Regulation of katanin activity in the ciliate Tetrahymena thermophila

Abstract

SummaryKatanin 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.

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

“…Such constructs enable expression/overexpression of γ‐tubulin as C‐terminally HA‐tagged fusions under the control of MTT1 promoter (Shang, Song et al, ) in the non‐essential BTU1 locus (Gaertig, Gao, Tishgarten, Clark, & Dickerson, ). To express GTU1‐HAp from its native locus, the pMTT1‐GTU1‐HA plasmid was digested with MluI and EcoRV restriction enzymes, and a fragment containing GTU1 ‐HA gene and BTU1 terminator was inserted into pNeo4‐KAT3‐3HA plasmid (Waclawek, Joachimiak, Hall, Fabczak, & Wloga, ). Next, the 3′UTR of GTU1 was amplified with primers adding the PstI and XhoI restriction sites (Supplementary Table S1), digested and inserted into the plasmid constructed in the previous step.…”

“…Such (Shang, Song et al, 2002) in the non-essential BTU1 locus (Gaertig, Gao, Tishgarten, Clark, & Dickerson, 1999). To express GTU1-HAp from its native locus, the pMTT1-GTU1-HA plasmid was digested with MluI and EcoRV restriction enzymes, and a fragment containing GTU1-HA gene and BTU1 terminator was inserted into pNeo4-KAT3-3HA plasmid (Waclawek, Joachimiak, Hall, Fabczak, & Wloga, 2017 For overexpression, transgenes were introduced into the paclitaxel-sensitive CU522 cells (Gaertig et al, 1999). Paclitaxel-resistant clones were isolated after 3-4 days of selection on SPP medium supplied with 20 μM paclitaxel (BioShop, Burlington, Canada).…”

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

“…To express C-terminally 3HA-tagged Kat2 in the native locus, 2.2 kb of the coding region and a 1.7 kb fragment of the 3 UTR of KAT2 were amplified and cloned into the appropriate plasmid, as previously described [27]. About 10 µg of the final plasmid was digested with MluI and XhoI and used for biolistic transformation.…”

“…For immunofluorescence analyses, cells were handled as drops on coverslips and fixed as previously described [27,28]. The primary antibodies were used as follows: monoclonal mouse anti-HA.…”

“…The primary antibodies were used as follows: monoclonal mouse anti-HA. 11 To analyze Kat2-HA localization at the ultrastructural level, the Kat2-HA-overexpressing cells were induced with 1 µg/mL CdCl 2 for 3 h and processed for immunoanalysis with anti-HA monoclonal antibodies using either classical TEM or cryofixation methods, as previously described [27]. All samples were analyzed using a JEM 1200 EX transmission electron microscope (JEOL Co, Tokyo, Japan).…”