Limits...
The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Rogers GC, Rusan NM, Roberts DM, Peifer M, Rogers SL - J. Cell Biol. (2009)

Bottom Line: We found that Plk4 binds to Slimb and is an SCF(Slimb) target.Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase.Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Show MeSH

Related in: MedlinePlus

Slimb regulates Plk4 levels on centrioles to control centriole number. (A) Asymmetrical Plk4 localization. Transient coexpression of Nlp-EGFP (Ito et al., 1996) as a cotransfection marker (green) labeling nuclei (arrow) and Plk4-EGFP (green). D-PLP (red) marks centrioles (arrowheads). Insets show centrioles at a higher magnification. (B–D) Mutating the Slimb-binding site stabilizes Plk4 on centrioles. (B) Cell cycle distributions after 24-h drug-induced S, G2, or mitotic arrest. Histograms are shown to scale and were assessed by HTM (5,000 cells/histogram). (C) Plk4-EGFP (green) only localizes to M-phase centrioles (arrowheads and insets) marked with mCherry–SAS-6 in live cells (red). Condensed DNA (blue) reveals mitotic cells. (D) Plk4-SBM–EGFP (green) localizes to centrioles during all cell cycle phases that were examined. Centrioles (arrowheads and insets) are marked with D-PLP in fixed cells (red). Nlp-EGFP (green nuclei, cytoplasmic during mitosis) is the cotransfection control. Bars: (A) 5 µm; (C and D) 2.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2654306&req=5

fig4: Slimb regulates Plk4 levels on centrioles to control centriole number. (A) Asymmetrical Plk4 localization. Transient coexpression of Nlp-EGFP (Ito et al., 1996) as a cotransfection marker (green) labeling nuclei (arrow) and Plk4-EGFP (green). D-PLP (red) marks centrioles (arrowheads). Insets show centrioles at a higher magnification. (B–D) Mutating the Slimb-binding site stabilizes Plk4 on centrioles. (B) Cell cycle distributions after 24-h drug-induced S, G2, or mitotic arrest. Histograms are shown to scale and were assessed by HTM (5,000 cells/histogram). (C) Plk4-EGFP (green) only localizes to M-phase centrioles (arrowheads and insets) marked with mCherry–SAS-6 in live cells (red). Condensed DNA (blue) reveals mitotic cells. (D) Plk4-SBM–EGFP (green) localizes to centrioles during all cell cycle phases that were examined. Centrioles (arrowheads and insets) are marked with D-PLP in fixed cells (red). Nlp-EGFP (green nuclei, cytoplasmic during mitosis) is the cotransfection control. Bars: (A) 5 µm; (C and D) 2.5 µm.

Mentions: Plk4 localizes to centrioles (Bettencourt-Dias et al., 2005). If Plk4 regulates the timing of centriole duplication, its centriole localization should be tightly regulated through the cell cycle. To assess this, we drove Plk4-EGFP using the weak SAS-6p, which is expressed throughout the cell cycle (unpublished data). Because of its low expression, identifying Plk4-EGFP–transfected cells was made possible by cotransfecting with the abundant nuclear protein nucleophosmin (Nlp)-EGFP (Ito et al., 1996). Notably, unlike mammalian Nlp, which localizes to centrosomes and suppresses their overduplication (Okuda et al., 2000; Wang et al., 2005), Drosophila Nlp does not localize to centrioles (Fig. S2 E), and nlp RNAi has no effect on centriole number (not depicted). We found that Plk4-EGFP was asymmetrically localized on D-PLP–labeled centrioles in interphase (Fig. 4 A), as seen in mammalian cells (Kleylein-Sohn et al., 2007). To assess whether Plk4 centriole localization is cell cycle regulated, we coexpressed Plk4-EGFP and mCherry–SAS-6 in cells chemically arrested during S, G2, or M phase (Fig. 4, B and C). Plk4 was undetectable in S phase–arrested cells, whereas levels on centrioles peaked during mitotic arrest (Fig. 4 C). Plk4-EGFP was observed on centrioles in some but not all cycling interphase cells (Fig. 4 A), a population presumably lost after prolonged interphase drug arrest. Thus, whereas Slimb localizes to centrioles during interphase, Plk4 on centrioles peaks during mitosis, and it is not detected there during S phase. This is consistent with the hypothesis that Slimb on centrioles can destabilize Plk4 during a period of the cell cycle when centriole duplication occurs.


The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Rogers GC, Rusan NM, Roberts DM, Peifer M, Rogers SL - J. Cell Biol. (2009)

Slimb regulates Plk4 levels on centrioles to control centriole number. (A) Asymmetrical Plk4 localization. Transient coexpression of Nlp-EGFP (Ito et al., 1996) as a cotransfection marker (green) labeling nuclei (arrow) and Plk4-EGFP (green). D-PLP (red) marks centrioles (arrowheads). Insets show centrioles at a higher magnification. (B–D) Mutating the Slimb-binding site stabilizes Plk4 on centrioles. (B) Cell cycle distributions after 24-h drug-induced S, G2, or mitotic arrest. Histograms are shown to scale and were assessed by HTM (5,000 cells/histogram). (C) Plk4-EGFP (green) only localizes to M-phase centrioles (arrowheads and insets) marked with mCherry–SAS-6 in live cells (red). Condensed DNA (blue) reveals mitotic cells. (D) Plk4-SBM–EGFP (green) localizes to centrioles during all cell cycle phases that were examined. Centrioles (arrowheads and insets) are marked with D-PLP in fixed cells (red). Nlp-EGFP (green nuclei, cytoplasmic during mitosis) is the cotransfection control. Bars: (A) 5 µm; (C and D) 2.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654306&req=5

fig4: Slimb regulates Plk4 levels on centrioles to control centriole number. (A) Asymmetrical Plk4 localization. Transient coexpression of Nlp-EGFP (Ito et al., 1996) as a cotransfection marker (green) labeling nuclei (arrow) and Plk4-EGFP (green). D-PLP (red) marks centrioles (arrowheads). Insets show centrioles at a higher magnification. (B–D) Mutating the Slimb-binding site stabilizes Plk4 on centrioles. (B) Cell cycle distributions after 24-h drug-induced S, G2, or mitotic arrest. Histograms are shown to scale and were assessed by HTM (5,000 cells/histogram). (C) Plk4-EGFP (green) only localizes to M-phase centrioles (arrowheads and insets) marked with mCherry–SAS-6 in live cells (red). Condensed DNA (blue) reveals mitotic cells. (D) Plk4-SBM–EGFP (green) localizes to centrioles during all cell cycle phases that were examined. Centrioles (arrowheads and insets) are marked with D-PLP in fixed cells (red). Nlp-EGFP (green nuclei, cytoplasmic during mitosis) is the cotransfection control. Bars: (A) 5 µm; (C and D) 2.5 µm.
Mentions: Plk4 localizes to centrioles (Bettencourt-Dias et al., 2005). If Plk4 regulates the timing of centriole duplication, its centriole localization should be tightly regulated through the cell cycle. To assess this, we drove Plk4-EGFP using the weak SAS-6p, which is expressed throughout the cell cycle (unpublished data). Because of its low expression, identifying Plk4-EGFP–transfected cells was made possible by cotransfecting with the abundant nuclear protein nucleophosmin (Nlp)-EGFP (Ito et al., 1996). Notably, unlike mammalian Nlp, which localizes to centrosomes and suppresses their overduplication (Okuda et al., 2000; Wang et al., 2005), Drosophila Nlp does not localize to centrioles (Fig. S2 E), and nlp RNAi has no effect on centriole number (not depicted). We found that Plk4-EGFP was asymmetrically localized on D-PLP–labeled centrioles in interphase (Fig. 4 A), as seen in mammalian cells (Kleylein-Sohn et al., 2007). To assess whether Plk4 centriole localization is cell cycle regulated, we coexpressed Plk4-EGFP and mCherry–SAS-6 in cells chemically arrested during S, G2, or M phase (Fig. 4, B and C). Plk4 was undetectable in S phase–arrested cells, whereas levels on centrioles peaked during mitotic arrest (Fig. 4 C). Plk4-EGFP was observed on centrioles in some but not all cycling interphase cells (Fig. 4 A), a population presumably lost after prolonged interphase drug arrest. Thus, whereas Slimb localizes to centrioles during interphase, Plk4 on centrioles peaks during mitosis, and it is not detected there during S phase. This is consistent with the hypothesis that Slimb on centrioles can destabilize Plk4 during a period of the cell cycle when centriole duplication occurs.

Bottom Line: We found that Plk4 binds to Slimb and is an SCF(Slimb) target.Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase.Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCF(Slimb) as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCF(Slimb) target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Show MeSH
Related in: MedlinePlus