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Molecular Biology

, Volume 53, Issue 3, pp 393–401 | Cite as

Inducible Expression of Ran1 and Its GDP- and GTP-Bound Mimetic Mutants Leads to Defects in Amitosis and Cytokinesis with Abnormal Cytoplasmic Microtubule Assembly

  • H. X. LiangEmail author
  • H. W. Liu
MOLECULAR CELL BIOLOGY
  • 23 Downloads

Abstract

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd2+ were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.

Keywords:

amitosis cytokinesis cytoplasmic microtubule GDP/GTP-bound Ran1-mimetic Ran GTPase 

Notes

FUNDING

This work was financially supported by grants from the National Natural Scientific Foundation of China [#31501124] and Key Research and Development Project of Shanxi Province (International Cooperation project no. 201803D421087).

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

REFERENCES

  1. 1.
    Osgood E.E., Chu I.T. 1948. The effect of urethane on the nuclear morphology of cells of the granulocyte series as observed in marrow cultures and leukemic blood. Blood. 3, 911‒917.Google Scholar
  2. 2.
    Stevens D., Schwenk E. 1959. Amitosis in a new ascites tumor. Experientia. 15, 470‒471.CrossRefGoogle Scholar
  3. 3.
    Reshmi S.C., Gollin S.M. 2005. Chromosomal instability in oral cancer cells. J. Dent. Res. 84, 107‒117.CrossRefGoogle Scholar
  4. 4.
    Wong L., Klionsky L., Wickert S., Merriam V., Orias E., Hamilton E.P. 2000. Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila. Genetics. 155, 1119‒1125.Google Scholar
  5. 5.
    Numata O., Fujiu K., Gonda K. 1999. Macronuclear division and cytokinesis in Tetrahymena. Cell Biol. Int. 23, 849‒57.CrossRefGoogle Scholar
  6. 6.
    Fujiu K., Numata O. 2000. Reorganization of microtubules in the amitotically dividing macronucleus of tetrahymena. Cell Motil. Cytoskeleton. 46, 17‒27.CrossRefGoogle Scholar
  7. 7.
    Cervantes M.D., Coyne R.S., Xi X., Yao M.C. 2006. The condensin complex is essential for amitotic segregation of bulk chromosomes, but not nucleoli, in the ciliate Tetrahymena thermophila. Mol. Cell Biol. 26, 4690‒4700.CrossRefGoogle Scholar
  8. 8.
    Rensen W.M., Mangiacasale R., Ciciarello M., Lavia P. 2008. The GTPase Ran: Regulation of cell life and potential roles in cell transformation. Front. Biosci. 13, 4097‒4121.CrossRefGoogle Scholar
  9. 9.
    Kalab P., Heald R. 2008. The RanGTP gradient—a GPS for the mitotic spindle. J. Cell Sci. 121, 1577‒1586.CrossRefGoogle Scholar
  10. 10.
    Gruss O.J., Carazo-Salas R.E., Schatz C.A., Guarguaglini G., Kast J., Wilm M., Le Bot N., Vernos I., Karsenti E., Mattaj I.W. 2001. Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell. 104, 83‒93.CrossRefGoogle Scholar
  11. 11.
    Hetzer M., Gruss O.J., Mattaj I.W. 2002. The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. Nat. Cell Biol. 4, E177‒184.CrossRefGoogle Scholar
  12. 12.
    Oh D., Yu C.H., Needleman D.J. 2016. Spatial organization of the Ran pathway by microtubules in mitosis. Proc. Natl. Acad. Sci. U. S. A. 113, 8729‒8734.CrossRefGoogle Scholar
  13. 13.
    Cesario J., McKim K.S. 2011. RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila. J. Cell Sci. 124, 3797‒3810.CrossRefGoogle Scholar
  14. 14.
    Moss D.K., Wilde A., Lane J.D. 2009. Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase. J. Cell Sci. 122, 644‒655.CrossRefGoogle Scholar
  15. 15.
    Carazo-Salas R.E., Guarguaglini G., Gruss O.J., Segref A., Karsenti E., Mattaj I.W. 1999. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature. 400, 178‒181.CrossRefGoogle Scholar
  16. 16.
    Ohkura H. 2015. Meiosis: An overview of key differences from mitosis. Cold Spring Harb. Perspect. Biol. 7, pii: a015859.CrossRefGoogle Scholar
  17. 17.
    Fleig U., Salus S.S., Karig I., Sazer S. 2000. The fission yeast ran GTPase is required for microtubule integrity. J. Cell Biol. 151, 1101‒1111.CrossRefGoogle Scholar
  18. 18.
    Yudin D., Fainzilber M. 2009. Ran on tracks: Cytoplasmic roles for a nuclear regulator. J. Cell Sci. 122, 587‒593.CrossRefGoogle Scholar
  19. 19.
    Liang H., Xu J., Zhao D., Tian H., Yang X., Liang A. Wang W. 2012. Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus. FEBS. J. 279, 2520‒2533.CrossRefGoogle Scholar
  20. 20.
    Prelich G. 2012. Gene overexpression: Uses, mechanisms, and interpretation. Genetics. 190, 841‒854.CrossRefGoogle Scholar
  21. 21.
    Mochizuki K., Fine N.A., Fujisawa T., Gorovsky M.A. 2002. Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell. 110, 689‒699.CrossRefGoogle Scholar
  22. 22.
    Bischoff F.R., Klebe C., Kretschmer J., Wittinghofer A., Ponstingl H. 1994. RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc. Natl. Acad. Sci. U. S. A. 91, 2587‒2591.CrossRefGoogle Scholar
  23. 23.
    Klebe C., Bischoff F.R., Ponstingl H., Wittinghofer A. 1995. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry. 34, 639‒647.CrossRefGoogle Scholar
  24. 24.
    Mahajan R., Delphin C., Guan T., Gerace L., Melchior F. 1997. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 88, 97‒107.CrossRefGoogle Scholar
  25. 25.
    Rose A., Meier I. 2001. A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc. Natl. Acad. Sci. U. S. A. 98,15377‒15382.CrossRefGoogle Scholar
  26. 26.
    Zhao D., Liang H., Xu J., Wang W. 2013. Ran Binding Protein 1 affects macronuclear and micronuclear division in Tetrahymena thermophila. Chin. J. Bio. Mol. Biol. 29, 330‒337.Google Scholar
  27. 27.
    Ren X., Xu J., Wang W. 2015. Localization and function of RanGTPase activating protein (RanGAP) from Tetrahymena thermophila. Chin. J. Bio. Mol. Biol. 31, 264‒273.Google Scholar
  28. 28.
    Moore W., Zhang C., Clarke P.R. 2002. Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells. Curr. Biol. 12, 1442‒1447.CrossRefGoogle Scholar
  29. 29.
    Duan W., Xu J., Liang A. 2014. Localization and function of regulator of chromosome condensation 1 from Tetrahymena thermophila. Chin. J. Bio. Mol. Biol. 30, 255‒263.Google Scholar
  30. 30.
    Silverman-Gavrila R.V. 2006. Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization. Mol. Biol. Cell. 17, 2069‒2080.CrossRefGoogle Scholar
  31. 31.
    Riddick G., Macara I.G. 2005. A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import. J. Cell Biol. 168, 1027‒1038.CrossRefGoogle Scholar
  32. 32.
    Arai R., Mabuchi I. 2002. F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 115, 887‒898.Google Scholar
  33. 33.
    Smith L.G. 2002. Plant cytokinesis: Motoring to the finish. Curr. Biol. 12, R206‒R208.CrossRefGoogle Scholar
  34. 34.
    Steiner A., Rybak K., Altmann M., McFarlane H.E., Klaeger S., Nguyen N., Facher E., Ivakov A., Wanner G., Kuster B., Persson S., Braun P., Hauser M.T., Assaad F.F. 2016. Cell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis. Plant J. 88, 531‒541.CrossRefGoogle Scholar
  35. 35.
    Wang H., Oliferenko S., Balasubramanian M.K. 2003. Cytokinesis: Relative alignment of the cell division apparatus and the mitotic spindle. Curr. Opin. Cell Biol. 15, 82‒87.CrossRefGoogle Scholar
  36. 36.
    Thery M., Bornens M. 2006. Cell shape and cell division. Curr. Opin. Cell Biol. 18, 648‒657.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  1. 1.Institute of Biomedical Engineering, Taiyuan University of TechnologyTaiyuan, ShanxiChina
  2. 2.College of Chemistry and Chemical Engineering, Taiyuan University of TechnologyTaiyuan, ShanxiChina

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