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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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lin-35/Rb mutants undergo additional rounds of centriole duplication.(A and B) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SPD-2 (green) to monitor centriole dynamics at the nuclear division. The panel (A) was obtained by staining animals approximately one hour after the nuclear division (t = 1 h), while (B) was acquired two hours after the division (t = 2 h). Asterisks indicate the intestinal nuclei and the arrowheads indicate SPD-2 foci. (C and D) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SAS-4 (green) to monitor centriole numbers after the nuclear division. The insets in (A–D) represent magnified views of regions highlighted by the white rectangles. Scale bar, 5 µm. (E) Quantification of SPD-2 or SAS-4 foci in intestinal nuclei in both wild type and lin-35/Rb mutants two hours after the first intestinal nuclear division. n = 75. (F) RT-PCR analysis of cell-specific transcripts from N2 and lin-35 (n745). elt-2 is intestinal specific, while htp-3 is expressed exclusively in the germ line. (G) The expression of spd-2, -5, zyg-1, -9, sas-4, -5, -6, tbg-1 and dlg-1 (control) [77] was quantified using RT-PCR from total or intestine-enriched mRNA from wild type (N2) or from lin-35 (n745) larvae before or after the first nuclear division, and 6–8 hours after the last nuclear division in lin-35 (n745) mutants. int., intestinal. bp, base pair.
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pone-0110958-g004: lin-35/Rb mutants undergo additional rounds of centriole duplication.(A and B) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SPD-2 (green) to monitor centriole dynamics at the nuclear division. The panel (A) was obtained by staining animals approximately one hour after the nuclear division (t = 1 h), while (B) was acquired two hours after the division (t = 2 h). Asterisks indicate the intestinal nuclei and the arrowheads indicate SPD-2 foci. (C and D) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SAS-4 (green) to monitor centriole numbers after the nuclear division. The insets in (A–D) represent magnified views of regions highlighted by the white rectangles. Scale bar, 5 µm. (E) Quantification of SPD-2 or SAS-4 foci in intestinal nuclei in both wild type and lin-35/Rb mutants two hours after the first intestinal nuclear division. n = 75. (F) RT-PCR analysis of cell-specific transcripts from N2 and lin-35 (n745). elt-2 is intestinal specific, while htp-3 is expressed exclusively in the germ line. (G) The expression of spd-2, -5, zyg-1, -9, sas-4, -5, -6, tbg-1 and dlg-1 (control) [77] was quantified using RT-PCR from total or intestine-enriched mRNA from wild type (N2) or from lin-35 (n745) larvae before or after the first nuclear division, and 6–8 hours after the last nuclear division in lin-35 (n745) mutants. int., intestinal. bp, base pair.

Mentions: Since the elimination of the centrioles occurs specifically during the L2 stage, we next wondered whether elimination might be under temporal control, or whether it is part of the endocycle program and therefore contingent on the transition from the mitotic divisions to the endocycles. To distinguish between these possibilities, we used a lin-35 (n745) mutant that is mutated for the C. elegans orthologue of Rb [43]. These animals repeat the nuclear divisions, thus giving rise to supernumerary intestinal nuclei prior to their eventual switch to the endocycle program later in L2 stage [44], but developmental timing is otherwise normal. Interestingly, although centriole numbers were unaffected in lin-35 (n745) animals during the L1 stage (data not shown), two SPD-2 foci per 4C nucleus were frequently visualized in lin-35/Rb mutants 1-2 hours after the first nuclear division (Figure 4A, 4E, S1D). Similarly, this was reflected in the number of SAS-4 foci during the L2 stage (Figure 4C, 4E), presumably as a consequence of failing to uncouple centriole duplication from the first endo-S phase. Some animals occasionally even possessed more than two foci (Figure 4B, 4D, 4E). Because we see numeral defects in lin-35/Rb mutants that do not initiate the endocycle program in a timely manner, our data suggest that centriole duplication will be re-licensed as long as cells undergo a mitotic nuclear division and do not execute the endocycle program. However, following the onset of the endocycle program after the nuclear division(s) have terminated, the cells become refractory to the endo-S and will eliminate the centrioles shortly thereafter. Taken together, our data suggest that the apparent uncoupling of centriole duplication from S phase and subsequent centriole elimination relies on the initiation of the endocycle program, and does not result as a consequence of prolonged or aberrant S-phase, or chronological developmental time per se.


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

Lu Y, Roy R - PLoS ONE (2014)

lin-35/Rb mutants undergo additional rounds of centriole duplication.(A and B) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SPD-2 (green) to monitor centriole dynamics at the nuclear division. The panel (A) was obtained by staining animals approximately one hour after the nuclear division (t = 1 h), while (B) was acquired two hours after the division (t = 2 h). Asterisks indicate the intestinal nuclei and the arrowheads indicate SPD-2 foci. (C and D) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SAS-4 (green) to monitor centriole numbers after the nuclear division. The insets in (A–D) represent magnified views of regions highlighted by the white rectangles. Scale bar, 5 µm. (E) Quantification of SPD-2 or SAS-4 foci in intestinal nuclei in both wild type and lin-35/Rb mutants two hours after the first intestinal nuclear division. n = 75. (F) RT-PCR analysis of cell-specific transcripts from N2 and lin-35 (n745). elt-2 is intestinal specific, while htp-3 is expressed exclusively in the germ line. (G) The expression of spd-2, -5, zyg-1, -9, sas-4, -5, -6, tbg-1 and dlg-1 (control) [77] was quantified using RT-PCR from total or intestine-enriched mRNA from wild type (N2) or from lin-35 (n745) larvae before or after the first nuclear division, and 6–8 hours after the last nuclear division in lin-35 (n745) mutants. int., intestinal. bp, base pair.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110958-g004: lin-35/Rb mutants undergo additional rounds of centriole duplication.(A and B) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SPD-2 (green) to monitor centriole dynamics at the nuclear division. The panel (A) was obtained by staining animals approximately one hour after the nuclear division (t = 1 h), while (B) was acquired two hours after the division (t = 2 h). Asterisks indicate the intestinal nuclei and the arrowheads indicate SPD-2 foci. (C and D) lin-35/Rb mutant larvae were stained with DAPI (red) and anti-SAS-4 (green) to monitor centriole numbers after the nuclear division. The insets in (A–D) represent magnified views of regions highlighted by the white rectangles. Scale bar, 5 µm. (E) Quantification of SPD-2 or SAS-4 foci in intestinal nuclei in both wild type and lin-35/Rb mutants two hours after the first intestinal nuclear division. n = 75. (F) RT-PCR analysis of cell-specific transcripts from N2 and lin-35 (n745). elt-2 is intestinal specific, while htp-3 is expressed exclusively in the germ line. (G) The expression of spd-2, -5, zyg-1, -9, sas-4, -5, -6, tbg-1 and dlg-1 (control) [77] was quantified using RT-PCR from total or intestine-enriched mRNA from wild type (N2) or from lin-35 (n745) larvae before or after the first nuclear division, and 6–8 hours after the last nuclear division in lin-35 (n745) mutants. int., intestinal. bp, base pair.
Mentions: Since the elimination of the centrioles occurs specifically during the L2 stage, we next wondered whether elimination might be under temporal control, or whether it is part of the endocycle program and therefore contingent on the transition from the mitotic divisions to the endocycles. To distinguish between these possibilities, we used a lin-35 (n745) mutant that is mutated for the C. elegans orthologue of Rb [43]. These animals repeat the nuclear divisions, thus giving rise to supernumerary intestinal nuclei prior to their eventual switch to the endocycle program later in L2 stage [44], but developmental timing is otherwise normal. Interestingly, although centriole numbers were unaffected in lin-35 (n745) animals during the L1 stage (data not shown), two SPD-2 foci per 4C nucleus were frequently visualized in lin-35/Rb mutants 1-2 hours after the first nuclear division (Figure 4A, 4E, S1D). Similarly, this was reflected in the number of SAS-4 foci during the L2 stage (Figure 4C, 4E), presumably as a consequence of failing to uncouple centriole duplication from the first endo-S phase. Some animals occasionally even possessed more than two foci (Figure 4B, 4D, 4E). Because we see numeral defects in lin-35/Rb mutants that do not initiate the endocycle program in a timely manner, our data suggest that centriole duplication will be re-licensed as long as cells undergo a mitotic nuclear division and do not execute the endocycle program. However, following the onset of the endocycle program after the nuclear division(s) have terminated, the cells become refractory to the endo-S and will eliminate the centrioles shortly thereafter. Taken together, our data suggest that the apparent uncoupling of centriole duplication from S phase and subsequent centriole elimination relies on the initiation of the endocycle program, and does not result as a consequence of prolonged or aberrant S-phase, or chronological developmental time per se.

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