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The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis

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Abstract

Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They have been implicated, together with their interacting minus-end directed motor protein KIFC5A, in the regulation of centriole duplication in mammalian cells. Here we show that Nubp1 and Nubp2 are integral components of centrioles throughout the cell cycle, recruited independently of KIFC5A. We further demonstrate their localization at the basal body of the primary cilium in quiescent vertebrate cells or invertebrate sensory cilia, as well as in the motile cilia of mouse cells and in the flagella of Chlamydomonas. RNAi-mediated silencing of nubp-1 in C. elegans causes the formation of morphologically aberrant and additional cilia in sensory neurons. Correspondingly, downregulation of Nubp1 or Nubp2 in mouse quiescent NIH 3T3 cells markedly increases the number of ciliated cells, while knockdown of KIFC5A dramatically reduces ciliogenesis. Simultaneous double silencing of Nubp1 + KIFC5A restores the percentage of ciliated cells to control levels. We document the normal ciliary recruitment, during these silencing regimes, of basal body proteins critical for ciliogenesis, namely CP110, CEP290, cenexin, Chibby, AurA, Rab8, and BBS7. Interestingly, we uncover novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which we find enriched at the basal body and recruited independently of the Nubps or KIFC5A. Our combined results for Nubp1, Nubp2, and KIFC5A and their striking effects on cilium formation suggest a central regulatory role for these proteins, likely involving CCT/TRiC chaperone activity, in ciliogenesis.

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Acknowledgments

We warmly thank our colleagues for kindly providing antibodies: Profs. Ryoko Kuriyama (University of Minnesota), Anand Swaroop (NIH, Bethesda), Isabelle Vernos (CRG, Barcelona), Johan Peränen (University of Helsinki), Joel Rosenbaum (Yale University) and Elizabeth Smith (Dartmouth College, NH). We are indebted to Ioanna Georgiou and Maria Christoforou (University of Cyprus) for cell-cycle analysis of Nubp1 and to Elena Panayiotou-Worth (Cyprus Institute of Neurology and Genetics) for providing us with mouse tracheal sections. This work was funded by the Research Promotion Foundation of Cyprus and Structural Funds from the EU (grant DIDAKTOR/DISEK/0308-05 to N.S.) and the Danish Natural Science Research Council (grants 09-070398 and 10-085373 to L.B.P.). E.K. received a FEBS short-term fellowship to visit the laboratory of N.T. for work with C. elegans.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Cyprus, University Avenue 1, 1678, Nicosia, Cyprus

    Elena Kypri, Andri Christodoulou, Giannis Maimaris, Costas Lysandrou & Niovi Santama

  2. Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Mette Lethan & Lotte B. Pedersen

  3. Institute of Molecular Biology and Biotechnology, Crete, Greece

    Maria Markaki & Nektarios Tavernarakis

  4. Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

    Carsten W. Lederer

  5. University of Bayreuth, Bayreuth, Germany

    Stefan Geimer

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  1. Elena Kypri
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  2. Andri Christodoulou
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Correspondence to Niovi Santama.

Additional information

E. Kypri, A. Christodoulou, and G. Maimaris contributed equally to the experimental work.

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Supplementary material 1 Detection of Nubp1 and Nubp2 in mouse NIH 3T3 cells. A–B. WB analysis of total protein extracts from NIH 3T3 fibroblasts, using affinity-purified rabbit anti-Nubp1 ab (A) and rat anti-Nubp2 ab (B), reveals unique bands consistent with the predicted Mr for Nubp1 (34.085 × 103) and Nubp2 (29.518 × 103). (EPS 1040 kb)

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Supplementary material 2 A. Localization of Nubp2 during the sub-phases of the mitosis in NIH 3T3 fibroblasts. NIH 3T3 cells were labeled with an ab against α-tubulin (red) and an ab against Nubp2 (green). DNA was visualized with Hoechst (blue). Scale bars 10 μm. B. Nubp1 and Nubp2 are co-localized. Example of co-localization of Nubp2 (red) and Nubp1 (green) in prometaphase. DNA was visualized with Hoechst (blue). Scale bars 10 μm. (EPS 17527 kb)

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Supplementary material 3 Nubp1 and Nubp2 localize at centrioles even after MT depolymerization, indicating that they are integral centriole components in NIH 3T3 cells. A1–A4. Double labeling with anti-α tubulin (red) and anti-Nubp1 (green), and counterstaining for DNA (blue), in untreated cells, serving as control samples. B1–B4. Equivalent panels following depolymerization of MT with nocodazole treatment. Efficient depolymerization is indicated by absence of MTs emanating from centrioles, where Nubp1 labeling is maintained. C1–C4. Control samples with double labeling for α-tubulin (red) and anti-Nubp2 (green), and counterstaining for DNA (blue). D1–D4. Nocodazole-treated equivalent samples, showing Nubp2 labeling in centrioles after MT depolymerization. Scale bars 10 μm. (EPS 10699 kb)

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Supplementary material 4 Nubp1 is localized in the axonemes of motile cilia and their basal bodies by immunofluorescence in mouse trachea sections. A1–A2. Hematoxylin/eosin-stained tracheal cross sections from adult mouse at low magnification (A1), showing its characteristic tissue architecture, and detail at higher magnification (A2) revealing the ciliated pseudostratified columnar tracheal epithelium, which was immunolabeled in panels B1–C3. The detail in panel A2 represents the area demarcated with a square and pointed at with an arrow in panel A1. The layer of motile cilia is indicated with black arrows in A2. B1–B3. Immunostaining of tracheal sections, similar to those in A2, using abs against acetylated-tubulin (red, revealing intense labeling of the axonemes in the arrayed motile cilia; B1), Nubp1 (green, revealing a characteristic thin line formed by the consecutive labeled basal bodies as well their corresponding labeled axonemes) and counterstaining for DNA (blue; B2). The overlay image is shown in B3. C1–C3. Equivalent magnified details from images B1–B3, corresponding to the area demarcated in the dotted square in panel B2. The line of labeled basal bodies is indicated by white arrowheads and labeled axonemes by yellow arrowheads. Abbreviations: bronch. bronchiole; bb basal bodies. Scale bars 250 μm (panel A1), 5 μm (panels A2, C1–C3), 10 μm (panels B1–B3). (EPS 18458 kb)

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Supplementary material 5 The localization of Nubp1, Nubp2, and KIFC5A to centrioles and basal bodies is not interdependent in NIH 3T3 cells. A1–B3. Double labeling of KIFC5A-silenced cells for γ-tubulin as a centriolar marker (red) and Nubp1 (green) and counterstaining for DNA (blue) in mitotic cells with multi-polar spindles (panels A1–A2) or in interphase cells with amplified centrioles (panels B1–B3) show correct recruitment of Nubp1 in centrioles in the absence of KIFC5A. C1–D3. Equivalent samples as for A1-B3 to show correct Nubp2 localization to centrioles in the absence of KIFC5A. E1–E2. Double labeling of a mitotic Nubp1-silenced cell for γ-tubulin (red) and Nubp1 (green) and counterstaining for DNA (blue) shows normal Nubp2 recruitment to centrioles. F1–F3. Correspondingly, double labeling for acetylated tubulin as an axonemal ciliary marker (red), Nubp1 (green) and DNA (blue) reveals localization of Nubp2 at the basal body in a Nubp1-silenced ciliated cell. All scale bars 10 μm; scale bar in panel F3 is 2.5 μm. (EPS 13506 kb)

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Supplementary material 6 Nubp1 is nuclear in interphase, enriched in centrioles throughout the cell cycle, including the mitotic subphases, and localizes to the basal body of the sensory cilium in Xenopus XL177 cells. XL177 cells were labeled with an ab against α-tubulin (red) and an ab against Nubp1 (green). DNA was visualized with Hoechst (blue). (Last row): In serum-deprived XL177 cells, labeled with abs to acetylated tubulin to mark the ciliary axoneme (red), Nubp1 (green), and counterstained for DNA (blue), Nubp1 is detected at the basal body and accompanying daughter centriole. Scale bars 10 μm. (EPS 16450 kb)

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Supplementary material 7 A combined silencing and induction of ciliogenesis protocol in NIH 3T3 cells. A. A combined silencing and induction of ciliogenesis protocol allowing the comparative study, in the absence of the depleted protein(s) of interest, of centriole dynamics and ciliogenesis in cycling cells (96 h) and growth-arrested cells (120 h). B1–C2. Control-silenced cells at 96 h (B1, B2) are cycling, as indicated by proliferation marker Ki67 (green) and DNA counterstaining (blue), but are quiescent at 120 h, as evidenced by the lack of Ki-67 signal (C1, C2). Images B1 and C1 were acquired with the same exposure (149.65 ms). D. A random panel of control-silenced cells, by way of an example, labeled for acetylated tubulin (red) and counterstained for DNA (blue) to confirm ciliogenesis, following serum deprivation. In addition to the primary cilium axoneme, acetylated tubulin-containing MTs are visible in some cells, the presence of which is well documented in current literature (reviewed by [102]; [103]). Scale bars 10 μm. (EPS 6638 kb)

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Supplementary material 8 Centriole arithmetics and number of nuclei per NIH 3T3 cell at 96 and 120 h in different silencing regimes. A1–C1. Quantification of the number of centrioles per cell in KIFC5A-, Nubp1-, or Nubp2-silenced populations, compared with corresponding control-silenced populations, at 96 h (growth conditions) and after induction of ciliogenesis by serum deprivation at 120 h (cell cycle arrest). A2–C2. Quantification of the number of nuclei per cell in exactly the same silenced populations as above (in corresponding pairs). In panels A1–C2, the averages from four independent experiments are shown and bars represent standard deviation (STD) values. Differences are significant (*), highly significant (**) or extremely significant (***), as indicated, according to two-tailed t test analysis. Abbreviations: Cont.: control silencing; K: KIFC5A-; N1: Nubp1-; N2: Nubp2-silencing. (EPS 581 kb)

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Supplementary material 9 Centriole arithmetics and number of nuclei in NIH 3T3 cells at 96 and 120 h in double KIFC5A + Nubp1 silencing. A1–A2. Quantification of the number of centrioles per cell (panel A1) or the number of nuclei per cell (panel A2) in doubly silenced KIFC5A + Nubp1 populations, compared with corresponding control-silenced populations, at 96 h (growth conditions) and after induction of ciliogenesis by serum deprivation at 120 h (cell cycle arrest). Shown are the averages from four independent experiments and bars represent standard deviation (STD) values. Differences are significant (*), highly significant (**) or extremely significant (***), as indicated, according to two-tailed t test analysis. The high incident of multinucleated cells observed for single KIFC5A or Nubp1 silencing was strongly maintained in double-silenced cells at 120 h (13.43 ± 4.55 % vs. 1.15 ± 1.12 %) and was statistically highly and extremely significant in 96 h (p = 0.0068) and 120 h (p = 0.00088) populations (panel A2). The average number of centrioles per cell was comparable in silenced and control populations (panel A1). Abbreviations: Cont.: control silencing; K + N1: KIFC5A + Nubp1 double silencing. (EPS 490 kb)

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Supplementary material 10 Protein levels for Nubp1 and Nubp2 are stable throughout the cell cycle in NIH 3T3 cells. A1–C1. Quantification of dimethyl-phospho H3 (panel A1), Nubp1 (panel B1) and Nubp2 protein expression (panel C1) in G1/S synchronized cultures. Values represent average normalized values (ratio of test protein expression to actin expression) from three independent experiments and bars represent standard deviation (STD) values. Differences are significant (*), highly significant (**) or extremely significant (***), as indicated, according to statistical evaluation by one-way ANOVA with Tukey’s test. A2–C3. Corresponding indicative WBs on which this analysis was based. Samples were collected as indicated between 0–14 h, following release from a double thymidine block. The distribution of signal for dimethyl-phospho H3 confirms efficient synchronization and the peak signal at 7 h indicates the timing of mitosis. (EPS 8285 kb)

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Supplementary material 11 Nubp1 and Nubp2 protein levels are downregulated upon serum withdrawal in NIH 3T3 cells. A1–B1. Quantification of Nubp1 (panel A1) or Nubp2 (panel B1) protein expression during a time course of serum deprivation in cultured cells. Samples were of cycling cells just prior to serum withdrawal (time point 1), at 12 h after serum withdrawal (point 2), at 24 h after serum withdrawal (point 3) and at 12 h after serum re-introduction (point 4). Values represent average normalized values (ratio of Nubp1 or Nubp2 expression to actin expression) from three independent experiments and bars represent standard deviation (STD) values. Differences were statistically evaluated, as indicated, by one-way ANOVA with Tukey’s test. The statistically significant (*) or highly significant (**) reduction of Nubp1 protein levels during cell cycle exit were down to 62.12 % and the extremely significant (***) reduction of Nubp2 protein levels down to 54.09 % of initial levels in cycling conditions. A2–B2. Corresponding indicative Western blots on which this analysis was based. (EPS 3106 kb)

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Supplementary material 12 Mother centriole-specific protein cenexin is localized in amplified centrioles of silenced NIH 3T3 cells and at the basal body of the cilium under the different silencing regimes. A–C. Silencing at 96 h (cycling cells). Double staining for γ-tubulin as a marker for centrioles (red) and for cenexin (green) and counterstaining for DNA (blue) in control-silenced cells (A), Nubp1- (B), and KIFC5A-silenced cells (C). In control-silenced cells, cenexin is detected in only one of the two centrioles: in the images the mother centriole is indicated with a yellow arrowhead and the daughter centriole with a red arrowhead. In silenced cells, most or all amplified centrioles are cenexin-positive. D–F. Silencing at 120 h (serum-deprived cells). Panels with double staining for acetylated-tubulin as a marker for the ciliary axoneme (red) and for cenexin (green) and counterstaining for DNA (blue) in control-silenced cells (D), Nubp1- (E), and Nubp2-silenced cells (F). Scale bars 10 μm; details in the accompanying small images are shown at double the magnification of their corresponding main image. (EPS 9651 kb)

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Supplementary material 13 CP110 and CEP290 are normally recruited to the centrosome and basal body under the different silencing regimes in NIH 3T3 cells. A1–A4. Silencing at 96 h (cycling cells). Double staining for γ-tubulin as a marker for centrioles (red) and for CP110 (green) and counterstaining for DNA (blue) in control-silenced cells (A1), Nubp1- (A2), Nubp2- (A3), and KIFC5A-silenced cells (A4). B1–B4. Silencing at 120 h (serum-deprived cells). Equivalent panels with double staining of silenced cells for acetylated-tubulin as a marker for the ciliary axoneme (red) and for CP110 (green) and counterstaining for DNA (blue). C1–C3. Silencing at 96 h (cycling cells). Double staining for γ-tubulin (red) and for CEP290 (green) and counterstaining for DNA (blue) in control-silenced cells (C1), Nubp1- (C2), and KIFC5A-silenced cells (C3). D1–D3. Silencing at 120 h (serum-deprived cells). Equivalent panels with double staining of silenced cells for acetylated-tubulin (red) and for CEP290 (green) and counterstaining for DNA (blue). Scale bars 10 μm; details in the accompanying small images are shown at double the magnification of their corresponding main image. (EPS 22850 kb)

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Supplementary material 14 Localization of Aurora A kinase (AurA) and the GTPase Rab8 in centrioles and basal body of the cilium under the different silencing regimes in NIH 3T3 cells. A1–C3. Silencing at 96 h (cycling cells). Double staining for α-tubulin as a marker for MTs in normal and multi-polar spindles (red) and for AurA (green) and counterstaining for DNA (blue) in control-silenced cells (A1–A3), Nubp1- (B1–B3), and KIFC5A-silenced cells (C1-C3). D–H. Silencing at 120 h (serum-deprived cells). Panels with double staining for acetylated-tubulin as a marker for the ciliary axoneme (red) and for Rab8 (green) in control-silenced cells (D), Nubp1- (E), Nubp2- (F), KIFC5A- (G) and double KIFC5A + Nubp1-silenced cells (H). Scale bars 10 μm; details in the accompanying small images are shown at double the magnification of their corresponding main image. (EPS 11860 kb)

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Supplementary material 15 Chaperones CCT8 and CCT4, and BBSome protein BBS7 are normally recruited to the basal body under the different silencing regimes in NIH 3T3 cells. A–E. Silencing at 120 h (serum-deprived cells). Panels with double staining for acetylated-tubulin as a marker for the ciliary axoneme (red), for CCT8 (green) and counterstaining for DNA (blue) in control-silenced cells (A), Nubp1- (B), Nubp2- (C), KIFC5A- (D) and double KIFC5A + Nubp1-silenced cells (E). F–G. Labeling for acetylated-tubulin (red), CCT4 (green) and counterstaining for DNA (blue) in control- (F), and Nubp1- silenced cells (G) at 120 h. H–K. Different silencing regimes, as indicated, at 120 h. Labeling for acetylated-tubulin (red), and BBS7 (green). Scale bars 10 μm; details in the accompanying small images are shown at double the magnification of their corresponding main image. (EPS 26062 kb)

Supplementary material 16 List of additional primary and secondary antibodies used in this work. (DOCX 110 kb)

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Supplementary material 17 Identification of proteins contained in Nubp1 immunoprecipitates (bands 1-7 in Fig. 8) by LC–MS/MS. (DOC 63 kb)

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Supplementary material 18 Summary of effects of different silencing regimes on ciliogenesis and centriole arithmetics, as presented in this work or by Christodoulou et al. 2006 [49]. The descriptions used in each case refer to statistically significant changes only (otherwise the term “unchanged” is used). (DOCX 41 kb)

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Kypri, E., Christodoulou, A., Maimaris, G. et al. The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis. Cell. Mol. Life Sci. 71, 517–538 (2014). https://doi.org/10.1007/s00018-013-1401-6

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