Significance and regulation of microtubule-severing in vivo
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Katanin is a microtubule-severing protein complex that oligomerizes on the surface of microtubules and disassembles microtubules by inducing an ATP hydrolysis-dependent conformational change in the polymer lattice. Katanin is conserved from unicellular to multicellular eukaryotic organisms. It is a heterodimer consisting of p60, a severing subunit and p80, a non-catalytic subunit. Spastin is another related microtubule-severing protein involved in human diseases known as spastic paraplegia. It is unknown how katanin and spastin-mediated disassembly of microtubules is regulated in vivo to ensure that specific microtubules undergo severing at correct locations. In addition, the significance of microtubule severing is not well understood. To understand the functional significance of microtubule severing in the context of several types of microtubular organelles, the ciliate Tetrahymena thermophila was used in the present study. We engineered strains of Tetrahymena completely lacking katanin and spastin subunits. We show that katanin is essential for the cytokinesis and assembly of cilia. However, spastin is not essential for cell viability. Absence of katanin-mediated severing activity inhibited the assembly of the central pair and elongation of peripheral doublet microtubules in cilia. Loss of katanin also resulted in excessive polymerization and stability of cortical and cytoplasmic microtubules. We thus show that the function of katanin depends on the microtubule type and subcellular environment. In the cell body, katanin reduces the mass and destabilizes the internal network of microtubules, whereas, it is required for the assembly of central pair as well as doublet microtubules inside the cilia. An overproduction of katanin disassembles ciliary and basal body microtubules in a non-random manner. Katanin preferentially severed polymodified (polyglutamylated and polyglycylated) microtubules in the axoneme, when overproduced. We also show that katanin regulates the levels of post-translation modifications on different types of microtubules. Previously, our laboratory showed that the polymodification sites present in ²-tubulin are required for cytokinesis and assembly of cilia. We now show that the lack of katanin function gives the same phenotype as the one resulting from polymodification sites mutations. In the light of our results, we propose that post-translationally modifications of microtubules act as marks to guide the function of katanin to ensure spatial control of katanin activity.