , , & mDia mediates Rho-regulated formation and orientation of stable microtubules. Nature Cell Biol. 3, 723–729 (2001).

  22. 22.

    et al. Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton. Blood 108, 3637–3645 (2006).

  23. 23.

    et al. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation. Blood 107, 1888–1891 (2006).

  24. 24.

    , , , & Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nature Cell Biol. 1, 136–143 (1999).

  25. 25.

    , , , & siRNA Selection Server: an automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 32, W130–W134 (2004).

  26. 26.

    , & Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc. Natl Acad. Sci. USA 101, 5494–5499 (2004).

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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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Author notes

    • Senthil Raja Jayapal

    Current address: Stem Cell Group 4, Genome Institute of Singapore #08-01, Genome, 60 Biopolis Street, Singapore 138672.

Affiliations

  1. Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Floor 6, Cambridge, MA 02142, USA.

    • Peng Ji
    • , Senthil Raja Jayapal
    •  & Harvey F. Lodish

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Contributions

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.

Corresponding author

Correspondence to Harvey F. Lodish.

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DOI

https://doi.org/10.1038/ncb1693

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