The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena.

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.

Multiple phosphorylation sites on γ-tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena.

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.

Regulation of katanin activity in the ciliate Tetrahymena thermophila.

Katanin is a microtubule severing protein that functions as a heterodimer composed of an AAA domain catalytic subunit, p60, and a regulatory subunit, a WD40 repeat protein, p80. Katanin-dependent severing of microtubules is important for proper execution of key cellular activities including cell division, migration, and differentiation. Published data obtained in Caenorhabditis elegans, Xenopus and mammals indicate that katanin is regulated at multiple levels including transcription, posttranslational modifications (of both katanin and microtubules) and degradation. Little is known about how katanin is regulated in unicellular organisms. Here we show that in the ciliated protist Tetrahymena thermophila, as in Metazoa, the localization and activity of katanin requires specific domains of both p60 and p80, and that the localization of p60, but not p80, is sensitive to the levels of microtubule glutamylation. A prolonged overexpression of either a full length, or a fragment of p80 containing WD40 repeats, partly phenocopies a knockout of p60, indicating that in addition to its activating role, p80 could also contribute to the inhibition of p60. We also show that the level of p80 depends on the 26S proteasome activity.

[Microtubule severing proteins - structure and activity regulation]. / Bialka tnace mikrotubule - struktura i regulacja aktywnosci.

Microtubule severing proteins, katanin, spastin and fidgetin cause local destabilization of the microtubules structure. This ATP-dependent activity leads to the shortening or disassembly of the existing microtubules. The generated short microtubule fragments may serve as templates to polymerize new microtubules and in consequence, the activity of the microtubule severing proteins leads to the reorganization of the microtubular cytoskeleton. This review summarizes current knowledge concerning structural organization of the microtubule severing proteins, the molecular mechanism of their action, factors that regulate the level of the katanin and spastin within the cells and their microtubule severing activity.

[Role of microtubule severing proteins in cytoskeleton reorganization]. / Rola bialek tnacych mikrotubule w reorganizacji cytoszkieletu.

ATP-dependent severing activity of microtubule severing proteins leads to the local destabilization of the microtubule structure and causes shortening or disassembly of the existing microtubules or formation of the numerous short microtubule fragments that serve as templates during new microtubule polymerization. Microtubule severing protein-dependent rearrangement of the microtubular cytoskeleton plays an important role in the numerous cellular processes including chromosome segregation during meiosis and mitosis, cells migration, dendrites and axon formation, cilia assembly and arrangement of the cortical microtubules in plant cells.