Fig. 4: Different POC1 interaction partners rely on different binding mechanisms. | Nature Communications

Fig. 4: Different POC1 interaction partners rely on different binding mechanisms.

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

Fig. 4

a RPE1 POC5−/− cells expressing different Dox-inducible versions of HA-tagged POC5 constructs were checked for centrosomal localisation by IF. Cells were stained against HA-POC5 (green) and γ-tubulin (red). Scale bars: 5 µm, magnification scale bars: 1 µm. b Quantification of (a). Percentage of interphase cells showing centrosomal and cytoplasmatic POC5 localisation. Data are presented as mean ± SD. N = 2 biologically independent experiments, n > 110 cells per cell line for each experiment. Source data are provided as a Source Data file. c Immunoblot of the cell lines from (a). The lower HA-immunoblot is a longer exposer of the upper one. GAPDH is used as a loading control. N = 2 biologically independent experiments. d Representative U-ExM images from intact centrioles of RPE1 POC5−/− cells expressing either full-length POC5 or POC5∆472-532 and stained against α-tubulin (grey) and γ-tubulin (red), M= merged channels. POC5∆472–532 cannot rescue the luminal γ-tubulin localisation. Scale bars: 100 nm. N = 3 biologically independent experiments. e Ensemble interaction map based on AlphaFold-Multimer predictions of an interaction between POC1A (light blue) or POC1B (green) and MDM1 (salmon) (see Materials and Methods—AlphaFold-Multimer predictions). Interactions predicted to be more robust, appear darker (black) and thicker. The coiled-coil regions of POC1A and POC1B and a C-terminal segment of MDM1 mediate mainly the interactions. aa: amino acids. f Representative FLAG IP from HEK293T cells expressing FLAG-tagged full-length or subdomains of either POC1A or POC1B together with HA-tagged MDM. Vinculin is used as input control. N = 3 biologically independent experiments. g Ensemble interaction map based on AlphaFold-Multimer predictions of an interaction between POC1A (light blue) or POC1B (green) and FAM161A (salmon). The WD40 domain as well as the coiled-coil regions of both POC1 proteins might be involved in the interaction. aa: amino acids. PAE plots and confidence scores for (e, g) are shown in Supplementary Figs. 9 and 10. h, i Representative HA IP from HEK293T cells expressing HA-tagged FAM161A and FLAG-tagged subdomains of POC1A or POC1B. GAPDH was used as input control. N = 3 biologically independent experiments.

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