Fig 1: POC1A and POC1B are luminal centriolar proteins with a similar domain architecture and centriolar localisation.a Schematic representation of POC1A and POC1B with the WD40 domain at the N-terminus and the coiled-coil (CC) at the C-terminus. b Ensembles of the 10 best-ranked AlphaFold2 predictions of POC1A and POC1B. Numbers 1–7 indicate WD40 domain blades. Colouring based on pLDDT score: the linker region (dotted line) has a lower confidence level than the WD40 and CC. c Conserved features between POC1A and POC1B in the regions with the higher confidence level. d U-ExM images showing the centriolar localisation of POC1A and POC1B in different cell cycle phases. Centrioles were stained with α-tubulin (grey) and POC1A or POC1B (red) antibodies. Scale bars: 100 nm. e Quantification of G1 cells from (d) measuring the coverage rate of POC1A and POC1B along the centriole. n = 26 (POC1A), 24 (POC1B) centrioles. f Schematic representation of POC1A (blue) and POC1B (green) along the centriole normalised to centrioles with a length of 500 nm. g Top view U-ExM images from centrioles stained with α-tubulin (grey) and POC1A or POC1B (red) antibodies. Scale bar: 100 nm. n = 28 (POC1A), 20 (POC1B) centrioles. h Diameter analysis of top view centrioles shown in (g). i U-ExM images from top view centrioles stained against α-tubulin (grey) and the indicated centriolar proteins. Scale bar: 100 nm. j Quantification of (g, i) measuring the distance of the respective proteins to the α-tubulin signal. n = 28 (POC1A), 20 (POC1B), 20 (POC5), 12 (CCDC15), 10 (FAM161A), 25 (MDM1), 16 (CEP44) centrioles. k Model of the protein organisation within the centriole, comprising the inner scaffold proteins. l HA-tagged POC1A and POC1B constructs and their ability to localise to centrosomes. m Representative IF images from one experiment of control cells expressing the constructs shown in (l). Cells were stained against HA (green) and γ-tubulin (red). Scale bars: 5 µm, magnification scale bars: 1 µm. N = 3 biologically independent experiments. e, h, j Data are presented as mean ± SD. All statistics were derived from two-tail unpaired t-test. Source data are provided as a Source Data file.
Fig 2: Different POC1 interaction partners rely on different binding mechanisms.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.
Supplier Page from Sino Biological, Inc. for Human MDM1 Gene ORF cDNA clone expression plasmid