Sarcoplasmic reticulum visualized via SERCA2

Movie. ​Z-stack of live hiPS cell colony expressing mEGFP-tagged SERCA2 protein. Cells were imaged in 3D on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. Scale bar, 5µm.

​​Movies. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged SERCA2 protein. Twelve days after the onset of differentiation, cells were plated on PEI and laminin coated glass and imaged in 3D on a spinning disk confocal microscope 29 days later (41 days total after the onset of differentiation). Cells were treated with 15 mM of the myosin inhibitor 2,3-Butanedione monoxime (BDM) to prevent beating during image acquisition. Movie starts at the bottom of the cells and ends at the top. Inset shows detail of SERCA2 in longitudinal sarcoplasmic reticulum (SR). Scale bars, 10 µm.

Movie. Time-lapse movie of live beating hiPSC-derived cardiomyocytes expressing mEGFP-tagged SERCA2 protein. Twelve days after the onset of differentiation, cells were plated on PEI and laminin coated glass and imaged on a spinning disk confocal microscope 8 days later (20 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset shows detail of SERCA2 in longitudinal sarcoplasmic reticulum (SR). Scale bars, 10 µm. Movie plays in real time.

Observations

• SERCA2 (sarco/endoplasmic reticulum calcium ATPase) proteins form pumps that use the energy from ATP hydrolysis to import calcium from the cytosol into the endoplasmic and/or sarcoplasmic reticula (ER and/or SR). This regulates cytosolic calcium concentration, important for many functions, including contractility in cardiomyocytes.
• In hiPSC, mEGFP-taged SERCA2 localizes to the nuclear periphery and to ER tubules. In contrast, while both mEGFP-tagged SERCA2 and Sec61-beta proteins localize to the ER membrane, Sec61 beta localizes to the nuclear periphery as well as both ER tubules and sheets. This difference in localization may reflect a preference for SERCA2 to localize to smooth ER over rough ER.
• In hiPSC-derived cardiomyocytes, mEGFP-tagged SERCA2 localizes to the nuclear periphery and to a tubular network throughout the rest of the cytoplasm. The linear alignment of SERCA2 labeling along presumable myofilaments suggests localization to longitudinal SR.
• mEGFP-tagged SERCA2 moves when cardiomyocytes beat, presumably moving along with the contraction and buckling of myofilaments.