Key result
Titin-mEos3.2 hiPSC-CMs rely on reversible photobleaching for most FRAP recovery unlike the eGFP model.
Why the study?
FRAP assumes irreversible photobleaching of fluorescent proteins after laser excitation, but whether reversible photobleaching occurs with mEos3.2 when studying titin dynamics was uncertain.
Reversible photobleaching of the mEos3.2 fluorescent protein accounts for the majority of titin recovery in hiPSC-CM models, warranting caution in interpreting FRAP data from specific fluorescent proteins.
Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.
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Cadar et al. (2019) studied Cardiac myofilament biology. mEos3.2 fluorescent protein vs. mouse titin-eGFP model was evaluated on Fluorescence recovery after photobleaching (FRAP). Reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model during FRAP, unlike the titin-eGFP model which showed no reversible photobleaching.
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