Interaction between CENP-C and CENP-A takes place at centromere positions.

<p>HEK293 cells transfected with plasmids expressing the chimeric protein combinations indicated at the top of each column A–E (A: N-GFP::HA::CENPC and HA::CENPA::C-GFP; B: N-GFP::HA::CENPC; C: HA::CENPA::C-GFP; D: N-GFP::HA::CENPCΔ890-943 and HA::CENPA::C-GFP; E: N-GFP::HA::CENPCΔ890-943). After fixation cells were immunolabelled with a monoclonal anti-HA antibody and polyclonal antibodies used as centromere markers (A and D: anti-CENP-B; B and E anti-CENP-A; C: anti-CENP-C). Row I shows complementing GFP (green); row II shows merge between complementing GFP and the centromeric marker (red); row III shows HA-tagged chimeric proteins (cyan); row IV shows merge between HA-tagged chimeric proteins and the centromeric marker; row V shows merge between centromeric marker and DNA (blue). F: diagrams of the chimeric proteins expressed in cells. G: quantification of colocalization between the green signal due to complementing GFP and the centromeric marker in cells that coexpress HA::CENPA::C-GFP and N-GFP::HA::CENPC full length or the truncated mutant N-GFP::HA::CENPCΔ890-943. 10 to 15 nuclei for each experiment were analyzed and the data expressed as ratio between GFP signal corresponding to centromeres and number of visible centromeres. Statistical significance have been checked with T-student test and indicated in the graph. The bright green punctate signals in panel AI indicate efficient GFP complementation in cells coexpressing N-GFP::HA::CENPC and HA::CENPA::C-GFP. The green signals in AI well correspond to centromere positions as indicated by merge in panel AII and to HA-tagged proteins shown in panels AIII-IV. The green signal shown by cells expressing respectively only N-GFP::HA::CENPC or HA::CENPA::C-GFP is very low (BI) or absent (CI) although the chimeric HA-tagged proteins are expressed. In cells expressing N-GFP::A::CENPCΔ890-943 and HA::CENPA::C-GFP few green signals are visible corresponding to centromere position (green and red in DI–II) suggesting lower interaction when CENP-C Mif2 homology domain III is deleted although N-GFP::HA::CENPCΔ890-943 localizes at centromeres (EIII–IV).