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Centrosome/Cell cycle uncoupling and elimination in the endoreduplicating intestinal cells of C. elegans.

Lu Y, Roy R - PLoS ONE (2014)

Bottom Line: The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage.Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity.On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Developmental Biology Research Initiative, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

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Phosphorylation of S357 on SPD-2 affects appropriate localization and stability.(A–C) spd-2 (oj29) animals carrying the SPD-2S357E variant were stained with DAPI (red) and anti-SPD-2 (green) in the L1, L1/L2 and L2. The asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (D) SPD-2 staining was monitored in intestinal cells and the percentage of intestinal nuclei that demonstrate nuclear-localized SPD-2 in strains carrying either wild type SPD-2 or SPD-2S357E variant were determined. (E) The frequency of SPD-2 persistence is quantified by counting the number of intestinal cells that continue to show any SPD-2 signal at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (F) Late L2 spd-2 (oj29) animals expressing the SPD-2WT or (G) the SPD-2S357E variant were stained with DAPI (red) and anti-SAS-4 (green). The number of SAS-4 foci were quantified and indicated in (H). The asterisks indicate the intestinal nuclei while arrowheads show SAS-4 foci. Error bar, standard deviation; n = 50; P<0.05 (t-test). Scale bar, 5 µm.
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pone-0110958-g006: Phosphorylation of S357 on SPD-2 affects appropriate localization and stability.(A–C) spd-2 (oj29) animals carrying the SPD-2S357E variant were stained with DAPI (red) and anti-SPD-2 (green) in the L1, L1/L2 and L2. The asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (D) SPD-2 staining was monitored in intestinal cells and the percentage of intestinal nuclei that demonstrate nuclear-localized SPD-2 in strains carrying either wild type SPD-2 or SPD-2S357E variant were determined. (E) The frequency of SPD-2 persistence is quantified by counting the number of intestinal cells that continue to show any SPD-2 signal at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (F) Late L2 spd-2 (oj29) animals expressing the SPD-2WT or (G) the SPD-2S357E variant were stained with DAPI (red) and anti-SAS-4 (green). The number of SAS-4 foci were quantified and indicated in (H). The asterisks indicate the intestinal nuclei while arrowheads show SAS-4 foci. Error bar, standard deviation; n = 50; P<0.05 (t-test). Scale bar, 5 µm.

Mentions: SPD-2 was previously reported to be phosphorylated on a consensus PLK phosphorylation site at Serine S357 [58]. We also identified S357 in our MS/MS analysis following an in vitro kinase assay performed with human PLK-1 and GST::SPD-2, although we were unable to detect the phosphorylation on S357 in vivo (Figure S5A and data not shown). Consistent with a functional role for this site, the phosphomimetic modification of Serine S357 (SPD-2S357E) caused SPD-2 to accumulate in aggregate-like puncta, paralleled by an increased frequency of cells with nuclear-localized SPD-2 prior to the nuclear division (Figure 6A, 6D), whereas the nuclear localization of wild type SPD-2 usually precedes its elimination during the L2 stage (Figure 2D). This SPD-2 accumulation has no apparent effect on centriole function in MTOC, since SPD-2 is normally present on the opposing poles of the condensing nuclei prior to nuclear division (Figure 6B). SPD-2S357E is still detectable in a number of intestinal cells even by the end of L2, long after SPD-2WT normally disappears (Figure 6C, 6E). This persistence of SPD-2S357E was not always reflected by the SAS-4 levels, but nonetheless 14% of the SPD-2S357E variant intestinal cells still possessed SAS-4 foci in the late L2 stage (Figure 6F-6H), suggesting that a portion of the centrioles are stabilized in this variant background. Therefore the phosphomimetic SPD-2S357E modification is sufficient to stabilize some SPD-2, causing it to accumulate in aggregate-like nuclear puncta. The same modification of SPD-2 also delays the elimination of some, but not all centrioles based on the persistence of the centriolar marker SAS-4.


Centrosome/Cell cycle uncoupling and elimination in the endoreduplicating intestinal cells of C. elegans.

Lu Y, Roy R - PLoS ONE (2014)

Phosphorylation of S357 on SPD-2 affects appropriate localization and stability.(A–C) spd-2 (oj29) animals carrying the SPD-2S357E variant were stained with DAPI (red) and anti-SPD-2 (green) in the L1, L1/L2 and L2. The asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (D) SPD-2 staining was monitored in intestinal cells and the percentage of intestinal nuclei that demonstrate nuclear-localized SPD-2 in strains carrying either wild type SPD-2 or SPD-2S357E variant were determined. (E) The frequency of SPD-2 persistence is quantified by counting the number of intestinal cells that continue to show any SPD-2 signal at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (F) Late L2 spd-2 (oj29) animals expressing the SPD-2WT or (G) the SPD-2S357E variant were stained with DAPI (red) and anti-SAS-4 (green). The number of SAS-4 foci were quantified and indicated in (H). The asterisks indicate the intestinal nuclei while arrowheads show SAS-4 foci. Error bar, standard deviation; n = 50; P<0.05 (t-test). Scale bar, 5 µm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215990&req=5

pone-0110958-g006: Phosphorylation of S357 on SPD-2 affects appropriate localization and stability.(A–C) spd-2 (oj29) animals carrying the SPD-2S357E variant were stained with DAPI (red) and anti-SPD-2 (green) in the L1, L1/L2 and L2. The asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (D) SPD-2 staining was monitored in intestinal cells and the percentage of intestinal nuclei that demonstrate nuclear-localized SPD-2 in strains carrying either wild type SPD-2 or SPD-2S357E variant were determined. (E) The frequency of SPD-2 persistence is quantified by counting the number of intestinal cells that continue to show any SPD-2 signal at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (F) Late L2 spd-2 (oj29) animals expressing the SPD-2WT or (G) the SPD-2S357E variant were stained with DAPI (red) and anti-SAS-4 (green). The number of SAS-4 foci were quantified and indicated in (H). The asterisks indicate the intestinal nuclei while arrowheads show SAS-4 foci. Error bar, standard deviation; n = 50; P<0.05 (t-test). Scale bar, 5 µm.
Mentions: SPD-2 was previously reported to be phosphorylated on a consensus PLK phosphorylation site at Serine S357 [58]. We also identified S357 in our MS/MS analysis following an in vitro kinase assay performed with human PLK-1 and GST::SPD-2, although we were unable to detect the phosphorylation on S357 in vivo (Figure S5A and data not shown). Consistent with a functional role for this site, the phosphomimetic modification of Serine S357 (SPD-2S357E) caused SPD-2 to accumulate in aggregate-like puncta, paralleled by an increased frequency of cells with nuclear-localized SPD-2 prior to the nuclear division (Figure 6A, 6D), whereas the nuclear localization of wild type SPD-2 usually precedes its elimination during the L2 stage (Figure 2D). This SPD-2 accumulation has no apparent effect on centriole function in MTOC, since SPD-2 is normally present on the opposing poles of the condensing nuclei prior to nuclear division (Figure 6B). SPD-2S357E is still detectable in a number of intestinal cells even by the end of L2, long after SPD-2WT normally disappears (Figure 6C, 6E). This persistence of SPD-2S357E was not always reflected by the SAS-4 levels, but nonetheless 14% of the SPD-2S357E variant intestinal cells still possessed SAS-4 foci in the late L2 stage (Figure 6F-6H), suggesting that a portion of the centrioles are stabilized in this variant background. Therefore the phosphomimetic SPD-2S357E modification is sufficient to stabilize some SPD-2, causing it to accumulate in aggregate-like nuclear puncta. The same modification of SPD-2 also delays the elimination of some, but not all centrioles based on the persistence of the centriolar marker SAS-4.

Bottom Line: The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage.Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity.On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Developmental Biology Research Initiative, McGill University, Montreal, Quebec, Canada.

ABSTRACT
The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

Show MeSH