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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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SPD-2 is ubiquitylated and its localization and stability are dependent on proteasome function.(A–F) Larvae were subjected to pbs-3(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1, L2 and L3 stages, respectively. (A), (C) and (E) show anti-SPD-2 alone. (G–H) Larvae were subjected to pbs-3(RNAi); spd-2(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1. (G) shows anti-SPD-2 alone. Asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (I) SPD-2 nuclear localization was monitored and the number of intestinal cells that demonstrate diffuse nuclear SPD-2 staining was compared in control and pbs-3(RNAi). (J) The effects of pbs-3(RNAi) on SPD-2 stability were quantified by determining the number of intestinal cells that express SPD-2 at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (K–M) SAS-4 levels were stained in the intestinal cells of late L2 pbs-3(RNAi) animals and were quantified as above (N). The asterisks indicate the intestinal nuclei, while arrowheads point to SAS-4 foci. Scale bar, 5 µm. n≥50; P<0.05 (t-test). (O) Homogenates obtained from animals expressing 3XFLAG tagged SPD-2 subjected to pbs-3(RNAi) or gfp(RNAi) (control), were incubated with anti-FLAG antisera and associated proteins were immunoprecipitated with Protein A-agarose. The pellets were blotted with anti-ubiquitin or anti-SPD-2 respectively. kDa, kilo Dalton.
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pone-0110958-g007: SPD-2 is ubiquitylated and its localization and stability are dependent on proteasome function.(A–F) Larvae were subjected to pbs-3(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1, L2 and L3 stages, respectively. (A), (C) and (E) show anti-SPD-2 alone. (G–H) Larvae were subjected to pbs-3(RNAi); spd-2(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1. (G) shows anti-SPD-2 alone. Asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (I) SPD-2 nuclear localization was monitored and the number of intestinal cells that demonstrate diffuse nuclear SPD-2 staining was compared in control and pbs-3(RNAi). (J) The effects of pbs-3(RNAi) on SPD-2 stability were quantified by determining the number of intestinal cells that express SPD-2 at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (K–M) SAS-4 levels were stained in the intestinal cells of late L2 pbs-3(RNAi) animals and were quantified as above (N). The asterisks indicate the intestinal nuclei, while arrowheads point to SAS-4 foci. Scale bar, 5 µm. n≥50; P<0.05 (t-test). (O) Homogenates obtained from animals expressing 3XFLAG tagged SPD-2 subjected to pbs-3(RNAi) or gfp(RNAi) (control), were incubated with anti-FLAG antisera and associated proteins were immunoprecipitated with Protein A-agarose. The pellets were blotted with anti-ubiquitin or anti-SPD-2 respectively. kDa, kilo Dalton.

Mentions: Recent data have implicated ubiquitin-mediated proteolytic degradation in the appropriate regulation of centrosomal components [59]–[61]. If SPD-2 abundance is controlled by ubiquitin-mediated degradation then the absence of essential ubiquitylation/proteasome components may also affect the elimination of the intestinal centrioles. proteasome b-subunit 3 (pbs-3) is essential for proteasome function [62] and in pbs-3(RNAi) animals, SPD-2 shows substantial nuclear accumulation (Figure 7A–7D, 7I), while SPD-2 signal is still detectable in the L3 stage, considerably later than in control animals (Figure 7E, 7F, 7J). Both the accumulation and persistence of SPD-2 are greatly reduced in pbs-3(RNAi); spd-2(RNAi) animals, confirming that the signals are indeed SPD-2-specific (Figure 7G, 7H). Similar effects on SPD-2 were also observed in pbs-5(RNAi) animals alone (data not shown), consistent with a role of proteasome-mediated degradation in the timely elimination of the centrioles after the intestinal cells commence endoreduplication. Intriguingly, in addition to the delayed centriole elimination phenotype, anti-SAS-4 staining revealed that centriole duplication was occasionally observed in pbs-3(RNAi) animals (Figure 7K–7N), which could reflect the stabilization of additional proteins involved in centriole duplication, or other key effectors of numeral regulation. Furthermore, by compromising proteasome function we were able to detect a higher molecular weight band in our FLAG-tagged SPD-2 immunoprecipitates. This band was recognized by both anti-SPD-2 and anti-Ubiquitin, consistent with the ubiquitylation of SPD-2 in vivo, which likely precedes its degradation (Figure 7O).


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

Lu Y, Roy R - PLoS ONE (2014)

SPD-2 is ubiquitylated and its localization and stability are dependent on proteasome function.(A–F) Larvae were subjected to pbs-3(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1, L2 and L3 stages, respectively. (A), (C) and (E) show anti-SPD-2 alone. (G–H) Larvae were subjected to pbs-3(RNAi); spd-2(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1. (G) shows anti-SPD-2 alone. Asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (I) SPD-2 nuclear localization was monitored and the number of intestinal cells that demonstrate diffuse nuclear SPD-2 staining was compared in control and pbs-3(RNAi). (J) The effects of pbs-3(RNAi) on SPD-2 stability were quantified by determining the number of intestinal cells that express SPD-2 at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (K–M) SAS-4 levels were stained in the intestinal cells of late L2 pbs-3(RNAi) animals and were quantified as above (N). The asterisks indicate the intestinal nuclei, while arrowheads point to SAS-4 foci. Scale bar, 5 µm. n≥50; P<0.05 (t-test). (O) Homogenates obtained from animals expressing 3XFLAG tagged SPD-2 subjected to pbs-3(RNAi) or gfp(RNAi) (control), were incubated with anti-FLAG antisera and associated proteins were immunoprecipitated with Protein A-agarose. The pellets were blotted with anti-ubiquitin or anti-SPD-2 respectively. kDa, kilo Dalton.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone-0110958-g007: SPD-2 is ubiquitylated and its localization and stability are dependent on proteasome function.(A–F) Larvae were subjected to pbs-3(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1, L2 and L3 stages, respectively. (A), (C) and (E) show anti-SPD-2 alone. (G–H) Larvae were subjected to pbs-3(RNAi); spd-2(RNAi) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1. (G) shows anti-SPD-2 alone. Asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (I) SPD-2 nuclear localization was monitored and the number of intestinal cells that demonstrate diffuse nuclear SPD-2 staining was compared in control and pbs-3(RNAi). (J) The effects of pbs-3(RNAi) on SPD-2 stability were quantified by determining the number of intestinal cells that express SPD-2 at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (K–M) SAS-4 levels were stained in the intestinal cells of late L2 pbs-3(RNAi) animals and were quantified as above (N). The asterisks indicate the intestinal nuclei, while arrowheads point to SAS-4 foci. Scale bar, 5 µm. n≥50; P<0.05 (t-test). (O) Homogenates obtained from animals expressing 3XFLAG tagged SPD-2 subjected to pbs-3(RNAi) or gfp(RNAi) (control), were incubated with anti-FLAG antisera and associated proteins were immunoprecipitated with Protein A-agarose. The pellets were blotted with anti-ubiquitin or anti-SPD-2 respectively. kDa, kilo Dalton.
Mentions: Recent data have implicated ubiquitin-mediated proteolytic degradation in the appropriate regulation of centrosomal components [59]–[61]. If SPD-2 abundance is controlled by ubiquitin-mediated degradation then the absence of essential ubiquitylation/proteasome components may also affect the elimination of the intestinal centrioles. proteasome b-subunit 3 (pbs-3) is essential for proteasome function [62] and in pbs-3(RNAi) animals, SPD-2 shows substantial nuclear accumulation (Figure 7A–7D, 7I), while SPD-2 signal is still detectable in the L3 stage, considerably later than in control animals (Figure 7E, 7F, 7J). Both the accumulation and persistence of SPD-2 are greatly reduced in pbs-3(RNAi); spd-2(RNAi) animals, confirming that the signals are indeed SPD-2-specific (Figure 7G, 7H). Similar effects on SPD-2 were also observed in pbs-5(RNAi) animals alone (data not shown), consistent with a role of proteasome-mediated degradation in the timely elimination of the centrioles after the intestinal cells commence endoreduplication. Intriguingly, in addition to the delayed centriole elimination phenotype, anti-SAS-4 staining revealed that centriole duplication was occasionally observed in pbs-3(RNAi) animals (Figure 7K–7N), which could reflect the stabilization of additional proteins involved in centriole duplication, or other key effectors of numeral regulation. Furthermore, by compromising proteasome function we were able to detect a higher molecular weight band in our FLAG-tagged SPD-2 immunoprecipitates. This band was recognized by both anti-SPD-2 and anti-Ubiquitin, consistent with the ubiquitylation of SPD-2 in vivo, which likely precedes its degradation (Figure 7O).

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