Figure 4. The TCP domain of C. elegans SAS-4 is required for its interaction with SAS-5, its localisation to centrioles, and for centriole assembly.
(A) Schematic illustration of the MosSCI system used for generating single-copy sas-4 transgene insertions. (B) A schematic illustration of the monopolar spindle assay for centriole duplication in C. elegans embryos. Panels show maximum intensity projections of representative fluorescence confocal z-series taken of sas-4(RNAi) embryos expressing either WT or ΔTCP SAS-4::GFP. Transgenic SAS-4WT::GFP localises to sharp foci representing the centrioles, whereas SAS-4ΔTCP::GFP localises diffusely to the pericentriolar material. Bar, 10 μM. (C) Graphs show the quantification of second division monopolar spindles (left) and embryonic viability (right) after sas-4(RNAi) and rescue with either a WT or ΔTCP sas-4::gfp transgene. (D) Panels show autoradiographs (top panel) and a Coomassie stained gel from a Ni-NTA pull-down experiment with 35S-labelled in vitro translated SAS-4 fragments (prey) and SAS-5-6xHis fragments (baits).