FLIM‐FRET‐based analysis of S100A11/annexin interactions in living cells

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)‐based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1‐containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM‐FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells. Proteins often fulfill their functions in interaction with other proteins within a protein complex. The calcium‐binding annexin proteins and S100 proteins interact with each other and are involved in cellular membrane dynamics, vesicle transport, and vesicle fusion. We have analyzed the interaction of different members of the annexin protein family with S100A10 and S100A11 using FLIM‐based FRET measurements.