[
International Worm Meeting,
2017]
Microtubules (MTs) are composed of a- and beta -tubulins, which are encoded by multiple genomic loci in most organisms. Despite implications of distinct properties of tubulin isotypes, whether and how they contribute to diverse MT functions in vivo remains unclear. In this study, we aimed to comprehensively analyze null phenotypes and expression patterns/levels of all tubulin isotypes ( nine a-tubulins and six beta -tubulins) in C.elegans, using CRISPR/Cas9-mediate genome editing. Firstly, we focused on two a-tubulins (TBA-1 and TBA-2) and two beta -tubulins (TBB-1and TBB-2) that are expressed in early embryos. Among beta -tubulins, TBB-2 was expressed two times higher than TBB-1. Substitution of isotype coding regions by CRISPR/Cas9 demonstrated that, under the same concentration, TBB-1-composed MTs had higher switching frequency between growth and shrinkage than TBB-2-composed MTs. The two a-tubulins distinctively affected growth rates of TBB-1-composed MTs. These results demonstrated that each isotype has distinct properties for MT dynamics, and alteration of ratio and concentration of isotypes distinctively modulates MT dynamics in vivo. Next, using GFP-insertion lines, we analyzed expression patterns and levels of each tubulin isotype in adult tissues. TBA-1, TBA-2, TBB-1, TBB-2 were ubiquitously expressed at a high level, whereas TBA-4 was expressed in many tissues except germline at a low level. On the other hand, MEC-12 (a), MEC-7 ( beta ) and TBB-4 were expressed in specific sets of neurons (as previously reported) at a much lower level than the expression levels of ubiquitous tubulin isotypes, and TBA-7 was expressed in intestine. MEC-12 and MEC-7 are known to be required for the function of touch receptor neurons and generating 15-protofilament microtubules. Thus, these results implicate that MTs are mainly constructed by ubiquitous tubulin isotypes and only a small fraction of tissue-specific tubulin isotypes (such as MEC-12 and MEC-7) can affect MT dynamics to confer tissue-specific MT behaviors. To further understand specific properties of tubulin isotypes, we plan to artificially alter isotype composition and examine their effects on MT dynamics
Onodera, Shizuka, Tsuchiya, Kenta, Nishida, Kei, Honda, Yu, Sugimoto, Asako, Ikeda, Masanori, Haruta, Nami, Tanaka, Kozo, Obinata, Hiroyuki
[
International Worm Meeting,
2021]
Most organisms have multiple alpha- and beta-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine alpha-tubulin isotypes and six beta-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and quantitatively analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-a-tubulins TBA-1 and TBA-2 and b-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Thus, MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes, and incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. Three alpha-tubulin isotypes (TBA-5, TBA-6, TBA-9) and one beta-tubulin isotype (TBB-4) were expressed in different subsets of ciliated sensory neurons. When each of the ciliated neuron-specific isotypes was ectopically expressed in early embryos, they were less efficiently incorporated into mitotic spindle MTs than broadly-expressed isotypes and affected MT dynamics. The low incorporation efficiency was slightly improved when the ciliated neuron-specific alpha-tubulins were co-expressed with TBB-4, implying that some specific combinations of alpha- and beta-tubulin isotypes form heterodimers preferably incorporated into MTs. The lower MT incorporation efficiencies of these ciliated neuron-specific tubulin isotypes were also observed by their heterologous expression in human cultured cells. Taken together, we speculate that a small amount of ciliated neuron-specific tubulins isotypes is incorporated into MTs mainly formed by broadly-expressed isotypes, which contribute to the cell-type-specific MT properties.