Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes
Autor: | Chee Chew Lim, Michael Gotthardt, Lili Wang, Joseph A. Balsamo, Charles C. Hong, Dan M. Roden, Bjorn C. Knollmann, Tromondae K. Feaster, Young-Jae Nam, Kevin R. Bersell, Young Wook Chun, Adrian G. Cadar, TingTing Hong, Zhentao Zhang |
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Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
Adult Male Sarcomeres Myofilament Fluorophore animal structures Physiology Recombinant Fusion Proteins Induced Pluripotent Stem Cells macromolecular substances 030204 cardiovascular system & hematology Sarcomere Cell Line 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Humans Connectin Myocytes Cardiac Induced pluripotent stem cell Microscopy Video biology Dynamics (mechanics) Fluorescence recovery after photobleaching Reproducibility of Results Cell Differentiation Cell Biology musculoskeletal system Photobleaching Kinetics Luminescent Proteins 030104 developmental biology chemistry Microscopy Fluorescence biology.protein Biophysics cardiovascular system Titin tissues Fluorescence Recovery After Photobleaching Research Article |
Zdroj: | Am J Physiol Cell Physiol |
ISSN: | 1522-1563 |
Popis: | 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. |
Databáze: | OpenAIRE |
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