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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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Stoichiometry and shape analysis of SPD-2, SPD-5, and the SPD-5/RSA-1/RSA-2 complex. (A) Cytoplasmic extracts prepared from C. elegans embryos were fractionated by size exclusion chromatography, and an immunoblot of the fractions (collected in 0.5-ml steps) probed for SPD-5, RSA-2, RSA-1, and SPD-2 is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their hydrodynamic radius, by the black arrowheads above the top lane. (B) Graph blotting the measured signal for each protein vs. elution volume. ­Standard calibration curves were repeated twice (dotted lines) and calculated from the position of the standard proteins (filled rectangles). (C) Svedberg coefficients for SPD-5, RSA-1, RSA-2, and SPD-2 were determined by rate zonal ultracentrifugation. An immunoblot of the 25 × 0.2 ml fractions obtained from a 5-ml 15–55% sucrose gradient is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their Svedberg coefficients, by the black arrowheads above the top lane. (D) Graph plotting the measured signal for each protein vs. fraction. The standard calibration curve (dotted line) was calculated from the position of the standard proteins (filled rectangles). (E) Immunoblot of RSA-2 coimmunoprecipitation experiment using RSA-2–specific antibodies on cytoplasmic extract, showing that SPD-5 and RSA-1 coimmunoprecipitate with RSA-2. 1) αRSA-2 antibody–coupled beads were incubated with fresh cytoplasmic extract; 2) the remaining extract from step 1 was reincubated with fresh αRSA-2 antibody–coupled beads to control for full depletion of RSA-2; 3) remaining supernatant from step 2; 4) blank beads were incubated with fresh cytoplasmic extract to control for unspecific binding of beads; 5) remaining supernatant from step 4.
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Figure 2: Stoichiometry and shape analysis of SPD-2, SPD-5, and the SPD-5/RSA-1/RSA-2 complex. (A) Cytoplasmic extracts prepared from C. elegans embryos were fractionated by size exclusion chromatography, and an immunoblot of the fractions (collected in 0.5-ml steps) probed for SPD-5, RSA-2, RSA-1, and SPD-2 is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their hydrodynamic radius, by the black arrowheads above the top lane. (B) Graph blotting the measured signal for each protein vs. elution volume. ­Standard calibration curves were repeated twice (dotted lines) and calculated from the position of the standard proteins (filled rectangles). (C) Svedberg coefficients for SPD-5, RSA-1, RSA-2, and SPD-2 were determined by rate zonal ultracentrifugation. An immunoblot of the 25 × 0.2 ml fractions obtained from a 5-ml 15–55% sucrose gradient is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their Svedberg coefficients, by the black arrowheads above the top lane. (D) Graph plotting the measured signal for each protein vs. fraction. The standard calibration curve (dotted line) was calculated from the position of the standard proteins (filled rectangles). (E) Immunoblot of RSA-2 coimmunoprecipitation experiment using RSA-2–specific antibodies on cytoplasmic extract, showing that SPD-5 and RSA-1 coimmunoprecipitate with RSA-2. 1) αRSA-2 antibody–coupled beads were incubated with fresh cytoplasmic extract; 2) the remaining extract from step 1 was reincubated with fresh αRSA-2 antibody–coupled beads to control for full depletion of RSA-2; 3) remaining supernatant from step 2; 4) blank beads were incubated with fresh cytoplasmic extract to control for unspecific binding of beads; 5) remaining supernatant from step 4.

Mentions: Size-exclusion chromatography revealed that SPD-2 migrated with a hydrodynamic radius (Rh) of ∼6.0 nm (Figure 2, A and B), and rate-zonal ultracentrifugation showed a peak fraction for SPD-2 with a Svedberg coefficient (Sexp) of 3.8 S (Figure 2, C and D). These values yield a calculated molecular weight of 95 kDa (monomeric molecular weight, 92 kDa) and a shape factor of 1.94 (Table 1), indicating that SPD-2 exists as an elongated monomer (Erickson et al., 2009).


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)

Stoichiometry and shape analysis of SPD-2, SPD-5, and the SPD-5/RSA-1/RSA-2 complex. (A) Cytoplasmic extracts prepared from C. elegans embryos were fractionated by size exclusion chromatography, and an immunoblot of the fractions (collected in 0.5-ml steps) probed for SPD-5, RSA-2, RSA-1, and SPD-2 is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their hydrodynamic radius, by the black arrowheads above the top lane. (B) Graph blotting the measured signal for each protein vs. elution volume. ­Standard calibration curves were repeated twice (dotted lines) and calculated from the position of the standard proteins (filled rectangles). (C) Svedberg coefficients for SPD-5, RSA-1, RSA-2, and SPD-2 were determined by rate zonal ultracentrifugation. An immunoblot of the 25 × 0.2 ml fractions obtained from a 5-ml 15–55% sucrose gradient is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their Svedberg coefficients, by the black arrowheads above the top lane. (D) Graph plotting the measured signal for each protein vs. fraction. The standard calibration curve (dotted line) was calculated from the position of the standard proteins (filled rectangles). (E) Immunoblot of RSA-2 coimmunoprecipitation experiment using RSA-2–specific antibodies on cytoplasmic extract, showing that SPD-5 and RSA-1 coimmunoprecipitate with RSA-2. 1) αRSA-2 antibody–coupled beads were incubated with fresh cytoplasmic extract; 2) the remaining extract from step 1 was reincubated with fresh αRSA-2 antibody–coupled beads to control for full depletion of RSA-2; 3) remaining supernatant from step 2; 4) blank beads were incubated with fresh cytoplasmic extract to control for unspecific binding of beads; 5) remaining supernatant from step 4.
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Figure 2: Stoichiometry and shape analysis of SPD-2, SPD-5, and the SPD-5/RSA-1/RSA-2 complex. (A) Cytoplasmic extracts prepared from C. elegans embryos were fractionated by size exclusion chromatography, and an immunoblot of the fractions (collected in 0.5-ml steps) probed for SPD-5, RSA-2, RSA-1, and SPD-2 is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their hydrodynamic radius, by the black arrowheads above the top lane. (B) Graph blotting the measured signal for each protein vs. elution volume. ­Standard calibration curves were repeated twice (dotted lines) and calculated from the position of the standard proteins (filled rectangles). (C) Svedberg coefficients for SPD-5, RSA-1, RSA-2, and SPD-2 were determined by rate zonal ultracentrifugation. An immunoblot of the 25 × 0.2 ml fractions obtained from a 5-ml 15–55% sucrose gradient is shown. Black arrowheads mark the center of the peak for each protein. The centers of the peaks for each standard protein are indicated, along with their Svedberg coefficients, by the black arrowheads above the top lane. (D) Graph plotting the measured signal for each protein vs. fraction. The standard calibration curve (dotted line) was calculated from the position of the standard proteins (filled rectangles). (E) Immunoblot of RSA-2 coimmunoprecipitation experiment using RSA-2–specific antibodies on cytoplasmic extract, showing that SPD-5 and RSA-1 coimmunoprecipitate with RSA-2. 1) αRSA-2 antibody–coupled beads were incubated with fresh cytoplasmic extract; 2) the remaining extract from step 1 was reincubated with fresh αRSA-2 antibody–coupled beads to control for full depletion of RSA-2; 3) remaining supernatant from step 2; 4) blank beads were incubated with fresh cytoplasmic extract to control for unspecific binding of beads; 5) remaining supernatant from step 4.
Mentions: Size-exclusion chromatography revealed that SPD-2 migrated with a hydrodynamic radius (Rh) of ∼6.0 nm (Figure 2, A and B), and rate-zonal ultracentrifugation showed a peak fraction for SPD-2 with a Svedberg coefficient (Sexp) of 3.8 S (Figure 2, C and D). These values yield a calculated molecular weight of 95 kDa (monomeric molecular weight, 92 kDa) and a shape factor of 1.94 (Table 1), indicating that SPD-2 exists as an elongated monomer (Erickson et al., 2009).

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