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Eukaryotic Cell, March 2006, p. 555-567, Vol. 5, No. 3
1535-9778/06/$08.00+0     doi:10.1128/EC.5.3.555-567.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Actin Gene ACT1 Is Required for Phagocytosis, Motility, and Cell Separation of Tetrahymena thermophila{dagger}

Norman E. Williams,1 Che-Chia Tsao,2 Josephine Bowen,2 Gery L. Hehman,1 Ruth J. Williams,1 and Joseph Frankel1*

Department of Biological Sciences, University of Iowa, Iowa City, Iowa,1 Department of Biology, University of Rochester, Rochester, New York2

Received 20 September 2005/ Accepted 2 December 2005

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1{Delta} mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1{Delta} cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1{Delta} cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.

* Corresponding author. Mailing address: Department of Biological Sciences, The University of Iowa, 143 Biology Bldg., Iowa City, IA 52242. Phone: (319) 335-1110. Fax: (319) 335-1069. E-mail: joseph-frankel{at}uiowa.edu.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, March 2006, p. 555-567, Vol. 5, No. 3
1535-9778/06/$08.00+0     doi:10.1128/EC.5.3.555-567.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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