Final Report Summary - CP ASSEMBLY IN CILIA (Role of the tubulin posttranslational modifications and microtubule severing protein, katanin in the cilia central pair assembly.)
The motile cilia scaffold is composed of 9 doublets of peripheral microtubules and two central microtubules. The assembly of central pair microtubules is abolished in cells with mutation or deletion of microtubule severing protein katanin, either p60 catalytic subunit or p80 regulatory subunit. Similar, assembly of central pair microtubules is affected in cells with modified levels of some tubulin posttranslational modifications or with tubulin mutations affecting the negative charge of tubulin tail.
We analyzed p60 and p80 katanin subunits in Tetrahymena cells and showed that molecular mechanisms that regulate katanin level and activity are evolutionary conserved. The domain analysis indicated that the N-terminal fragment of Tetrahymena p60 ortholog, KAT1p is sufficient to associate with microtubules and that this association is enhanced if both katanin subunit are expressed. Interestingly, the prolonged overexpression of p80 Tetrahymena ortholog, KAT3p results in inhibition of cytokinesis leading to formation of multinuclear monster cells similar as in KAT1 knockouts. Further analysis indicated that expression of only KAT3p fragment containing WD40 motifs is sufficient to phenocopy KAT1-KO.
In mammalian cells katanin preferentially severs acetylated microtubules. The in vitro assay showed that ciliary microtubules isolated from alpha tubulin acetyltransferase overexpressing cells are slightly faster fragmented than from wild type cells. The elevated level of tubulin acetylation has no effect on katanin localization. However, when katanin subunits were expressed in cells with hyperglutamylated microtubules, the KAT1p (but not KAT3p) was mislocalized and depleted from cilia and basal bodies.
We also found the evidence that in Tetrahymena cells the level of katanin subunits is regulated by 26S-dependent protein degradation.
Because some of ours initial hypothesis were negatively verified, we extended analysis and investigated the role of katanin p60 homolog, KATNAL2. Analyses justified by our preliminary data showing that in Tetrahymena cells KATNAL2 ortholog, KAT2p was present in cilia and co-localized not only with peripheral microtubule doublets but also with central pair microtubules. Thus we hoped to shed more light on the mechanisms that regulate central pair microtubules assembly.
The analysis of the role of katanin and tubulin PTMs in CP assembly is one of two cilia-oriented
scientific projects explored by my research group established within the Laboratory of Cell Movements Physiology, in the Nencki Institute of Experimental Biology, Warsaw, Poland. During MC IRG implementation I obtained next scientific degree, habilitation (2013) in open International Competition I was selected a new Lab Leader in the Nencki Institute and beginning July 1st, 2015, I established the Laboratory of Cytoskeleton and Cilia Biology.