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Eukaryotic Cell, October 2004, p. 1217-1226, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1217-1226.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A ß-Tubulin Mutation Selectively Uncouples Nuclear Division and Cytokinesis in Tetrahymena thermophila

Joshua J. Smith ,1,{dagger},{ddagger} J. Sebastian Yakisich,2,{dagger} Geoffrey M. Kapler,2 Eric S. Cole,3 and Daniel P. Romero1*

Department of Pharmacology, Medical School, University of Minnesota, Minneapolis,1 Department of Biology, St. Olaf College, Northfield, Minnesota,3 Department of Medical Biochemistry and Genetics, Texas A&M University Health Science Center, College Station, Texas2

Received 3 December 2003/ Accepted 23 June 2004

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell—the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant ß-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.

* Corresponding author. Mailing address: Department of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455. Phone: (612) 624-8997. Fax: (612) 625-8408. E-mail: romero{at}med.umn.edu.

{dagger} J.J.S. and J.S.Y. contributed equally to this study.

{ddagger} Present address: Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908.

Eukaryotic Cell, October 2004, p. 1217-1226, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1217-1226.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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