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Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division.

Müller-Taubenberger A, Kastner PM, Schleicher M, Bolourani P, Weeks G - BMC Cell Biol. (2014)

Bottom Line: A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain.In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig Maximilian University of Munich, Schillerstr, 42, 80336 Munich, Germany. amueller@lrz.uni-muenchen.de.

ABSTRACT

Background: Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans.

Results: In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.

Conclusions: Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC- cells, RasG- cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.

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Cells lacking NdrC show centrosomal aberrations. A. In comparison to wild-type, B, C multinucleated ndrC- cells frequently show supernumerous centrosomes. D. Histogram depicting the percentage of normal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1) centrosomes in wild-type and ndrC- cells. E, F. Visualization of mitotic spindles in fixed ndrC- cells by immunolabeling with anti-α-tubulin antibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs synchronously in multinucleate cells (E), as well as in multinucleate cells with supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B), (C), (E), and (F).
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Figure 3: Cells lacking NdrC show centrosomal aberrations. A. In comparison to wild-type, B, C multinucleated ndrC- cells frequently show supernumerous centrosomes. D. Histogram depicting the percentage of normal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1) centrosomes in wild-type and ndrC- cells. E, F. Visualization of mitotic spindles in fixed ndrC- cells by immunolabeling with anti-α-tubulin antibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs synchronously in multinucleate cells (E), as well as in multinucleate cells with supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B), (C), (E), and (F).

Mentions: Inspection of multinucleated ndrC- cells, fixed and stained with TO-PRO-3 for visualization of nuclei and immunostained with anti-α-tubulin antibodies to visualize microtubules and centrosomes, revealed a high percentage of mutant cells carrying increased numbers of centrosomes (Figure 3). Frequently, the supernumerous centrosomes were not connected to the nuclei (Figure 3B). To quantify the disruption of centrosome integrity, the ratio of centrosomes visualized by anti-α-tubulin staining to nuclei visualized by DAPI staining, was determined for wild-type and ndrC- cells (Figure 3D). About one third (31%) of the ndrC- cells had more than one centrosome per nucleus, compared to only 0.6% in wild-type cells. ndrC- cells with evenly distributed centrosomes underwent mitosis in a synchronized manner (concerted mitosis) (Figure 3E), but although the increased ratio of centrosomes per nuclei did not affect the ability of cells to perform concerted mitosis, the unattached surplus centrosomes were not capable of forming mitotic spindles (Figure 3F).


Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division.

Müller-Taubenberger A, Kastner PM, Schleicher M, Bolourani P, Weeks G - BMC Cell Biol. (2014)

Cells lacking NdrC show centrosomal aberrations. A. In comparison to wild-type, B, C multinucleated ndrC- cells frequently show supernumerous centrosomes. D. Histogram depicting the percentage of normal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1) centrosomes in wild-type and ndrC- cells. E, F. Visualization of mitotic spindles in fixed ndrC- cells by immunolabeling with anti-α-tubulin antibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs synchronously in multinucleate cells (E), as well as in multinucleate cells with supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B), (C), (E), and (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4120859&req=5

Figure 3: Cells lacking NdrC show centrosomal aberrations. A. In comparison to wild-type, B, C multinucleated ndrC- cells frequently show supernumerous centrosomes. D. Histogram depicting the percentage of normal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1) centrosomes in wild-type and ndrC- cells. E, F. Visualization of mitotic spindles in fixed ndrC- cells by immunolabeling with anti-α-tubulin antibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs synchronously in multinucleate cells (E), as well as in multinucleate cells with supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B), (C), (E), and (F).
Mentions: Inspection of multinucleated ndrC- cells, fixed and stained with TO-PRO-3 for visualization of nuclei and immunostained with anti-α-tubulin antibodies to visualize microtubules and centrosomes, revealed a high percentage of mutant cells carrying increased numbers of centrosomes (Figure 3). Frequently, the supernumerous centrosomes were not connected to the nuclei (Figure 3B). To quantify the disruption of centrosome integrity, the ratio of centrosomes visualized by anti-α-tubulin staining to nuclei visualized by DAPI staining, was determined for wild-type and ndrC- cells (Figure 3D). About one third (31%) of the ndrC- cells had more than one centrosome per nucleus, compared to only 0.6% in wild-type cells. ndrC- cells with evenly distributed centrosomes underwent mitosis in a synchronized manner (concerted mitosis) (Figure 3E), but although the increased ratio of centrosomes per nuclei did not affect the ability of cells to perform concerted mitosis, the unattached surplus centrosomes were not capable of forming mitotic spindles (Figure 3F).

Bottom Line: A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain.In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig Maximilian University of Munich, Schillerstr, 42, 80336 Munich, Germany. amueller@lrz.uni-muenchen.de.

ABSTRACT

Background: Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans.

Results: In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.

Conclusions: Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC- cells, RasG- cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.

Show MeSH
Related in: MedlinePlus