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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.

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Slimb localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.
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fig2: Slimb localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.

Mentions: We hypothesized that if Slimb regulates centriole duplication, it might localize to centrioles. To test this, we generated antisera specifically recognizing Slimb (Fig. 2, A and B). Although mostly cytoplasmic, Slimb was enriched on D-PLP–labeled centrioles in interphase S2 cells (Fig. 2 C). In contrast, Slimb immunostaining at centrioles was significantly reduced by Slimb depletion (Fig. S2 D). To confirm this localization, we examined an S2 stable line expressing both Slimb-EGFP and mCherry–SAS-6 (a centriole protein; Rusan and Peifer, 2007). Like Slimb, Slimb-EGFP was enriched at mCherry–SAS-6–labeled centrioles in live interphase cells (Fig. 2 D). Furthermore, a fraction of both endogenous and EGFP-tagged Slimb copurified with centrioles isolated using sucrose gradient centrifugation (Fig. 2 F). These data demonstrate that Slimb associates with centrioles. In addition, we noted that Slimb occasionally appeared asymmetrically positioned on centrioles in live cells (Fig. 2 D and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200808049/DC1). However, centrioles in these cells are not stationary but instead constantly spin and move apparently randomly throughout the cell (Rogers et al., 2008). Thus, this perceived asymmetry in Slimb localization on centrioles is likely the result of a delay in image acquisition using live cell microscopy.


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 localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Slimb localizes to centrioles. (A) Immunoblot of affinity-purified anti-Slimb antibody against an S2 cell lysate. (B) slimb RNAi depletes protein by 98%. (C and C′) Immunostaining of Slimb (green, monochrome) and D-PLP centrioles (red) in interphase S2 cells. (D and D′) Stable S2 line expressing Slimb-EGFP (green, monochrome) and mCherry–SAS-6 centrioles in live interphase cells. (C–D′) Centrioles (arrowheads) are shown at a higher magnification (insets). (E) Endogenous Slimb and Slimb-EGFP cosediment with centrioles purified from S2 cells on a 20–70% sucrose gradient. Fractions were immunoblotted for D-PLP, GFP, and Slimb. Asterisks denote peak centriole-containing fractions. (F) Slimb (green, arrowheads) immunolocalizes to D-PLP centrioles (red) after 24-h drug-induced S-, G2-, or M-phase arrest. Histograms (to scale) of DNA content assessed by HTM (5,000 cells/histogram). Condensed DNA (blue) reveals a mitotic cell. Insets show centrioles at a higher magnification. (G) Graph of endogenous Slimb levels after 24-h drug-induced cell cycle arrest as determined from quantitative anti-Slimb immunoblots (below graph). (B and G) α-Tubulin was used as a loading control. Bars, 5 µm.
Mentions: We hypothesized that if Slimb regulates centriole duplication, it might localize to centrioles. To test this, we generated antisera specifically recognizing Slimb (Fig. 2, A and B). Although mostly cytoplasmic, Slimb was enriched on D-PLP–labeled centrioles in interphase S2 cells (Fig. 2 C). In contrast, Slimb immunostaining at centrioles was significantly reduced by Slimb depletion (Fig. S2 D). To confirm this localization, we examined an S2 stable line expressing both Slimb-EGFP and mCherry–SAS-6 (a centriole protein; Rusan and Peifer, 2007). Like Slimb, Slimb-EGFP was enriched at mCherry–SAS-6–labeled centrioles in live interphase cells (Fig. 2 D). Furthermore, a fraction of both endogenous and EGFP-tagged Slimb copurified with centrioles isolated using sucrose gradient centrifugation (Fig. 2 F). These data demonstrate that Slimb associates with centrioles. In addition, we noted that Slimb occasionally appeared asymmetrically positioned on centrioles in live cells (Fig. 2 D and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200808049/DC1). However, centrioles in these cells are not stationary but instead constantly spin and move apparently randomly throughout the cell (Rogers et al., 2008). Thus, this perceived asymmetry in Slimb localization on centrioles is likely the result of a delay in image acquisition using live cell microscopy.

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