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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 lose their capacity to recruit γ-tubulin following the intestinal nuclear division that precedes the onset of endoreduplication.(A–H) Wild type larvae were stained for γ-tubulin (TBG-1) before, during, and after the intestinal nuclear division. (A), (C), (E) and (G) represent the TBG-1 only; (B), (D), (F), and (H) TBG-1 with DAPI. The asterisks indicate the intestinal nuclei and the square bracket highlights a pair of sister intestinal nuclei. The circles in (F) and (H) highlight germ cells with γ-tubulin foci. (I–L) High resolution micrograph of cells following division during the L1 stage. (I and K) show TBG-1 signal only; (J and L) TBG-1 with DAPI. (I and J), dividing P cell in the ventral hypodermis; (K and L), germ line precursor cell. Square brackets highlight sister cells. (M and N) Similar staining was performed in cul-4 (gk434) homozygous animals. All cells were stained with DAPI (red) and anti-γ-tubulin (green). Scale bar, 5 µm.
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pone-0110958-g003: Centrioles lose their capacity to recruit γ-tubulin following the intestinal nuclear division that precedes the onset of endoreduplication.(A–H) Wild type larvae were stained for γ-tubulin (TBG-1) before, during, and after the intestinal nuclear division. (A), (C), (E) and (G) represent the TBG-1 only; (B), (D), (F), and (H) TBG-1 with DAPI. The asterisks indicate the intestinal nuclei and the square bracket highlights a pair of sister intestinal nuclei. The circles in (F) and (H) highlight germ cells with γ-tubulin foci. (I–L) High resolution micrograph of cells following division during the L1 stage. (I and K) show TBG-1 signal only; (J and L) TBG-1 with DAPI. (I and J), dividing P cell in the ventral hypodermis; (K and L), germ line precursor cell. Square brackets highlight sister cells. (M and N) Similar staining was performed in cul-4 (gk434) homozygous animals. All cells were stained with DAPI (red) and anti-γ-tubulin (green). Scale bar, 5 µm.

Mentions: One of the major functions of the centrosome is to organize the mitotic spindle during mitosis through recruitment of the γ-tubulin complex and other components of the PCM. The changes in centriole duplication and stability during the mitosis-endocycle transition led us to examine if any functional modification of the centrioles might also occur as a consequence of this process. Previous studies indicated that γ-tubulin recruitment is subject to cell cycle-dependent regulation in mitotic cells [37]. During centrosome maturation γ-tubulin accumulates around the centrioles, resulting in substantially enlarged γ-tubulin foci at metaphase. The intensity of γ-tubulin gradually returns to baseline levels at the onset of the next interphase [37]. We observed similar baseline levels of γ-tubulin expression during the L1 interphase (Figure 3A, 3B). The intensity of the γ-tubulin foci increases substantially when the metaphase chromosomes become discernable (Figure 3C, 3D; n = 15). During anaphase, most of the centrosomal γ-tubulin rapidly disperses prior to the onset of the following S-phase (Figure 3C, 3D). The anaphase dispersal of γ-tubulin is not due to the disappearance of the centriole (Figure S3A, a-a” and b-b”), since centriole elimination only begins later during the L2 stage (Figure 2J). Moreover, the γ-tubulin levels around the centrioles never recover thereafter (Figure 3E–3H). This change likely compromises the ability of the centrioles to function as a MTOC in intestinal cells following this stage (Figure S3B). This anaphase dispersal of γ-tubulin does not occur in the mitotically proliferating cells in the ventral hypodermis (Figure 3I, 3J), or in the germ cells that are simultaneously undergoing mitotic divisions (Figure 3K, 3L). Not surprisingly, centrioles still act as an MTOC in these mitotic cells (Figure S3C). Overall, our observations indicate that just subsequent to the intestinal cell nuclear division that occurs at late L1 stage, the γ-tubulin that is associated with the centriole disperses, thereafter compromising the ability of the centriole to act as a MTOC.


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

Lu Y, Roy R - PLoS ONE (2014)

Centrioles lose their capacity to recruit γ-tubulin following the intestinal nuclear division that precedes the onset of endoreduplication.(A–H) Wild type larvae were stained for γ-tubulin (TBG-1) before, during, and after the intestinal nuclear division. (A), (C), (E) and (G) represent the TBG-1 only; (B), (D), (F), and (H) TBG-1 with DAPI. The asterisks indicate the intestinal nuclei and the square bracket highlights a pair of sister intestinal nuclei. The circles in (F) and (H) highlight germ cells with γ-tubulin foci. (I–L) High resolution micrograph of cells following division during the L1 stage. (I and K) show TBG-1 signal only; (J and L) TBG-1 with DAPI. (I and J), dividing P cell in the ventral hypodermis; (K and L), germ line precursor cell. Square brackets highlight sister cells. (M and N) Similar staining was performed in cul-4 (gk434) homozygous animals. All cells were stained with DAPI (red) and anti-γ-tubulin (green). Scale bar, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110958-g003: Centrioles lose their capacity to recruit γ-tubulin following the intestinal nuclear division that precedes the onset of endoreduplication.(A–H) Wild type larvae were stained for γ-tubulin (TBG-1) before, during, and after the intestinal nuclear division. (A), (C), (E) and (G) represent the TBG-1 only; (B), (D), (F), and (H) TBG-1 with DAPI. The asterisks indicate the intestinal nuclei and the square bracket highlights a pair of sister intestinal nuclei. The circles in (F) and (H) highlight germ cells with γ-tubulin foci. (I–L) High resolution micrograph of cells following division during the L1 stage. (I and K) show TBG-1 signal only; (J and L) TBG-1 with DAPI. (I and J), dividing P cell in the ventral hypodermis; (K and L), germ line precursor cell. Square brackets highlight sister cells. (M and N) Similar staining was performed in cul-4 (gk434) homozygous animals. All cells were stained with DAPI (red) and anti-γ-tubulin (green). Scale bar, 5 µm.
Mentions: One of the major functions of the centrosome is to organize the mitotic spindle during mitosis through recruitment of the γ-tubulin complex and other components of the PCM. The changes in centriole duplication and stability during the mitosis-endocycle transition led us to examine if any functional modification of the centrioles might also occur as a consequence of this process. Previous studies indicated that γ-tubulin recruitment is subject to cell cycle-dependent regulation in mitotic cells [37]. During centrosome maturation γ-tubulin accumulates around the centrioles, resulting in substantially enlarged γ-tubulin foci at metaphase. The intensity of γ-tubulin gradually returns to baseline levels at the onset of the next interphase [37]. We observed similar baseline levels of γ-tubulin expression during the L1 interphase (Figure 3A, 3B). The intensity of the γ-tubulin foci increases substantially when the metaphase chromosomes become discernable (Figure 3C, 3D; n = 15). During anaphase, most of the centrosomal γ-tubulin rapidly disperses prior to the onset of the following S-phase (Figure 3C, 3D). The anaphase dispersal of γ-tubulin is not due to the disappearance of the centriole (Figure S3A, a-a” and b-b”), since centriole elimination only begins later during the L2 stage (Figure 2J). Moreover, the γ-tubulin levels around the centrioles never recover thereafter (Figure 3E–3H). This change likely compromises the ability of the centrioles to function as a MTOC in intestinal cells following this stage (Figure S3B). This anaphase dispersal of γ-tubulin does not occur in the mitotically proliferating cells in the ventral hypodermis (Figure 3I, 3J), or in the germ cells that are simultaneously undergoing mitotic divisions (Figure 3K, 3L). Not surprisingly, centrioles still act as an MTOC in these mitotic cells (Figure S3C). Overall, our observations indicate that just subsequent to the intestinal cell nuclear division that occurs at late L1 stage, the γ-tubulin that is associated with the centriole disperses, thereafter compromising the ability of the centriole to act as a MTOC.

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