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

Item Type:Article
Title:Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes
Creators Name:Cadar, A.G. and Feaster, T.K. and Bersell, K.R. and Wang, L. and Hong, T.T. and Balsamo, J.A. and Zhang, Z. and Chun, Y.W. and Nam, Y.J. and Gotthardt, M. and Knollmann, B.C. and Roden, D.M. and Lim, C.C. and Hong, C.C.
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 (hiPSC) to knock-in mEos3.2, into the C-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. 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 hours, 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.
Keywords:FRAP, hiPSC-CM, mEos3.2, Sarcomere, Titin
Source:American Journal of Physiology Cell Physiology
Publisher:American Physiological Society
Page Range:C163-C173
Date:January 2020
Additional Information:Copyright © 2020 the American Physiological Society
Official Publication:https://doi.org/10.1152/ajpcell.00107.2019
External Fulltext:View full text on PubMed Central
PubMed:View item in PubMed

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