Category: Sarcomere

Sarcomeric thick filaments via MLC-2v

2/20/2019

Movie. High magnification Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2v. 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 28 days later (40 days total after the onset of differentiation). Cells were treated with 20 mM of the myosin inhibitor 2,3-butanedione monoxime (BDM) to prevent beating during image acquisition. Inset is a 3x enlargement of the boxed region to show detail of MLC-2v in myofibrils. Movie starts at the bottom of the cells and ends at the top. Scale bar, 20 µm.

Movie. Time-lapse in high magnification movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2v 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 28 days later (40 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset is a 3x enlargement of the boxed region to show detail of MLC-2v in myofibrils. Movie plays in real time.Scale bar, 20 µm.

Observations

MLC-2v is the cardiac ventricular isoform of the Myosin Light Chain 2 (MLC-2) protein. It localizes to the thick filament of the sarcomere where it binds to the myosin heavy chain and functions to regulate myosin-based contractility. The expression of MLC-2v is developmentally regulated; it is frequently used as a marker of cardiac development due to its up-regulation with ventricular development.
In hiPSC-derived cardiomyocytes, we observed mEGFP-tagged MLC-2v in a striated appearance along myofilaments, reflecting its localization to the thick filaments of sarcomeres, and absence from the Z-disk and I-band. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.

Sarcomere M-line visualized via Titin

8/1/2018

Movie. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin 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 23 days later (35 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 titan in myofibrils. Scale bars, 10 µm.

Movie. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin 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 23 days later (35 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 titan in myofibrils. Scale bars, 10 µm. Movie plays in real time.

Observations

Titin is the largest known protein. It stretches across the sarcomere from the Z-disk (at its N-terminus) to the M-line (at its C-terminus). Titin functions like a spring when under tension with the contraction of sarcomeres. Titin also plays a structural role in regulating the length of the thick filament within sarcomeres.
In hiPSC-derived cardiomyocytes, titin tagged at its C-terminus with mEGFP localizes to thin lines that striate myofilaments, consistent with localization to the M-line of sarcomeres.
In hiPSC-derived cardiomyocytes from the mEGFP tagged Titin line, the fluorescent signal localizes to thin lines that striate myofilaments. While Titin spans the entire length from the Z-disk to the M-line, the much smaller mEGFP only highlights the location of the tagged Titin C-terminus. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.

Sarcomere thick filaments visualized via MLC-2a

8/1/2018

Movie. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2a. 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 20 days later (32 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 MLC-2a in myofibrils. Scale bars, 10 µm.

Movie. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2a 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 19 days later (31 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 MLC-2a in myofibrils. Scale bars, 10 µm. Movie plays in real time.

Observations

MLC-2a is the cardiac atrial isoform of the Myosin Light Chain 2 (MLC-2) protein. It localizes to the thick filament of the sarcomere where it functions to regulate myosin-based contractility. The expression of MLC-2a is developmentally regulated; it is frequently used as a marker of cardiac development due to its down-regulation with ventricular development.
In hiPSC-derived cardiomyocytes, we observed mEGFP-tagged MLC-2a in a striated appearance along myofilaments, reflecting its localization to the thick filaments of sarcomeres, and absence from the Z-disk and I-band. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.

Sarcomere visualized via Troponin I, slow skeletal muscle

4/24/2018

Movie. Timelapse movie of sarcomeres in beating cells. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged troponin I. Cells were continuously imaged on a spinning-disk confocal microscope with a 100ms exposure time. Inset shows detail of sarcomeres in myofibrils. Scale bars, 5 µm and 10 µm for inset and larger field respectively. Movie plays in real time.

Observations

Troponin I, slow skeletal muscle labels the thin filament of sarcomeres (actin-based), the contractile apparatus in muscle cells, and regulates myosin-based contraction. Sarcomeres align end-to-end to form myofibrils, striated filaments that generate force when sarcomeres contract.
Troponin I is absent from Z-disks, which results in a striated appearance along a myofibril. The spacing of this striation shortens as the cardiomyocytes beat. Sometimes buckling of the myofibril can be seen during beating.

Cell Structure Observations

Sarcomeric thick filaments via MLC-2v

Sarcomere M-line visualized via Titin

Sarcomere thick filaments visualized via MLC-2a

Sarcomere visualized via Troponin I, slow skeletal muscle

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