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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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Centrioles no longer duplicate in endocycling cells prior to their elimination.(A) SPD-2::GFP signal can be seen throughout the germ line until oogenesis in (a), while in (b) it is undetectable in the intestinal cells in adult hermaphrodites. a′ and b′ are high magnification images of the GFP signal in the field identified by the two white rectangles a and b. (B–F) and (G–I) SPD-2 and SAS-4 foci are detectable up to the L1/L2 transition. The signal is no longer detectable by the L3 stage. The nuclear localized SPD-2 is absent following spd-2(RNAi) in (F). Animals were stained with DAPI (red) and anti-SPD-2 (green) in (B–F) or anti-SAS-4 (green) in (G–I). Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. The insets represent magnified SPD-2 or SAS-4 signal of the highlighted regions (white rectangles). The circles in (E) and (I) highlight the germ cells with either SPD-2 foci or SAS-4 foci. (J) Quantification of centriole numbers and described in (B–E) and (G–I) based on SPD-2 or SAS-4 detection. n≥56 for each stage. (K) Centriole duplication is uncoupled from the endocycles in the lateral hypodermal V cells in the late L1 stage. The centriole appears to be uncoupled from S-phase in the anterior endoreduplicating daughter cell but remains coupled to DNA replication in the mitotic posterior daughter. The square brackets indicate the daughter cells from a common mother V cell, while arrowheads indicate the centrioles. The inset is a magnified view of the region delineated by the white rectangle. A, anterior; P, posterior. hyp7 marks the hyp7 nucleus. (L and M) Supernumerary centrioles are detected in HU-treated L1 animals (M) but not in the control (L). The inset is a magnified SPD-2 signal of the region delineated by the white rectangle. Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. (N and O) Centriole duplication occurs once in response to S phase, but centrioles do not overduplicate and are not eliminated during un-quantized DNA re-replication. Heterozygous in (N) and homozygous cul-4 (gk434) mutants in (O) were stained with DAPI (red) and SPD-2 (green) respectively. The insets show the SPD-2 signal in the regions outlined by the white rectangles. Arrowheads indicate centrioles. Scale bar, 5 µm.
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pone-0110958-g002: Centrioles no longer duplicate in endocycling cells prior to their elimination.(A) SPD-2::GFP signal can be seen throughout the germ line until oogenesis in (a), while in (b) it is undetectable in the intestinal cells in adult hermaphrodites. a′ and b′ are high magnification images of the GFP signal in the field identified by the two white rectangles a and b. (B–F) and (G–I) SPD-2 and SAS-4 foci are detectable up to the L1/L2 transition. The signal is no longer detectable by the L3 stage. The nuclear localized SPD-2 is absent following spd-2(RNAi) in (F). Animals were stained with DAPI (red) and anti-SPD-2 (green) in (B–F) or anti-SAS-4 (green) in (G–I). Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. The insets represent magnified SPD-2 or SAS-4 signal of the highlighted regions (white rectangles). The circles in (E) and (I) highlight the germ cells with either SPD-2 foci or SAS-4 foci. (J) Quantification of centriole numbers and described in (B–E) and (G–I) based on SPD-2 or SAS-4 detection. n≥56 for each stage. (K) Centriole duplication is uncoupled from the endocycles in the lateral hypodermal V cells in the late L1 stage. The centriole appears to be uncoupled from S-phase in the anterior endoreduplicating daughter cell but remains coupled to DNA replication in the mitotic posterior daughter. The square brackets indicate the daughter cells from a common mother V cell, while arrowheads indicate the centrioles. The inset is a magnified view of the region delineated by the white rectangle. A, anterior; P, posterior. hyp7 marks the hyp7 nucleus. (L and M) Supernumerary centrioles are detected in HU-treated L1 animals (M) but not in the control (L). The inset is a magnified SPD-2 signal of the region delineated by the white rectangle. Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. (N and O) Centriole duplication occurs once in response to S phase, but centrioles do not overduplicate and are not eliminated during un-quantized DNA re-replication. Heterozygous in (N) and homozygous cul-4 (gk434) mutants in (O) were stained with DAPI (red) and SPD-2 (green) respectively. The insets show the SPD-2 signal in the regions outlined by the white rectangles. Arrowheads indicate centrioles. Scale bar, 5 µm.

Mentions: We monitored the levels of two centriolar proteins in the intestinal cells throughout postembryonic development: SPD-2, which is associated both with the centriole and the PCM, and a highly conserved centriolar component called SAS-4 that is associated exclusively with centrioles [13], [33]. We first fused SPD-2 to GFP and found that it is most prominently expressed in the distal, mitotic region of the adult germ line (Figure 2A, a and a′), yet was notably absent from the adult intestinal cells (Figure 2A, b and b′), suggesting that SPD-2 was either not expressed in the adult intestinal lineage, and/or it was eliminated during development.


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

Lu Y, Roy R - PLoS ONE (2014)

Centrioles no longer duplicate in endocycling cells prior to their elimination.(A) SPD-2::GFP signal can be seen throughout the germ line until oogenesis in (a), while in (b) it is undetectable in the intestinal cells in adult hermaphrodites. a′ and b′ are high magnification images of the GFP signal in the field identified by the two white rectangles a and b. (B–F) and (G–I) SPD-2 and SAS-4 foci are detectable up to the L1/L2 transition. The signal is no longer detectable by the L3 stage. The nuclear localized SPD-2 is absent following spd-2(RNAi) in (F). Animals were stained with DAPI (red) and anti-SPD-2 (green) in (B–F) or anti-SAS-4 (green) in (G–I). Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. The insets represent magnified SPD-2 or SAS-4 signal of the highlighted regions (white rectangles). The circles in (E) and (I) highlight the germ cells with either SPD-2 foci or SAS-4 foci. (J) Quantification of centriole numbers and described in (B–E) and (G–I) based on SPD-2 or SAS-4 detection. n≥56 for each stage. (K) Centriole duplication is uncoupled from the endocycles in the lateral hypodermal V cells in the late L1 stage. The centriole appears to be uncoupled from S-phase in the anterior endoreduplicating daughter cell but remains coupled to DNA replication in the mitotic posterior daughter. The square brackets indicate the daughter cells from a common mother V cell, while arrowheads indicate the centrioles. The inset is a magnified view of the region delineated by the white rectangle. A, anterior; P, posterior. hyp7 marks the hyp7 nucleus. (L and M) Supernumerary centrioles are detected in HU-treated L1 animals (M) but not in the control (L). The inset is a magnified SPD-2 signal of the region delineated by the white rectangle. Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. (N and O) Centriole duplication occurs once in response to S phase, but centrioles do not overduplicate and are not eliminated during un-quantized DNA re-replication. Heterozygous in (N) and homozygous cul-4 (gk434) mutants in (O) were stained with DAPI (red) and SPD-2 (green) respectively. The insets show the SPD-2 signal in the regions outlined by the white rectangles. Arrowheads indicate centrioles. Scale bar, 5 µm.
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pone-0110958-g002: Centrioles no longer duplicate in endocycling cells prior to their elimination.(A) SPD-2::GFP signal can be seen throughout the germ line until oogenesis in (a), while in (b) it is undetectable in the intestinal cells in adult hermaphrodites. a′ and b′ are high magnification images of the GFP signal in the field identified by the two white rectangles a and b. (B–F) and (G–I) SPD-2 and SAS-4 foci are detectable up to the L1/L2 transition. The signal is no longer detectable by the L3 stage. The nuclear localized SPD-2 is absent following spd-2(RNAi) in (F). Animals were stained with DAPI (red) and anti-SPD-2 (green) in (B–F) or anti-SAS-4 (green) in (G–I). Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. The insets represent magnified SPD-2 or SAS-4 signal of the highlighted regions (white rectangles). The circles in (E) and (I) highlight the germ cells with either SPD-2 foci or SAS-4 foci. (J) Quantification of centriole numbers and described in (B–E) and (G–I) based on SPD-2 or SAS-4 detection. n≥56 for each stage. (K) Centriole duplication is uncoupled from the endocycles in the lateral hypodermal V cells in the late L1 stage. The centriole appears to be uncoupled from S-phase in the anterior endoreduplicating daughter cell but remains coupled to DNA replication in the mitotic posterior daughter. The square brackets indicate the daughter cells from a common mother V cell, while arrowheads indicate the centrioles. The inset is a magnified view of the region delineated by the white rectangle. A, anterior; P, posterior. hyp7 marks the hyp7 nucleus. (L and M) Supernumerary centrioles are detected in HU-treated L1 animals (M) but not in the control (L). The inset is a magnified SPD-2 signal of the region delineated by the white rectangle. Arrowheads indicate centrioles, while asterisks indicate intestinal nuclei. (N and O) Centriole duplication occurs once in response to S phase, but centrioles do not overduplicate and are not eliminated during un-quantized DNA re-replication. Heterozygous in (N) and homozygous cul-4 (gk434) mutants in (O) were stained with DAPI (red) and SPD-2 (green) respectively. The insets show the SPD-2 signal in the regions outlined by the white rectangles. Arrowheads indicate centrioles. Scale bar, 5 µm.
Mentions: We monitored the levels of two centriolar proteins in the intestinal cells throughout postembryonic development: SPD-2, which is associated both with the centriole and the PCM, and a highly conserved centriolar component called SAS-4 that is associated exclusively with centrioles [13], [33]. We first fused SPD-2 to GFP and found that it is most prominently expressed in the distal, mitotic region of the adult germ line (Figure 2A, a and a′), yet was notably absent from the adult intestinal cells (Figure 2A, b and b′), suggesting that SPD-2 was either not expressed in the adult intestinal lineage, and/or it was eliminated during development.

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