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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 SPD-2 affects numeral regulation of centrioles in the intestinal cells.(A) Diagram of SPD-2 and its potential phosphorylated sites. Numbers represent amino acid position. S, Serine. T, Threonine. Orange numbers: predicted consensus PLK phosphorylation site. Blue numbers: predicted consensus CDK phosphorylation site. Blue or Orange S indicates experimentally-confirmed phosphorylated Serine [58], [78]. (B–D) and (E–G) Early L2 spd-2 (oj29) animals carrying transgenic WT or the S545-variant SPD-2 following the intestinal nuclear division. DAPI (red) and SPD-2 (green) in (B–D) or SAS-4 (green) in (E–G). Asterisks indicate the intestinal nuclei and arrowheads show SPD-2 or SAS-4 foci. The insets show high magnification of the regions within the white rectangles. Scale bar, 5 µm. (H) The frequency of centriole duplication failure is represented by quantifying undivided intestinal nuclei associated with single SPD-2 or SAS-4 foci. (I) The frequency of supernumerary centriole duplication is indicated by the number of divided intestinal nuclei with more than one SPD-2 or SAS-4 focus after the nuclear division. Error bar, standard deviation; n≥75; P<0.05 (t-test). (K) Mass spectrometric analysis of SPD-2. +80 indicates the phosphorylated amino acid and the arrow highlights S545 in red.
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pone-0110958-g005: Phosphorylation of SPD-2 affects numeral regulation of centrioles in the intestinal cells.(A) Diagram of SPD-2 and its potential phosphorylated sites. Numbers represent amino acid position. S, Serine. T, Threonine. Orange numbers: predicted consensus PLK phosphorylation site. Blue numbers: predicted consensus CDK phosphorylation site. Blue or Orange S indicates experimentally-confirmed phosphorylated Serine [58], [78]. (B–D) and (E–G) Early L2 spd-2 (oj29) animals carrying transgenic WT or the S545-variant SPD-2 following the intestinal nuclear division. DAPI (red) and SPD-2 (green) in (B–D) or SAS-4 (green) in (E–G). Asterisks indicate the intestinal nuclei and arrowheads show SPD-2 or SAS-4 foci. The insets show high magnification of the regions within the white rectangles. Scale bar, 5 µm. (H) The frequency of centriole duplication failure is represented by quantifying undivided intestinal nuclei associated with single SPD-2 or SAS-4 foci. (I) The frequency of supernumerary centriole duplication is indicated by the number of divided intestinal nuclei with more than one SPD-2 or SAS-4 focus after the nuclear division. Error bar, standard deviation; n≥75; P<0.05 (t-test). (K) Mass spectrometric analysis of SPD-2. +80 indicates the phosphorylated amino acid and the arrow highlights S545 in red.

Mentions: Using a bioinformatic Group-based Prediction System (GPS) [52]–[53], we identified a number of predicted phosphorylation sites on SPD-2 (Figure 5A). In order to experimentally test whether these amino acids affect centriole fate in the endocycling intestinal cells we mutated each of these sites to either Alanine (A) or Glutamic Acid (E) to convert the wild type SPD-2 sites into non-phosphorylable or phosphomimetic variants, respectively. The variants, or the wild type SPD-2, were integrated as single copies into the genome [54] and the resulting transgenic animals were crossed into the spd-2 (oj29) mutant background [55].


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

Lu Y, Roy R - PLoS ONE (2014)

Phosphorylation of SPD-2 affects numeral regulation of centrioles in the intestinal cells.(A) Diagram of SPD-2 and its potential phosphorylated sites. Numbers represent amino acid position. S, Serine. T, Threonine. Orange numbers: predicted consensus PLK phosphorylation site. Blue numbers: predicted consensus CDK phosphorylation site. Blue or Orange S indicates experimentally-confirmed phosphorylated Serine [58], [78]. (B–D) and (E–G) Early L2 spd-2 (oj29) animals carrying transgenic WT or the S545-variant SPD-2 following the intestinal nuclear division. DAPI (red) and SPD-2 (green) in (B–D) or SAS-4 (green) in (E–G). Asterisks indicate the intestinal nuclei and arrowheads show SPD-2 or SAS-4 foci. The insets show high magnification of the regions within the white rectangles. Scale bar, 5 µm. (H) The frequency of centriole duplication failure is represented by quantifying undivided intestinal nuclei associated with single SPD-2 or SAS-4 foci. (I) The frequency of supernumerary centriole duplication is indicated by the number of divided intestinal nuclei with more than one SPD-2 or SAS-4 focus after the nuclear division. Error bar, standard deviation; n≥75; P<0.05 (t-test). (K) Mass spectrometric analysis of SPD-2. +80 indicates the phosphorylated amino acid and the arrow highlights S545 in red.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110958-g005: Phosphorylation of SPD-2 affects numeral regulation of centrioles in the intestinal cells.(A) Diagram of SPD-2 and its potential phosphorylated sites. Numbers represent amino acid position. S, Serine. T, Threonine. Orange numbers: predicted consensus PLK phosphorylation site. Blue numbers: predicted consensus CDK phosphorylation site. Blue or Orange S indicates experimentally-confirmed phosphorylated Serine [58], [78]. (B–D) and (E–G) Early L2 spd-2 (oj29) animals carrying transgenic WT or the S545-variant SPD-2 following the intestinal nuclear division. DAPI (red) and SPD-2 (green) in (B–D) or SAS-4 (green) in (E–G). Asterisks indicate the intestinal nuclei and arrowheads show SPD-2 or SAS-4 foci. The insets show high magnification of the regions within the white rectangles. Scale bar, 5 µm. (H) The frequency of centriole duplication failure is represented by quantifying undivided intestinal nuclei associated with single SPD-2 or SAS-4 foci. (I) The frequency of supernumerary centriole duplication is indicated by the number of divided intestinal nuclei with more than one SPD-2 or SAS-4 focus after the nuclear division. Error bar, standard deviation; n≥75; P<0.05 (t-test). (K) Mass spectrometric analysis of SPD-2. +80 indicates the phosphorylated amino acid and the arrow highlights S545 in red.
Mentions: Using a bioinformatic Group-based Prediction System (GPS) [52]–[53], we identified a number of predicted phosphorylation sites on SPD-2 (Figure 5A). In order to experimentally test whether these amino acids affect centriole fate in the endocycling intestinal cells we mutated each of these sites to either Alanine (A) or Glutamic Acid (E) to convert the wild type SPD-2 sites into non-phosphorylable or phosphomimetic variants, respectively. The variants, or the wild type SPD-2, were integrated as single copies into the genome [54] and the resulting transgenic animals were crossed into the spd-2 (oj29) mutant background [55].

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