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. 2017 Jun 1;169(6):1078–1089.e13. doi: 10.1016/j.cell.2017.05.030

Figure 5.

Figure 5

The PReM Domain Interacts with CM2 and Forms Large, Micron-Scale Complexes Whose Assembly Is Promoted by Plk1-Mediated Phosphorylation

(A) Schematic illustration of the PReM domain showing the internal LZ (taken from the LZ:CM2 crystal structure) and surrounding sequences that are predicted to be helical (Jones, 1999) (blue bars). The ten Ser/Thr residues potentially phosphorylated by Polo are indicated by red asterisks; the larger asterisk represents the S567 residue used to raise the phospho-specific antibody.

(B and C) SEC-MALS analysis of MBP-PReM (blue) and either CM2 (B) or CM2-T1133E (C) (orange) and MBP-PReM+CM2 (B) or MBP-PReM+CM2-T1133E (C) (green).

(D) Chart quantifies and micrographs show examples of the micron-scale complexes visible by fluorescence microscopy when PReM-GFP is mixed with either WT (left) or mutant (example shown T1133E, right) forms of CM2. Error bars indicate SD. Both proteins are at a final concentration of 20 μM.

(E) Micrographs show a FRAP analysis of protein turnover in the PReM-GFP:CM2 complexes (as seen in [D]). Complexes were imaged (t = −2 min), a small area was photobleached (t = 0 min), and fluorescence recovery monitored (t = 20 min).

(F) Western dot-blot shows that PReM-GFP is phosphorylated by purified Plk1 in vitro, allowing the phospho-specific Cnn-pS567 antibody to recognize the protein; the same blot was probed with anti-GFP antibodies to confirm equal loading of PReM-GFP.

(G) Graph quantifies the visible area of the PReM-GFP:CM2 complexes (both proteins at a final concentration of 10 μM) at various time points after mixing when the PReM-GFP protein has been pre-treated with Plk1 (red line) or with buffer control (gray line). Error bars indicate SD (n = three independent experimental replicates; note that one outlier time point in one experiment that was ∼10 times brighter than all the others was excluded from this analysis).

(H) Micrographs show how anti-Cnn-p567 antibodies (red) preferentially recognize the inner region of the centrosome and are largely absent from the more peripheral regions (in some cases highlighted with arrows) recognized by antibodies that recognize total Cnn (green). See also Figure S6. Scale bars = 10 μm (A), 5 μm (E), and 3 μm (H).