Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes

Abstract

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.

Cardiac sarcomere mechanics in health and disease

“…Considering the impressive molecular weights of titin and the plethora of possible isoforms, it could be tempting to think that titin protein homeostasis is a static and slow process. However, titin dynamics studies revealed an unexpected level of protein mobility in and out of sarcomeres (da Silva et al 2011 ; Rudolph et al 2019 ; Cadar et al 2020 ). Pulse-chase experiments demonstrated that the protein half-life of titin is approximately 70 h in cultured skeletal muscle cells (Isaacs et al 1989 ).…”

Imaging of existing and newly translated proteins elucidates mechanisms of sarcomere turnover

“…Several fluorescent molecules, including GFP, were shown to exhibit photoswitching 39 , and such reversible photoswitching may appear as mimicking diffusion. Indeed, one study that used fluorescently tagged TTN found extensive reversible photobleaching that accounted for most of the TTN fluorescence recovery 40 . Our experiments did not reveal any recovery of OG Halo-tagged sarcomeric proteins, even when we bleached at a submaximal level.…”

“…Indeed, one study that used fluorescently tagged TTN found extensive reversible photobleaching that accounted for most of the TTN fluorescence recovery 40 . Our experiments did not reveal any recovery of OG Halo-tagged sarcomeric proteins, even when we bleached at a submaximal level.…”

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy

“…Human induced pluripotent stem cell (iPSC) lines were generated from healthy controls using an episomal approach and validated previously. [9][10][11] All protocols were approved by the Vanderbilt University Institutional Review Board and informed consent was given.…”