Figure 3. Endoglin missense mutants form homodimers and heterodimers with wild-type endoglin as demonstrated by BiFC.

In order to investigate the possible assembly of mutant and wild-type (wt) homo- and heterodimers, the Bimolecular Fluorescence Complementation method was applied. The BiFC constructs represent the different endoglin protein variants to which either the N-terminal part of the EYFP protein, N172 (aa 1–172), or the C-terminal part of the EYFP protein, 173C (aa 173–238) was fused to the C-terminus of the respective endoglin variant. In the case of dimerization of an endoglin-N172 and endoglin-173C protein, the EYFP becomes recomplemented and EYFP fluorescence is restored. CHO cells were co-transfected with different endoglin BiFC construct combinations, as indicated. For the visualization of the heterodimers (row 1+3), both possible BiFC combinations are illustrated, in which the two BiFC fragments N172 and 173C fused to the two different endoglin variants were interchanged. The right column of the image shows only endoglin^wt/wt homodimers, as interchanging of BiFC fragments does not apply.