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Coiled-coil domain containing protein 124 is a novel centrosome and midbody protein that interacts with the Ras-guanine nucleotide exchange factor 1B and is involved in cytokinesis.

Telkoparan P, Erkek S, Yaman E, Alotaibi H, Bayık D, Tazebay UH - PLoS ONE (2013)

Bottom Line: Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein.Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected.We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.

ABSTRACT
Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.

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Depletion of Ccdc124 in HeLa cells by RNAi leads to cytokinesis failure.(A) HeLa cells were transfected with either esiRNAs or shRNA vectors (Sh1, Sh2, Sh3) targeting Ccdc124, cell lysates were collected at 48 hrs post-transfection, and immunoblotted with antisera to Ccdc124. Where indicated, Ccdc124 expression vector (CMV-Ccdc124) was cotransfected with gene-specific esiRNAs in order to rescue the cellular effect of Ccdc124 depletion. Scrambled control transfections were indicated (Scr). Calnexin expression was monitored as loading control. (B) Immunostainings of endogenous Ccdc124 in cells transfected with Ccdc124-specific esiRNA, or with scrambled control esiRNA were carried out with anti-Ccdc124 Ab. Costainings with γ-tubulin antisera have indicated subcellular positions of MTOCs (C) Cells described and analyzed in (A) were scored for bi- and multinucleation (n = 5± SD). (D) Representative micrographs of Ccdc124 depleted multinuclear or control esiRNA treated normal dividing cells described in (C). Bars represent 10 µm.
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pone-0069289-g004: Depletion of Ccdc124 in HeLa cells by RNAi leads to cytokinesis failure.(A) HeLa cells were transfected with either esiRNAs or shRNA vectors (Sh1, Sh2, Sh3) targeting Ccdc124, cell lysates were collected at 48 hrs post-transfection, and immunoblotted with antisera to Ccdc124. Where indicated, Ccdc124 expression vector (CMV-Ccdc124) was cotransfected with gene-specific esiRNAs in order to rescue the cellular effect of Ccdc124 depletion. Scrambled control transfections were indicated (Scr). Calnexin expression was monitored as loading control. (B) Immunostainings of endogenous Ccdc124 in cells transfected with Ccdc124-specific esiRNA, or with scrambled control esiRNA were carried out with anti-Ccdc124 Ab. Costainings with γ-tubulin antisera have indicated subcellular positions of MTOCs (C) Cells described and analyzed in (A) were scored for bi- and multinucleation (n = 5± SD). (D) Representative micrographs of Ccdc124 depleted multinuclear or control esiRNA treated normal dividing cells described in (C). Bars represent 10 µm.

Mentions: In order to obtain insight into the biological function of Ccdc124, we assessed the subcellular localization of endogenous Ccdc124 by using generated or commercial anti-Ccdc124 antibodies in cellular immunofluorescence assays. When asynchronusly growing HeLa cells were subjected to a preliminary immunofluorescence analysis by using an anti-mid-Ccdc124 antibody recognizing the central part of the protein (between residues 100–150), subcellular dot-like structures reminiscent of centrosomal staining patterns were detected (results not shown, but please see Fig. 2, below). We then synchronized the cells at G2/M stage of the cell cycle by the MT polymerization inhibitor nocodazole following double-thymidine treatments (Fig. S2, see Methods s1), and followed cell cycle stage-dependent subcellular localization of endogenous Ccdc124 by immunofluorescence assays using Ccdc124 N-terminal epitope (residues 1 to 24), mid-region, or C-terminal epitope (residues 173 to 223)-specific antibodies. Independent of the Ccdc124 antibody used, these studies further indicated centrosome colocalization of Ccdc124 with γ-tubulin at interphase, prophase, metaphase, and anaphase stages, albeit it was relatively diffused to the pericentrosomal region at anaphase (Fig. 2A). Similar results were obtained when subcellular localization of an N-terminus flag-tagged version of Ccdc124 was monitored by immunofluorescence stainings using anti-flag antibodies on cells transfected with the corresponding vector construct (Fig. 2B). Moreover, endogenous centrosome immunostainings with anti-Ccdc124 Abs were very significantly reduced in response to Ccdc124 depletion by esiRNAs targeting its expression (see below, Fig. 4B), further supporting the notion that Ccdc124 is a novel centrosome protein. Centrosome localization of endogenous Ccdc124 was also observed in Retinal Pigment Epithelial cells (RPE1) containing another centrosomal marker, GFP-Centrin [26] (results not shown). Interestingly, at telophase and in cytokinesis Ccdc124 dissociates from centrosomes and relocalizes to the midzone, subsequently accumulating at the midbody at cytokinesis as assessed by its colocalization with the midzone-specific γ-tubulin (Fig. 2A–B, and Fig. 3A, C), or by its positioning at the midbody marked by the empty mid-space in α-tubulin stainings (Fig. 3B). Immunofluorescence studies with peptide competition assays further indicated that the Ccdc124 signal detected at the midbody was specific, as anti-N-ter-Ccdc124 antibodies pre-treated with the epitope peptide failed to recognize Ccdc124 at the midbody (Fig. 3C).


Coiled-coil domain containing protein 124 is a novel centrosome and midbody protein that interacts with the Ras-guanine nucleotide exchange factor 1B and is involved in cytokinesis.

Telkoparan P, Erkek S, Yaman E, Alotaibi H, Bayık D, Tazebay UH - PLoS ONE (2013)

Depletion of Ccdc124 in HeLa cells by RNAi leads to cytokinesis failure.(A) HeLa cells were transfected with either esiRNAs or shRNA vectors (Sh1, Sh2, Sh3) targeting Ccdc124, cell lysates were collected at 48 hrs post-transfection, and immunoblotted with antisera to Ccdc124. Where indicated, Ccdc124 expression vector (CMV-Ccdc124) was cotransfected with gene-specific esiRNAs in order to rescue the cellular effect of Ccdc124 depletion. Scrambled control transfections were indicated (Scr). Calnexin expression was monitored as loading control. (B) Immunostainings of endogenous Ccdc124 in cells transfected with Ccdc124-specific esiRNA, or with scrambled control esiRNA were carried out with anti-Ccdc124 Ab. Costainings with γ-tubulin antisera have indicated subcellular positions of MTOCs (C) Cells described and analyzed in (A) were scored for bi- and multinucleation (n = 5± SD). (D) Representative micrographs of Ccdc124 depleted multinuclear or control esiRNA treated normal dividing cells described in (C). Bars represent 10 µm.
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Related In: Results  -  Collection

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pone-0069289-g004: Depletion of Ccdc124 in HeLa cells by RNAi leads to cytokinesis failure.(A) HeLa cells were transfected with either esiRNAs or shRNA vectors (Sh1, Sh2, Sh3) targeting Ccdc124, cell lysates were collected at 48 hrs post-transfection, and immunoblotted with antisera to Ccdc124. Where indicated, Ccdc124 expression vector (CMV-Ccdc124) was cotransfected with gene-specific esiRNAs in order to rescue the cellular effect of Ccdc124 depletion. Scrambled control transfections were indicated (Scr). Calnexin expression was monitored as loading control. (B) Immunostainings of endogenous Ccdc124 in cells transfected with Ccdc124-specific esiRNA, or with scrambled control esiRNA were carried out with anti-Ccdc124 Ab. Costainings with γ-tubulin antisera have indicated subcellular positions of MTOCs (C) Cells described and analyzed in (A) were scored for bi- and multinucleation (n = 5± SD). (D) Representative micrographs of Ccdc124 depleted multinuclear or control esiRNA treated normal dividing cells described in (C). Bars represent 10 µm.
Mentions: In order to obtain insight into the biological function of Ccdc124, we assessed the subcellular localization of endogenous Ccdc124 by using generated or commercial anti-Ccdc124 antibodies in cellular immunofluorescence assays. When asynchronusly growing HeLa cells were subjected to a preliminary immunofluorescence analysis by using an anti-mid-Ccdc124 antibody recognizing the central part of the protein (between residues 100–150), subcellular dot-like structures reminiscent of centrosomal staining patterns were detected (results not shown, but please see Fig. 2, below). We then synchronized the cells at G2/M stage of the cell cycle by the MT polymerization inhibitor nocodazole following double-thymidine treatments (Fig. S2, see Methods s1), and followed cell cycle stage-dependent subcellular localization of endogenous Ccdc124 by immunofluorescence assays using Ccdc124 N-terminal epitope (residues 1 to 24), mid-region, or C-terminal epitope (residues 173 to 223)-specific antibodies. Independent of the Ccdc124 antibody used, these studies further indicated centrosome colocalization of Ccdc124 with γ-tubulin at interphase, prophase, metaphase, and anaphase stages, albeit it was relatively diffused to the pericentrosomal region at anaphase (Fig. 2A). Similar results were obtained when subcellular localization of an N-terminus flag-tagged version of Ccdc124 was monitored by immunofluorescence stainings using anti-flag antibodies on cells transfected with the corresponding vector construct (Fig. 2B). Moreover, endogenous centrosome immunostainings with anti-Ccdc124 Abs were very significantly reduced in response to Ccdc124 depletion by esiRNAs targeting its expression (see below, Fig. 4B), further supporting the notion that Ccdc124 is a novel centrosome protein. Centrosome localization of endogenous Ccdc124 was also observed in Retinal Pigment Epithelial cells (RPE1) containing another centrosomal marker, GFP-Centrin [26] (results not shown). Interestingly, at telophase and in cytokinesis Ccdc124 dissociates from centrosomes and relocalizes to the midzone, subsequently accumulating at the midbody at cytokinesis as assessed by its colocalization with the midzone-specific γ-tubulin (Fig. 2A–B, and Fig. 3A, C), or by its positioning at the midbody marked by the empty mid-space in α-tubulin stainings (Fig. 3B). Immunofluorescence studies with peptide competition assays further indicated that the Ccdc124 signal detected at the midbody was specific, as anti-N-ter-Ccdc124 antibodies pre-treated with the epitope peptide failed to recognize Ccdc124 at the midbody (Fig. 3C).

Bottom Line: Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein.Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected.We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.

ABSTRACT
Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.

Show MeSH
Related in: MedlinePlus