Figure 1.

Design of a calpain-sensitive marker, pYSCS. (A) Using the pEGFP-C3 vector (CLONTECH), EYFP and ECFP were fused in-frame with an intervening sequence from the μ-calpain cleavage site in α-spectrin. A PDZ cognate sequence that binds to PSD95 was added at the 3′ end to facilitate retention of the expressed protein at postsynaptic sites. (B) Expression of pYSCS yielded a fusion protein from which FRET could be visualized in situ after excitation of the donor ECFP fluorophore. Separation of ECFP and EYFP by μ-calpain abolishes FRET, resulting in decreased EYFP fluorescence and a concomitant increase of ECFP fluorescence.