Role of Tubulin Isoforms in Basal Body Duplication

The role of various tubulin isoforms in basal body duplication has been addressed by mutational analysis and gene silencing in Chlamydomonas, Trypanosoma brucei and Paramecium tetrauleis. Gamma (y)-tubulin plays an important role in all microtubule organizing centers [32]. Its role in basal body duplication has been addressed using gene silencing in Trypanosoma [33] and in Paramecium [34]. The experiments in Trypanosoma also suggest a role for y-tubulin in assembly of the central pair of microtubules in the flagellar axoneme. This result is consistent with the previous localization of y-tubulin to the interior of Chlamydomonas basal bodies [35]. In Paramecium, reduction in y-tubulin results in defects in basal body duplication. Examination of the proximal end of the triplet microtubules in the tomographic reconstructions of Chlamydomonas using the IMOD software, which allows one to rotate the reconstructed image, shows closed microtubule ends [5]. These closed ends have been associated with the presence of y-tubulin and the gamma-tubulin ring complex proteins [36].

Delta (5)-tubulin, which is present in most organisms with triplet microtubules in their centrioles, does not appear to play a primary role in basal body duplication

[37]. When this gene is deleted in Chlamydomonas, basal bodies are able to duplicate but fail to form triplet microtubules along most of the length of the basal body. The majority of the basal body blades contain A and B subfibers. At the distal end of the basal body where the transitional fibers are found, a short stretch of triplet microtubules is observed [5, 21].

Epsilon (e)-tubulin, which is also present in most organisms with triplet microtubules in their centrioles, is likely to have a more profound role in basal body duplication. This gene is essential in Chlamydomonas. When the amount of e-tubulin is reduced [38] or a truncated form is made ([39] and unpublished data), short incomplete basal bodies result. In Chlamydomonas, these incomplete basal bodies are 40-90 nm in length and are primarily composed of singlet microtubules [40]. In Paramecium, such basal bodies have singlet, doublet, and triplet microtubules

[38]. Similarly, Xenopus extracts that have been immunodepleted for e-tubulin fail to assemble centrin-containing centrioles [41]. It is likely therefore that e-tubulin plays a key role in basal body duplication.

Recently, eta (h)-tubulin has been found in Paramecium [42] as well as in databases for Ciona, and Chlamydomonas. Two mutations in Paramecium suggest that h-tubulin has an important role in basal body duplication. The temperature-sensitive h-tubulin mutation, sml9, fails to duplicate basal bodies upon shift to the restrictive temperature. Already assembled basal bodies appear to be nearly wildtype, but new ones fail to assemble. This is consistent with the notion that the assembled h-tubulin remains stable at the restrictive temperature or that h -tubulin provides a scaffolding function only during basal body duplication. Antibodies to h-tubulin in Paramecium are not available to discriminate between these two mechanisms.

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