Basal Body Duplication and Maintenance Require One Member of theTetrahymena thermophilaCentrin Gene Family

Abstract

Centrins, small calcium binding EF-hand proteins, function in the duplication of a variety of microtubule organizing centers. These include centrioles in humans, basal bodies in green algae, and spindle pole bodies in yeast. The ciliate Tetrahymena thermophila contains at least four centrin genes as determined by sequence homology, and these have distinct localization and expression patterns. CEN1's role at the basal body was examined more closely. The Cen1 protein localizes primarily to two locations: one is the site at the base of the basal body where duplication is initiated. The other is the transition zone between the basal body and axoneme. CEN1 is an essential gene, the deletion of which results in the loss of basal bodies, which is likely due to defects in both basal body duplication and basal body maintenance. Analysis of the three other centrins indicates that two of them function at microtubule-rich structures unique to ciliates, whereas the fourth is not expressed under conditions examined in this study, although when artificially expressed it localizes to basal bodies. This study provides evidence that in addition to its previously known function in the duplication of basal bodies, centrin is also important for the integrity of these organelles.

Functional diversification of centrins and cell morphological complexity

“…Several further discrete localisations have also been identified, in cilia (Gonda et al, 2004;Guerra et al, 2003;LeDizet and Piperno, 1995) and ciliary-derived organelles of sensory cells (Giessl et al, 2006), at the contractile vacuole pores in Tetrahymena (Stemm-Wolf et al, 2005) and at the Golgi in Trypanosoma (He et al, 2005;Selvapandiyan et al, 2007). Interestingly, if species differ widely in their number of centrin genes (one in yeast, four in mammals, seven in Leishmania, at least ten in Tetrahymena and 49 in Paramecium), when more than a single gene is present, a particular localisation or function may involve either a single or different centrin isotypes and conversely, a particular isotype may show different localisations or functions.…”

“…They have long been shown to localise at two major sites: microtubule organising centres (MTOCs), for example, spindle pole bodies, centrosomes or basal bodies and MTOC-associated arrays of contractile fibres where they were first identified (Salisbury et al, 1984). In MTOCs, centrin is a cytologically discrete and functionally important component, required for their duplication, segregation and positioning (Baum et al, 1986;Koblenz et al, 2003;Paoletti et al, 1996;Ruiz et al, 2005;Salisbury et al, 2002;Stemm-Wolf et al, 2005;Wright et al, 1989;Wright et al, 1985). Remarkably, in acentriolar organisms, which form basal bodies only at a specific stage of their life cycle, such as in Naegleria or Marsilea, centrin synthesis strictly correlates with assembly of basal bodies (Klink and Wolniak, 2001;Levy et al, 1996).…”

Centrosomes as signalling centres

“…Mammalian centrin-2 is considered the orthologue of the S. cerevisiae Cdc31 gene product, which is clearly required for SPB duplication [141]. Likewise, centrins are essential for the formation of basal bodies in ciliates [142,143]. Whether or not centrin-2 is required for centrosome duplication in human cells is controversial, but there is no question that this protein is a genuine component of vertebrate centrosomes [144][145][146][147].…”

ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies

“…Once two strains of different mating types were constructed, they were mated and the subsequent progeny lacked ETU1 in the macronucleus (somatic nucleus), thus creating the etu1D strain. Staining of basal bodies using centrin 1 (Cen1), a basal body marker (Stemm-Wolf et al, 2005), revealed a decrease in the basal body density within cortical rows upon removal of the ETU1 gene product (supplementary material Fig. S3A-C).…”

“…All primary antibodies were diluted in 1% BSA in phosphate buffered saline (PBS). The Tetrahymena Cen1 antibody (Stemm-Wolf et al, 2005) was diluted 1:2000. The monoclonal Atu1 antibody (provided by Joe Frankel, University of Iowa, Iowa City, IA) was diluted 1:50.…”