Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia.
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ABSTRACT:
SUBMITTER: Sun L
PROVIDER: S-EPMC7717695 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Interactions between microtubule (MT) interacting and trafficking (MIT) domains and their binding proteins are important for the accurate progression of many cellular processes that require the AAA+ ATPase machinery. Therefore, knowledge on the structural basis of MIT domain interactions is crucial for understanding the molecular mechanisms underlying AAA+ ATPase function. Katanin is a MT-severing AAA+ ATPase that consists of p60 and p80 subunits. Although, the hexameric p60 subunit is active alone, its association with the p80 subunit greatly enhances both the MT-binding and -severing activities of katanin. However, the molecular mechanism of how the p80 subunit contributes to katanin function is currently unknown. Here, we structurally and functionally characterized the interaction between the two katanin subunits that is mediated by the p60-MIT domain and the p80 C-terminal domain (p80-CTD). We show that p60-MIT and p80-CTD form a tight heterodimeric complex, whose high-resolution structure we determined by X-ray crystallography. Based on the crystal structure, we identified two conserved charged residues that are important for p60-MIT:p80-CTD complex formation and katanin function. Moreover, p60-MIT was compared with other MIT domain structures and similarities are discussed.
| S-EPMC5668312 | biostudies-literature
Project description:BACKGROUND:Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown. METHODS:Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electrophoresis, and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines. RESULTS:In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity. CONCLUSIONS:Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.
| S-EPMC3179562 | biostudies-literature
Project description:ObjectiveThis study aimed to investigate the effect of Katanin p60 on the neurite growth and collateral formation in the hippocampus.MethodsGene cloning was performed to construct the Katanin p60 eukaryotic vector. The microtubule cutting effect and protein expression of Katanin p60 were investigated in 293T cells. Then, these vectors were transfected into hippocampal neurons of rats, and the effects of Katanin p60 on the neurite growth and collateral formation were observed.ResultsIn the present study, we successfully constructed Katanin p60-GFP recombinant plasmids. After transfecting into 293T cells, the Katanin p60 was over-expressed in these cells, the mesh-like structure of microtubules was disrupted, the residual microtubules circled the nucleus, the expression microtubule proteins reduced, and the tapered protrusions disappeared. In hippocampal neurons with Katanin p60 over-expression, the neural neurite growth was obvious, and a lot of dendrites arose from cell bodies. In cells without Katanin p60 expression, the neurites were small, and the number and length of dendrites reduced significantly when compared with Katanin p60 over-expressing cells (P < 0.05). In addition, in Katanin p60 over-expressing cells, the number of collaterals from the neurites and dendrites increased markedly when compared with cells without Katanin p60 expression (P < 0.05).ConclusionKatanin p60 can promote the neurite growth and collateral formation of hippocampal neurons.
| S-EPMC4211748 | biostudies-literature
Project description:The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.
| S-EPMC3841192 | biostudies-literature
Project description:Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific ?-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia.
| S-EPMC5698134 | biostudies-literature
Project description:The in vivo significance of microtubule severing and the mechanisms governing its spatial regulation are not well understood. In Tetrahymena, a cell type with elaborate microtubule arrays, we engineered null mutations in subunits of the microtubule-severing complex, katanin. We show that katanin activity is essential. The net effect of katanin on the polymer mass depends on the microtubule type and location. Although katanin reduces the polymer mass and destabilizes the internal network of microtubules, its activity increases the mass of ciliary microtubules. We also show that katanin reduces the levels of several types of post-translational modifications on tubulin of internal and cortical microtubules. Furthermore, katanin deficiencies phenocopy a mutation of beta-tubulin that prevents deposition of polymodifications (glutamylation and glycylation) on microtubules. We propose that katanin preferentially severs older, post-translationally modified segments of microtubules.
| S-EPMC2064628 | biostudies-literature
Project description:The proper regulation of factors involved in mitosis is crucial to ensure normal cell division. Levels and activities of proteins are regulated in many ways, one of which is ubiquitin-mediated protein degradation. E3 ubiquitin ligases are involved in targeting specific substrates for degradation by facilitating their ubiquitination. In seeking to elucidate additional biological roles for Cul3 we performed a two-hybrid screen and identified Ctb9/KLHDC5 as a Cul3-interacting protein. Overexpression of Ctb9/KLHDC5 resulted in an increase in microtubule density as well as persistent microtubule bridges between post-mitotic cells. Conversely, down-regulation of Ctb9/KLHDC5 showed a pronounced reduction in microtubule density. Based on these observations, we examined the interactions between Cul3, Ctb9/KLHDC5, and the microtubule-severing protein, p60/katanin. Here we show that p60/katanin interacts with a complex consisting of Cul3 and Ctb9/KLHDC5, which results in ubiquitin laddering of p60/katanin. Also, Cul3-deficient cells or Ctb9/KLHDC5-deficient cells show an increase in p60/katanin levels, indicating that Cul3/Ctb9/KLHDC5 is required for efficient p60/katanin removal. We demonstrate a novel regulatory mechanism for p60/katanin that occurs at the level of targeted proteolysis to allow normal mitotic progression in mammalian cells.
| S-EPMC2670170 | biostudies-literature
Project description:BackgroundThis study aimed to assess the correlation of katanin P60 expression with clinical characteristics and survival profiles of surgical non-small cell lung cancer (NSCLC) patients.MethodsTwo hundred and sixty-five primary NSCLC patients treated by surgical resection were retrospectively viewed. The expression of katanin P60 in the tumor specimen was detected by the immunohistochemical (IHC) staining assay. Preoperative clinical data were collected from patients' medical records, and survival data were extracted from follow-up records.ResultsThere were 127 (47.9%) and 138 (52.1%) patients with katanin P60-low expression and -high expression, respectively; in addition, patients presenting katanin P60-high+, -high++, and -high+++ expression were 62 (23.4%), 63 (23.8%), and 13 (4.9%), respectively. Katanin P60 expression was correlated with lymph node (LYN) metastasis and advanced TNM stage but not pathological grade, tumor size, carcinoembryonic antigen (CEA) level or other non-tumor features in NSCLC patients. Regarding survival profiles, disease-free survival (DFS) and overall survival (OS) were both the lowest in katanin P60-high+++ expression patients, followed with katanin P60-high++ patients, katanin P60-high+ patients, and the highest in katanin P60-low expression patients. Further analysis illustrated that katanin P60-high expression was an independent predictive factor for unfavorable DFS and OS in NSCLC patients.ConclusionsKatanin P60 presents potential as a biomarker for lymph node metastasis and prognosis in NSCLC patients.
| S-EPMC7339556 | biostudies-literature
Project description:Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.
| S-EPMC4485387 | biostudies-literature
Project description:Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant katanin p60. Our results demonstrate that targeting an active version of katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.
| S-EPMC7286084 | biostudies-literature