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RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity.

Shiratsuchi G, Takaoka K, Ashikawa T, Hamada H, Kitagawa D - EMBO J. (2014)

Bottom Line: Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes.Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity.We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles.

View Article: PubMed Central - PubMed

Affiliation: Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima Shizuoka, Japan.

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Ectopic centriolar protein complexes can be the sites of microtubule nucleationA Schematic of microtubule regrowth assay during mitosis in RBM14-depleted U2OS cells.B–E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30–60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).
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fig05: Ectopic centriolar protein complexes can be the sites of microtubule nucleationA Schematic of microtubule regrowth assay during mitosis in RBM14-depleted U2OS cells.B–E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30–60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).

Mentions: Since the ectopic centriolar protein complexes induced by RBM14 depletion could recruit PCM components (Fig 1E), we next tested whether they are potent to nucleate microtubules. Given that formation of the centriolar protein complexes frequently resulted in pseudo-bipolar spindle formation in which an abnormal number of centriolar protein foci clustered into spindle poles, we sought to establish an experimental setup in which it is possible to decluster the centriolar protein complexes and pre-existing centrioles and evaluate their ability to nucleate microtubules in mitosis. To this end, we carried out a microtubule regrowth assay using mitotic U2OS cells (Fig 5A). As expected, in control mitotic cells, we detected efficient microtubule regrowth and subsequent spindle formation within 30 min after cold treatment (Fig 5B). Importantly, we found that, upon RBM14 depletion, the ectopic centriolar protein complexes with PCM cloud formed extra spindle poles, which often induced formation of multipolar spindles at 30 min after cold treatment (Fig 5C–E). 46 ± 5% of the centriolar protein complexes appeared to harbor microtubule nucleation activity in mitosis (n = 150 in triplicate). However, we observed that extra small spindle poles in RBM14-depleted cells tended to be clustered into a pseudo-bipolar spindle assay at 60 min after cold treatment (Fig 5C). Taken together, these results indicate that the ectopic centriolar protein complexes induced by RBM14 depletion are at least in part potent to nucleate microtubules.


RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity.

Shiratsuchi G, Takaoka K, Ashikawa T, Hamada H, Kitagawa D - EMBO J. (2014)

Ectopic centriolar protein complexes can be the sites of microtubule nucleationA Schematic of microtubule regrowth assay during mitosis in RBM14-depleted U2OS cells.B–E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30–60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291483&req=5

fig05: Ectopic centriolar protein complexes can be the sites of microtubule nucleationA Schematic of microtubule regrowth assay during mitosis in RBM14-depleted U2OS cells.B–E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30–60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).
Mentions: Since the ectopic centriolar protein complexes induced by RBM14 depletion could recruit PCM components (Fig 1E), we next tested whether they are potent to nucleate microtubules. Given that formation of the centriolar protein complexes frequently resulted in pseudo-bipolar spindle formation in which an abnormal number of centriolar protein foci clustered into spindle poles, we sought to establish an experimental setup in which it is possible to decluster the centriolar protein complexes and pre-existing centrioles and evaluate their ability to nucleate microtubules in mitosis. To this end, we carried out a microtubule regrowth assay using mitotic U2OS cells (Fig 5A). As expected, in control mitotic cells, we detected efficient microtubule regrowth and subsequent spindle formation within 30 min after cold treatment (Fig 5B). Importantly, we found that, upon RBM14 depletion, the ectopic centriolar protein complexes with PCM cloud formed extra spindle poles, which often induced formation of multipolar spindles at 30 min after cold treatment (Fig 5C–E). 46 ± 5% of the centriolar protein complexes appeared to harbor microtubule nucleation activity in mitosis (n = 150 in triplicate). However, we observed that extra small spindle poles in RBM14-depleted cells tended to be clustered into a pseudo-bipolar spindle assay at 60 min after cold treatment (Fig 5C). Taken together, these results indicate that the ectopic centriolar protein complexes induced by RBM14 depletion are at least in part potent to nucleate microtubules.

Bottom Line: Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes.Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity.We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles.

View Article: PubMed Central - PubMed

Affiliation: Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima Shizuoka, Japan.

Show MeSH
Related in: MedlinePlus