A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells

Abstract

Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KAT-NAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

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

“…In murine fibroblasts (NIH3T3), kidney epithelial cells (mIMCD3), and HeLa cells, Katnal2 localizes within the cytoplasm, where it partly overlaps with microtubules of the interphase network, mitotic spindle, midbody, and centrioles. Additionally, Katnal2 was detected in primary cilia along their entire length [5,6]. Similar Katnal2 localization was observed in Xenopus XL177 cells assembling primary cilia [7].…”

“…The overexpression of human GFP-Katnal2 in HeLa cells did not change the microtubule signal, suggesting that Katnal2 does not sever microtubules [6]. On the other hand, in mammalian cells with depleted Katnal2, tubulin acetylation was elevated, suggesting the increased longevity of microtubules [5]. However, in Tetrahymena cells lacking Kat2-an ortholog of Katnal2-hyperacetylated microtubules were not observed and the phenotype of the knockout cells was not detectably altered [4].…”

“…In the unicellular parasites Trypanosoma brucei and Leishmania major, ectopically expressed Katnal2 localizes to flagella, especially at the base and the tip, and its expression reduces the length of flagella [8]. The ShRNA-induced silencing of Katnal2 in mammalian cells results in the formation of additional centrioles, a multipolar mitotic spindle, defects in cytokinesis, and reduced ciliogenesis [5]. The assembly of fewer and shorter cilia was also observed in multiciliated cells of Xenopus embryonic epidermis with depleted Katnal2 [7].…”

SPEF2 functions in microtubule-mediated transport in elongating spermatids to ensure proper male germ cell differentiation

“…However, the overall structure of the basal body appeared normal. Interestingly, in vitro studies have shown that depletion of the p80 or p60 katanin subunit also causes centriole duplication (Hu et al, 2014;Ververis et al, 2016). The mechanism via which SPEF2 or katanin is involved in centriole duplication remains unknown.…”

Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines

“…The katanin p80 B1-like protein displays sequence similarity to katanin p80, but lacks the WD40 repeats 17 . Katanin-like 1 and 2 are distinct proteins that are closely related to katanin p60 but as yet have not been extensively characterized 18; 19; 20 .…”