Fig. 7: An inner luminal protein network ensures the formation of the inner scaffold and the integrity of centrioles. | Nature Communications

Fig. 7: An inner luminal protein network ensures the formation of the inner scaffold and the integrity of centrioles.

From: An interaction network of inner centriole proteins organised by POC1A-POC1B heterodimer crosslinks ensures centriolar integrity

Fig. 7

Representative negative stain Electron Microscopy (EM) micrograph from purified human wild type POC5-Centrin2 (a) or POC5Δ153-184-Centrin2 (b) complex expressed in insect cells. The numbers in the left upper corner of the 2D class averages indicate particle numbers. Scale bars: 100 nm and 10 nm. N = 1 biologically independent experiment. c Comparison of wild type POC5-Centrin2 and POC5Δ153-184-Centrin2 complexes. Left: negative stain 2D classes, scale bars and particles numbers are given. Right: negative stain EM 3D reconstructions of wild type POC5-Centrin2 (grey) and POC5Δ153-184-Centrin2 (blue). POC5Δ153-184-Centrin2 particles show a structure shorter than the wild type. d U-ExM images of POC5−/− cells expressing the HA-tagged mutant POC5Δ153-184 and stained against HA (magenta), γ-tubulin (green), α-tubulin (grey), M = merged channels. Scale bar: 100 nm. N = 2 biologically independent experiments. e The rod-like POC5-Centrin2 negative stain EM 3D reconstruction (salmon) presented in (a, c) was fitted into a published1 subtomogram average of a microtubule triplet from Paramecium tetraurelia (emd-4926). f U-ExM images of control and POC1A/B−/− cells expressing POC1A or POC1B tagged at the N-terminus with EGFP and stained against EGFP (green) and α-tubulin (grey). Scale bar: 100 nm. g Quantification of centrioles shown in (f). The distance between the EGFP signal and α-tubulin was measured from top view centrioles and compared to the distance exhibited when stained with an antibody detecting epitopes at the C-terminus of the POC1 proteins (labelled as POC1A and POC1B, respectively). The data set for centrioles stained with the antibodies detecting the M/C-portions of the POC1 proteins was shown in Fig. 1j and is included in the graph of (g) for better comparison. Data are presented as mean ± SD. Statistics were derived from two-tail unpaired t-test. n of Control cell lines: 28 (POC1A), 18 (EGFP-POC1A), 20 (POC1B), 12 (EGFP-POC1B) centrioles. n of POC1A/B−/− cell lines: 17 (EGFP-POC1A), 14 (EGFP-POC1B) centrioles. Source data are provided as a Source Data file. h Model of the inner scaffold protein network within the centriole. See Discussion for details.

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