Tubulin Isotypic Diversity in Mammalian Cilia

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Authors
Dossou, Starlette Jessica Yehinou
Issue Date
2011-08-15
Type
Dissertation
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en_US
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Abstract
Multiple well-conserved α and β tubulin isotypes have been identified in mammals. These isotypes are encoded by different genes and are selectively expressed in mammalian cells. The tubulin isotypes are also subjected to several post-translational modifications, including the polymeric modifications polyglycylation and polyglutamylation, whose functions remain unknown. The C terminal region of the tubulin isotypes serves as the site for these post-translational modifications, as well as the region where these isotypes are most variable. The axoneme motif, a region at the C-terminus of β tubulin isotypes, has been proposed to be essential for the assembly and beating of the axoneme. The presence of multiple tubulin isotypes and PTMs contribute to a diverse pool of tubulin that will be referred to as tubulin isotypic diversity. This diversity also leads to fundamental questions about the need of such a diverse pool of tubulin in mammals. In this dissertation, several assays were used to analyze the distribution and significance of multiple tubulin isotypes. First, the distribution of tubulin post-translational modifications was characterized in ciliated mammalian epithelial tissues using immunohistochemistry and primary cell cultures. This distribution was then characterized at the sub-cellular level by analyzing the distribution of these modifications in the two microtubule populations (central pair and outer doublet microtubules) of mammalian tracheal cilia through Western blot, immunogold electron microscopy and immunohistochemistry. The distribution of β tubulin isotypes between central pair and outer doublet microtubules was also characterized in the same manner. Finally, the speculated role of the axoneme motif in binding to dynein during ciliary beating was investigated using two assays. First, the effect of synthetic heptapeptides that mimic the axoneme motifs of β tubulin isotypes was analyzed in microtubule sliding assays with Tetrahymena cilia. The affinity of dynein for binding the heptapeptides was then measured in co-precipitation assays with dynein arms extracted from mammalian tracheal cilia. In these studies, I found that monomeric glycylation was universal in ciliated epithelial tissues as well as in tracheal cell cultures. Polymeric glycylation, however, was rare. At the sub-cellular level, monomeric glycylation was again predominant in both central pair and outer doublet microtubules. Polymeric glycylation was rare in both microtubule populations. Polymeric glutamylation was also predominant in both central pair and outer doublet microtubules. The β tubulin isotypes, βI and βIV tubulin, were predominant in outer doublet microtubules but limited in central pair microtubules. The opposite was true for βV tubulin, which was predominant in central pair microtubules but limited in outer doublet microtubules. Finally, at the protein level, dynein bound heptapeptides mimic the axoneme motifs of βI, βIII and βIV tubulin but the affinity of binding differed for each peptide. For the first time, I demonstrate that a segregated distribution of tubulin post-translational modifications and β tubulin isotypes exists at a sub-cellular level. I also demonstrate that the axoneme motifs of β tubulin isotypes play a direct role in the binding of tubulin and dynein but the affinity of this binding differs between the isotypes. I conclude that the distribution of isotypes and modifications are specific in the axoneme and may be essential for regulating function by specifying the interaction of dynein and tubulin.
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Creighton University
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Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
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