Cortical Localization of α- and γ-Tubulin and the Assembly of Cortical Microtubule Cytoskeleton in Hypotrichous Ciliate Euplotes eurystomus

Xin Sheng,

Yan Sheng,

Yuehua Liu,

Junhua Wang

This study aimed to investigate the assembly characteristics of ciliature and cortical microtubules and the localization of tubulins in different depths and regions of the cortex. The hypotrichous ciliates have closely arranged cilia and a highly complex microtubular system. Direct fluorescence and immunofluorescence labeling were used to observe ciliary organelles and cortical microtubular cytoskeleton in Euplotes eurystomus. An immunofluorescence analysis demonstrated that α-tubulin localized to the ventral and dorsal ciliary organelles and their associated microtubules, while γ-tubulin localized to the basal bodies of ciliary organelles, macronuclear membrane, and excretory pore of a contractile vacuole in the interphase. A direct fluorescence analysis showed that the ciliature and cortical microtubules in the deep cortex were more clearly marked by fluorescent taxoid (FLUTAX). Interestingly, α- and γ-tubulins also colocalized to the ringlike ciliary base–associated microtubules of dorsal kineties. The short microtubular bundles between the bases of transverse cirri could be marked by FLUTAX and γ-tubulin rather than α-tubulin, suggesting that tubulins in cortical microtubules in E. eurystomus varied, and the differentiation of cortical microtubules in the hypotrichous ciliate tended to be versatile. Also, during morphogenesis, γ-tubulin also localized to the base of ciliary primordium, where new basal bodies were formed, suggesting that the endocellular position of γ-tubulin in ciliates was related to basal bodies and regulated by the cell cycle. This study might help understand the assembly characteristics and tubulin composition of microtubules in different depths and regions of the cortex in hypotrichous ciliate E. eurystomus.
Słowa kluczowe: basal body, ciliature microtubular organelles, E. eurystomus, FLUTAX, immunofluorescence, γ-tubulin

Allen R. D. (1967) Fine structure, reconstruction and possible fuctions of components of the cortex of Tetrahymena pyriformis. J. Eukaryot. Microbiol14: 553–565

Allen R. D. (1969) The morphogenesis of basal bodies and accessory structure of the cortex of the ciliated protozoan Tetrahymena pyriformisJ. Cell Biol40: 716–733

Arregui L., Muñoz-Fontela C., Serrano S., Barasoain I., Guinea A. (2002) Direct visualization of the microtubular cytoskeleton of ciliate protozoa with a fluorescent taxoid. J. Eukaryot. Microbiol49: 312–318

Beisson J., Wright M. (2003) Basal body/centriole assembly and continuity. Curr. Opin. Cell Biol15: 96–104

Binarová P., Cenklová V., Hause B., Kubátová E., Lysák M., Dolezel J., Bögre L., Dráber P. (2000) Nuclear gamma-tubulin during acentriolar plant mitosis. Plant Cell 12: 433–442

Díaz J. F., Strobe R., Engelborghs Y., Souto A. A., Andreu J. M. (2000) Molecular recognition of Taxol by microtubules: Kinetics and thermodynamics of binding of Fluorescent Taxol derivatives to an exprosed site. J. Biol. Chem275: 265–276

Elmendorf G. H., Dawson C. S., McCaffery M. J. (2003) The cytoskeleton of Giardia lambliaInt. J. Parasitol33: 3–28

Evangelio J. A., Abal M., Barasoain I., Souto A. A., Lillo M. P., Acuña A. U., Amat-Guerri F., Andreu J. M. (1998) Fluorescent taxoids as probes of the microtubule cytoskeletonCytoskeleton 39: 73–90

Fuller S. D., Gowen B. E., Reinsch S., Sawyer A., Buendia B., Wepf R., Karsenti E. (1995) The core of the mammalian centriole contains gamma-tubulin. Curr. Biol. 5: 1384–1393

Gaeritg J. (2000) Molecular mechanisms of microtubular organelle assembly in Tetrahymena. J. Eukaryot. Microbiol47: 185–190

Gu F. K. (1991) Introduction to protozoology. Higher Education Press, Peking.

Gu F. K., Zou S. F., Li Y. S., Ni B. (2003) Scanning electron microscopic observations on the ventral cortical cytoskeleton of Euplotes harpa (Protozoa, Ciliophora). Acta Zool. Sinica 49: 514–521

Iftode F., Clérot J. C., Levilliers N., Bré M. H. (2000) Tubulin polyglycylation: a morphogenetic marker in ciliates. Biol. Cell 92: 615–628

Iftode F., Fleury-Aubusson A. (2003) Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission. Biol. Cell 95: 39–51

Kilburn C. L., Pearson C. G., Romijn P. E., Meehl J. B., Giddings T. H., Culver B. P., Yates J. R., Winey M. (2007) New Tetrahymena basal body protein components identify basal body domain structure. J. Cell Biol178: 905–912

Klotz C., Ruiz F., Loubresse N. G. D., Wright M., Dupuid-Williams P., Beisson J. (2003) Gamma-tubulin and MTOCs in Paramecium. Protist 154: 193–209

Kovács P., Csaba G., Pállinger É., Czaker R. (2007) Effects of taxol treatment on the microtubular system and mitochondria of TetrahymenaCell Biol. Int31: 724–732

Lajoie-Mazenc I., Tollon Y., Detraves C., Julian M., Moisand A., Gueth-Hallonet C., Debec A., Salles-Passador I., Puget A., Mazarguil H. (1994) Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle. J. Cell Sci107: 2825–2837

Lechtreck F. K., Geimer S. (2000) Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates. Cell Motil. Cytoskel47: 219–235

Liang A., Heckmann K. (1993) The macronuclear γ-tubulin-encoding gene of Euplotes octocarinatus contains two introns and an in-frame TGA. Gene 136: 319–322

Liang A., Ruiz F., Heckmann K., Klotz C., Tollon Y., Beisson J., Wright M. (1996) Gamma-tubulin is permanently associated with basal bodies in ciliates. Eur. J. Cell Biol70: 331–338

Libusová L., Dráber P. (2006) Mutiple tubulin forms in ciliated protozoan Tetrymena and Paramecium species. Protoplasma 22765–76

Lou H. L., Gao W., Ni B., Gu F. K. (2007) Morphology and morphogenesis of the ciliature microtubular organelles in the ventral cortex of Paraurostyla weissei (Hyportrichida, Ciliophora). Acta Zool. Sinica 53: 742–749

Loubresse N. G. D., Riuz F., Beisson J., Klotz C. (2001) Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies. BMC Cell Biol2: 1–7

Lynn D., Small E. B. (1981) Protist kinetids: Structual conservatism, kinetid structure, and ancestral states. Biosystems 14: 377–385

Marshall W. F., Rosenbaum J. L. (2003) Tubulin superfamily – Giving Birth to Triplets. Curr. Biol. 13: R55–R56

Marziale F., Pucciarelli S., Ballarini P., Melki R., Uzun A., Llyin V. A., Detrich H. W., Miceli C. (2008) Different roles of two γ-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: Remodelling of interaction surfaces may enhance microtubule nucleation at low temperature. FEBS J275: 5367–5382

Moritz M., Braunfeld M. B., Sedat J. W., Alberts B., Agard D. A. (1995) Microtubule nucleation by γ-tubulin-containing rings in the centrosome. Nature 378: 638–640

Oakley C. E., Oakley B. R. (1989) Identification of a γ-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulansNature 338: 662–664

Olins D. E., Olins A. L., Robert-Nicoud M., Jovin T. M., Wehland J., Weber K. (1989) Differential distribution of α-tubulin isotypes in E. eurystomus determined by confocal immunofluorescence microscopy. Biol. Cell 66: 235–246

Oz S., Ivashkopachima Y., Gozes I. (2012). The ADNP derived peptide, NAP modulates the tubulin pool: implication for neurotrophic and neuroprotective activities. PloS One 7: 1411–1411

Ruiz F., Beisson J., Rossier J., Dupuis-Williams P. (1999) Basal body duplication in Paramecium requires γ-tubulin. Curr. Biol. 9: 43–46

Sambrook J., Fritsch F., Maniatis T. (1992) Molecular Clone: a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, NY.

Scott V., Sherwin T., Gull K. (1997) Gamma-tubulin in Trypanosomes: Molecular characterization and location to multiple and diverse microtubule organizing centres. J. Cell Sci110: 157–168

Shang Y. H., Li B., Gorovsky M. A. (2002) Tetrahymena thermophila contains a conventional γ-tubulin that is differentially required for the maintenance of different microtubule-organizing centers. J. Cell Biol158: 1195–1206

Sheng X., Zeng H., Zhang M., Yun M. X., Yin F., Gu F. K. (2011) Influences of the interference of the γ-tubulin gene expression on the morphology and microtubules of ciliate E. eurystomusZool. Sci28: 476–481

Shu H. B., Li Z., Palacios M. J., Li Q., Joshi H. C. (1995) A transient associated of gamma-tubulin at the midbody is required for the completion of cytokinesis during the mammaliam-cell division. J. Cell Sci108: 2955–2962

Tan M., Heckmann K. (1998) The two γ-tubulin-encoding genes of the ciliate Euplotes crassus differ in their sequences, codon usage, transcription initiation sites and poly (A) addition sites. Gene 210: 53–60

Zhou S. J., Yin F., Sheng X., Gu F. K. (2008) Morphology and morphogenesis of the ciliature microtubular organelles in the ventral cortex of Pseudourostyla cristata (Hyportrichida , Ciliophora). Acta Zool. Sinica 54: 299–308

Zhu H. (1984) The contractile vacuole. Bull Biol4: 3–5

Czasopismo w darmowym dostępie.

Czasopismo ukazuje się w sposób ciągły on-line.
Pierwotną formą czasopisma jest wersja elektroniczna.