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The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Wueseke O, Bunkenborg J, Hein MY, Zinke A, Viscardi V, Woodruff JB, Oegema K, Mann M, Andersen JS, Hyman AA - Mol. Biol. Cell (2014)

Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

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Identification of cytoplasmic interactors of GFP::SPD-2 and SPD-5::GFP using label-free quantification mass spectrometry. (A) Western blot showing depletion of endogenous SPD-5 or SPD-2 after 24 h of RNAi treatment. SPD-5 is fully removed from N2 worms after 24 h of RNAi. Endogenous SPD-2 levels in OD824 (GFP::SPD-2-rr) are reduced to 25% after 24 h of spd-2-rr RNAi, whereas GFP::SPD-2-rr levels increase threefold. (B) Embryonic viability of N2, OD824 (GFP::SPD-2rr), and TH327 (SPD-5::GFP codon optimized to cai 0.65) embryos after 24 h of treatment with spd-5 or spd-2-rr RNAi. Both transgenes were found to restore embryo viability when endogenous protein was removed. (C, D) Identification of cytoplasmic interactors of SPD-5::GFP and GFP::SPD-2. SPD-5::GFP or GFP::SPD-2 was immunopurified from C. elegans embryo extracts, and the coimmunoprecipitated proteins were identified via label-free quantification mass spectrometry. TBG-1::GFP immunoprecipitates were used as a control. Experiments were repeated in triplicate. Results are plotted as volcano plots, with the enrichment of identified peptides in sample vs. control plotted on the x-axis and the significance of enrichment on the y-axis. p values and enrichments were calculated as described previously (Hubner et al., 2010). Black circles indicate nonenriched centrosome proteins; black dots indicate the control protein, as well as significant interactors enriched in the sample.
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Figure 1: Identification of cytoplasmic interactors of GFP::SPD-2 and SPD-5::GFP using label-free quantification mass spectrometry. (A) Western blot showing depletion of endogenous SPD-5 or SPD-2 after 24 h of RNAi treatment. SPD-5 is fully removed from N2 worms after 24 h of RNAi. Endogenous SPD-2 levels in OD824 (GFP::SPD-2-rr) are reduced to 25% after 24 h of spd-2-rr RNAi, whereas GFP::SPD-2-rr levels increase threefold. (B) Embryonic viability of N2, OD824 (GFP::SPD-2rr), and TH327 (SPD-5::GFP codon optimized to cai 0.65) embryos after 24 h of treatment with spd-5 or spd-2-rr RNAi. Both transgenes were found to restore embryo viability when endogenous protein was removed. (C, D) Identification of cytoplasmic interactors of SPD-5::GFP and GFP::SPD-2. SPD-5::GFP or GFP::SPD-2 was immunopurified from C. elegans embryo extracts, and the coimmunoprecipitated proteins were identified via label-free quantification mass spectrometry. TBG-1::GFP immunoprecipitates were used as a control. Experiments were repeated in triplicate. Results are plotted as volcano plots, with the enrichment of identified peptides in sample vs. control plotted on the x-axis and the significance of enrichment on the y-axis. p values and enrichments were calculated as described previously (Hubner et al., 2010). Black circles indicate nonenriched centrosome proteins; black dots indicate the control protein, as well as significant interactors enriched in the sample.

Mentions: To identify potential cytoplasmic interactions of the PCM proteins SPD-2 and SPD-5, we used label-free-quantitative mass spectrometry. For proper quantification of protein amounts using this technique, the immunoprecipitated proteins must carry the same tag. We therefore used green fluorescent protein (GFP)–tagged versions of SPD-2 and SPD-5 (GFP::SPD-2 and SPD-5::GFP, respectively), which rescue depletion of the corresponding endogenous proteins and therefore are functional (Figure 1, A and B). First, we used ultracentrifugation to generate centrosome-free cytoplasmic extracts from transgenic C. elegans embryos expressing GFP::SPD-2 and SPD-5::GFP, similar to what was described by Gopalakrishnan et al. (2011). GFP::SPD-2 or SPD-5::GFP was then immunoprecipitated from these extracts using bead-coupled GFP antibodies and eluted, and then the coprecipitating proteins were identified by mass spectrometry; each experiment was repeated three times. The enrichment and significance of the enrichment of an identified interactor were assessed via label-free quantification using control protein (TBG-1::GFP) immunoprecipitates as described by Hubner et al. (2010). Analysis of SPD-5::GFP immunoprecipitates revealed that, in addition to SPD-5 (2022-fold enriched in comparison to the control), the most enriched proteins were RSA-2 (63-fold enriched) and RSA-1 (29-fold enriched) (Figure 1C), two known regulatory subunits of the protein phosphatase 2A (PP2A) complex (Schlaitz et al., 2007). This result is consistent with previous LAP::RSA-1 immunoprecipitations from centrosome-containing whole-worm extracts by Schlaitz et al. (2007). A novel interaction was found between SPD-5 and the RNA-binding protein RNP-6 (25-fold enriched above simulated noise level; see Materials and Methods), which shows a postembryonic lethal phenotype upon depletion (Sönnichsen et al., 2005). However, since RNP-6 depletion did not affect centrosomes during the first cell division (unpublished data), its role was not investigated further. Of importance, SPD-2, the centrosome-related kinases PLK-1 and AIR-1, and the centriole proteins SAS-4/-5/-6 were not detected in the immunoprecipitates. This data suggest that SPD-5 molecules interact with RSA-1, RSA-2, and RNP-6 in cytoplasmic embryo extracts.


The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.

Wueseke O, Bunkenborg J, Hein MY, Zinke A, Viscardi V, Woodruff JB, Oegema K, Mann M, Andersen JS, Hyman AA - Mol. Biol. Cell (2014)

Identification of cytoplasmic interactors of GFP::SPD-2 and SPD-5::GFP using label-free quantification mass spectrometry. (A) Western blot showing depletion of endogenous SPD-5 or SPD-2 after 24 h of RNAi treatment. SPD-5 is fully removed from N2 worms after 24 h of RNAi. Endogenous SPD-2 levels in OD824 (GFP::SPD-2-rr) are reduced to 25% after 24 h of spd-2-rr RNAi, whereas GFP::SPD-2-rr levels increase threefold. (B) Embryonic viability of N2, OD824 (GFP::SPD-2rr), and TH327 (SPD-5::GFP codon optimized to cai 0.65) embryos after 24 h of treatment with spd-5 or spd-2-rr RNAi. Both transgenes were found to restore embryo viability when endogenous protein was removed. (C, D) Identification of cytoplasmic interactors of SPD-5::GFP and GFP::SPD-2. SPD-5::GFP or GFP::SPD-2 was immunopurified from C. elegans embryo extracts, and the coimmunoprecipitated proteins were identified via label-free quantification mass spectrometry. TBG-1::GFP immunoprecipitates were used as a control. Experiments were repeated in triplicate. Results are plotted as volcano plots, with the enrichment of identified peptides in sample vs. control plotted on the x-axis and the significance of enrichment on the y-axis. p values and enrichments were calculated as described previously (Hubner et al., 2010). Black circles indicate nonenriched centrosome proteins; black dots indicate the control protein, as well as significant interactors enriched in the sample.
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Figure 1: Identification of cytoplasmic interactors of GFP::SPD-2 and SPD-5::GFP using label-free quantification mass spectrometry. (A) Western blot showing depletion of endogenous SPD-5 or SPD-2 after 24 h of RNAi treatment. SPD-5 is fully removed from N2 worms after 24 h of RNAi. Endogenous SPD-2 levels in OD824 (GFP::SPD-2-rr) are reduced to 25% after 24 h of spd-2-rr RNAi, whereas GFP::SPD-2-rr levels increase threefold. (B) Embryonic viability of N2, OD824 (GFP::SPD-2rr), and TH327 (SPD-5::GFP codon optimized to cai 0.65) embryos after 24 h of treatment with spd-5 or spd-2-rr RNAi. Both transgenes were found to restore embryo viability when endogenous protein was removed. (C, D) Identification of cytoplasmic interactors of SPD-5::GFP and GFP::SPD-2. SPD-5::GFP or GFP::SPD-2 was immunopurified from C. elegans embryo extracts, and the coimmunoprecipitated proteins were identified via label-free quantification mass spectrometry. TBG-1::GFP immunoprecipitates were used as a control. Experiments were repeated in triplicate. Results are plotted as volcano plots, with the enrichment of identified peptides in sample vs. control plotted on the x-axis and the significance of enrichment on the y-axis. p values and enrichments were calculated as described previously (Hubner et al., 2010). Black circles indicate nonenriched centrosome proteins; black dots indicate the control protein, as well as significant interactors enriched in the sample.
Mentions: To identify potential cytoplasmic interactions of the PCM proteins SPD-2 and SPD-5, we used label-free-quantitative mass spectrometry. For proper quantification of protein amounts using this technique, the immunoprecipitated proteins must carry the same tag. We therefore used green fluorescent protein (GFP)–tagged versions of SPD-2 and SPD-5 (GFP::SPD-2 and SPD-5::GFP, respectively), which rescue depletion of the corresponding endogenous proteins and therefore are functional (Figure 1, A and B). First, we used ultracentrifugation to generate centrosome-free cytoplasmic extracts from transgenic C. elegans embryos expressing GFP::SPD-2 and SPD-5::GFP, similar to what was described by Gopalakrishnan et al. (2011). GFP::SPD-2 or SPD-5::GFP was then immunoprecipitated from these extracts using bead-coupled GFP antibodies and eluted, and then the coprecipitating proteins were identified by mass spectrometry; each experiment was repeated three times. The enrichment and significance of the enrichment of an identified interactor were assessed via label-free quantification using control protein (TBG-1::GFP) immunoprecipitates as described by Hubner et al. (2010). Analysis of SPD-5::GFP immunoprecipitates revealed that, in addition to SPD-5 (2022-fold enriched in comparison to the control), the most enriched proteins were RSA-2 (63-fold enriched) and RSA-1 (29-fold enriched) (Figure 1C), two known regulatory subunits of the protein phosphatase 2A (PP2A) complex (Schlaitz et al., 2007). This result is consistent with previous LAP::RSA-1 immunoprecipitations from centrosome-containing whole-worm extracts by Schlaitz et al. (2007). A novel interaction was found between SPD-5 and the RNA-binding protein RNP-6 (25-fold enriched above simulated noise level; see Materials and Methods), which shows a postembryonic lethal phenotype upon depletion (Sönnichsen et al., 2005). However, since RNP-6 depletion did not affect centrosomes during the first cell division (unpublished data), its role was not investigated further. Of importance, SPD-2, the centrosome-related kinases PLK-1 and AIR-1, and the centriole proteins SAS-4/-5/-6 were not detected in the immunoprecipitates. This data suggest that SPD-5 molecules interact with RSA-1, RSA-2, and RNP-6 in cytoplasmic embryo extracts.

Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

Show MeSH