Fig. 7
From: Direct binding of CEP85 to STIL ensures robust PLK4 activation and efficient centriole assembly
The interaction between CEP85 and STIL is essential for PLK4 activation and PLK4-induced centriole overduplication. a, b Effect of CEP85 mutations on centriole overduplication. U-2 OS cells conditionally expressing Myc-PLK4 and constitutively expressing GFP-CEP85 (WT and mutant) were used to perform the PLK4-induced centriole overduplication assays. Cells were labelled with DAPI and the indicated antibodies. Scale bar 10 μm, white boxes indicate the magnified region. b Quantification showing the percentage of cells with over four centrioles (n = 100/experiment, three independent experiments). c Western blot analysis indicating the Myc-PLK4 and GFP-CEP85 protein levels using the PLK4 assays described in A. Cyclin A was used as a cell cycle marker and α-tubulin served as a loading control. d Quantification of protein levels shown in C (α-tubulin normalized, n = 2/experiment, six independent experiments). e Effect of CEP85 mutations on PLK4 activation. The PLK4-induced centriole overduplication assays were performed as described in a. Selected images showing Myc-PLK4 and PLK4 pS305 labelling. Scale bar 10 μm, white boxes indicate the magnified region. f, g The graph indicates the relative levels of Myc-PLK4 and the relative ratio of pS305/PLK4 at centrosomes. (n = 100/experiment, three independent experiments). h A model for how CEP85 operates in the centriole duplication pathway. CEP85 acts downstream of CEP192, CEP152 and PLK4 in centriole duplication. Direct binding of CEP85 to STIL stabilises STIL and facilitates its recruitment to centrosomes. This further facilitates robust PLK4 activation and subsequent centriole assembly. Two-tailed t-test was performed for all p-values, all error bars represent SD, and asterisks for p-values are **p < 0.01 and *p < 0.05
