Fig 2: HBR of CEP215 mediates HSET binding exclusively, whereas its CM1 domain is responsible for multiple interactions.(a) Table depicts summary of TAP-tagged cell lines. The panel below shows the expression of protein products from CEP215-TAP, CEP215?HBR-TAP and CEP215?CM1-TAP cell lines. (b) CEP215-containing protein complexes were affinity purified from CEP215-TAP, CEP215?HBR-TAP and CEP215?CM1-TAP cells, followed by western blotting for indicated antibodies. (c) Binding of MBP-CEP215 (1–300) to microtubules was assayed using microtubule spin-down in the presence of tubulin (-taxol) or taxol-stabilized (+taxol) microtubules. MBP served as a negative control. Following centrifugation supernatants (S) and pellets (P) were loaded on gel and stained with Coomassie blue. (d) Microtubule spin-down assays were performed from lysates of CEP215-TAP, CEP215?HBR-TAP and CEP215?CM1-TAP cells in the presence of tubulin (-taxol) or taxol-stabilized microtubules (+taxol). Following centrifugation supernatants (S) and pellets (P) were subjected to western blotting. Antibodies for immunoblotting are indicated. Arrowhead marks the panel depicting the reduction of CEP215(?CM1)-TAP binding to microtubules. (e) Pericentrosomal CEP215 particles are visualized in DMSO- and MG132-treated WT, CEP215?HBR and CEP215?CM1 cells. Cells were stained for CEP215 (green) and ?-tubulin (red). DNA is in blue. Arrow highlights a particle. Insets show higher magnification of CEP215 and ?-tubulin stainings corresponding to framed areas. Scale bar, 4 µm. (f) Graphs show quantitation of pericentrosomal CEP215 particles as percentage of mitotic cells in the presence of DMSO or MG132. P values of paired t-tests (*P<0.05, **P<0.005); n=3 biological replicates. Error bars correspond to standard deviation. (g) DMSO- and MG132-treated WT cells were stained for CEP215 (green) and HSET (red). DNA is in blue. Scale bar, 4 µm. Insets show higher magnification of CEP215 and HSET stainings corresponding to framed areas. Note co-localization of the two proteins on pericentrosomal particles.

Fig 3: CEP215 and HSET interact through vertebrate-specific binding domains.(a) Left panel depicts the workflow for separation of TAP-CEP215-bound complexes on a 5–40% sucrose gradient. Western blots of sucrose fractions probed with antibodies as indicated. (b) Whole-cell extracts (WCE) of HeLa cells expressing FLAG-tagged CEP215 fragments were subjected to FLAG pull-down followed by western blotting with the indicated antibodies. (c) WCE of CEP215-depleted HeLa cells expressing Bioease-tagged CEP215 fragments as indicated were subjected to streptavidin (strep) pull-down followed by western blotting with the indicated antibodies. (d) WCE of HeLa cells expressing Bioease-tagged HSET fragments were subjected to streptavidin (strep) pull-down followed by western blotting with the indicated antibodies. (e) HSET and CEP215 bind directly. Graph depicts qualitative analysis of binding between MBP-tagged CEP215 fragments (substrates) and GST-tagged HSET fragments (ligands) using surface plasmon resonance plotted as relative response units. GST and MBP proteins were used as controls. MBP shows background response for each analyte. Values for three technical replicates are shown. Error bars correspond to standard deviation. (f) Sequences of HBR of CEP215 and aa1–150 of HSET have been analysed across 97 organisms (Supplementary Fig. 2d). Dark grey cells indicate high sequence conservation within HBR of CEP215 and aa1–150 of HSET. Light grey cells depict lesser conservation of aa1–150 of HSET. Compared with human HSET aa1–150, invertebrates showed an average sequence identity of 12% in contrast to 54% among vertebrates. White cells depict the absence of HBR in CEP215 orthologues. Numbers in parentheses represent the number of organisms per class for which CEP215 and/or HSET sequences are available. (g) WCE of mitotic HeLa cells were subjected to immunoprecipitation by an anti-CEP215 antibody or random IgG (con) followed by western blotting with the indicated antibodies. (h) WCE of Drosophila Dmel2 cells were subjected to immunoprecipitation by an anti-centrosomin (Cnn) antibody followed by western blotting with the indicated antibodies.

Fig 4: Immunofluorescence co-localization experiment. Top: control experiment is performed in absence of DNA molecules. Kinesin KIFC1 bound to the microtubules is visualized in the FITC channel (image on the left). On the right is the FRET channel to confirm that there is no fluorescence passage from KIFC1. Bottom: DNA experiment is performed by using a DNA marked with Cy3. On the left is the KIFC1 in the FITC channel. On the right we observe the DNA molecules excited by the secondary antibody bound to KIFC1. Scale bar is 10 µm.

Fig 5: CEP215 and HSET promote centrosome association with mitotic spindle poles and centrosome clustering in human breast cancer cells.(a) Western blots of whole-cell extracts of BT-549 cells prepared 72 h after siRNA transfections. Untreated (unt) cells are included as controls. Antibodies for immunoblotting are indicated. (b) Still frames from a time-lapse experiment show mitosis in BT-549 cells untreated (unt) or treated with control (siCon), CEP215 (siCEP215) or HSET (siHSET) siRNAs. Graph depicts the number of multipolar anaphases in cells treated with the indicated siRNAs. Total number of mitoses analysed per treatment is shown. (c) Graph shows viability of untreated (unt) or siRNA-treated BT-549 cells as a function of relative light units (RLU) using CellTiter-Glo assay. n=3 biological replicates, where error bars denote standard deviation and statistical significance was computed using paired t-test. (d) Images of siRNA-treated BT-549 cells stained for the centriolar marker CPAP (green) and a-tubulin (red). DNA is in blue. Arrows mark detached centrosomes. Scale bar, 6 µm. (e) Graphs show quantifications of centrosome and spindle phenotypes (as depicted in schematics) in siRNA-treated BT-549 cells. Two-way ANOVA followed by Tukey's test were performed (*P<0.05, **P<0.005); n=3 biological replicates. Error bars correspond to standard deviation. (f) Schematic representation of the proposed function of CEP215 at the centrosome–spindle pole interface. Briefly, through HBR CEP215 captures HSET- bound minus ends of k-fibres and interpolar microtubules, thereby anchoring these at the centrosome. CEP215 may also capture dynein-associated microtubules through the CM1 domain.