FIGURE 5.

Discrimination of different types of dimeric interactions among three potential interaction partners using fluorescence lifetime-based measurements. (A) Representative images showing the color-coded fluorescence lifetime of mTurquoise2 before bleaching (unbleached), after the first bleaching step (mCherry bleached) and after the second bleaching step (YPet bleached). (B) Change in average amplitude weighted lifetime of the cells during the two-step bleaching protocol (n = 6). After mCherry bleaching, the fluorescence lifetime increased in TY&TC, TC&YC and TY&TC&YC samples, but remained constant in TY&YC. After YPet bleaching, the fluorescence lifetime strongly increased in TY&TC, TY&YC and TY&TC&YC, but only moderately in TC&YC. (C,D) Relative change in fluorescence lifetime of mTurquoise2 after mCherry bleaching [(C), first bleaching step] and the additional relative change in fluorescence lifetime after YPet bleaching [(D), second bleaching step] (n = 6). Gray lines indicate threshold values to discriminate FRET-specific from non-specific lifetime changes of the donor. (E) Updated color-coded response patterns and decision matrix of fluorescence lifetime-based measurements. For each direction of FRET, a Yes-response (occurrence of specific FRET) is indicated by a green and a No-response (no or non-specific FRET) is indicated by a red square. Without information about YC-related FRET, these patterns cannot be used to discriminate dimeric from trimeric and double-dimeric interactions as it was shown for the intensity-based patterns. (F) However, the different basal fluorescence lifetimes of the combination of all three dimers versus each of the FRET triplet constructs allow for their discrimination of multi-dimeric and trimeric interactions which was not possible from the intensity-based pattern.