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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.

Nucleolus visualized via nucleophosmin

4/24/2018

Movie. Z-stack and timelapse movie of nucleoli. The left panel shows a Z-stack of a live hiPS cell colony expressing mEGFP-tagged nucleophosmin. Cells were imaged in 3D on a spinning-disk confocal microscope. Right panel is the same image as the left but with contrast enhanced to visualize dimmer localization in mitotic cells. Movie starts at the bottom of the cells and ends at the top. Scale bar = 5µm.

Movie. Z-stack and timelapse movie of nucleoli. Timelapse movie of a live hiPS cell colony expressing mEGFP-tagged nucleophosmin. Cells were imaged on a spinning-disk confocal microscope every 3 min. Image is a maximum intensity projection through the volume of the cells. Frames were histogram matched to adjust for photobleaching. Movie plays at 900x real time. Scale bar = 5 µm.

Observations

Nucleophosmin marks the granular component of the nucleolus, the nuclear subcompartment where ribosomes are made.
During much of interphase the nucleolus exists in one to several large, textured clusters within the nucleus of hiPS cells. During cell division, the nucleolus appears to ‘melt’ and then dissociate. After cell division, the nucleolus reassembles, first into small particles and progressing into the larger textured clusters observed during interphase.
The mEGFP-tagged nucleophosmin exhibits many similarities with the mEGF-tagged fibrillarin, another marker of the nucleolus. However, it also exhibits notable differences, including its specific localization within the nucleolus, as each line marks a different component of the nucleus. The granular component of the nucleolus where nucleophosmin resides forms hollow-looking spheres, giving the nucleolus a mesh-like appearance when imaged with this marker.

Plasma Membrane visualized via CAAX domain of K-Ras

4/24/2018

Figure 1. Timelapse movie of plasma membrane. Timelapse movie of a live hiPS cell colony expressing the CAAX domain of K-Ras tagged with mTagRFP-T. Cells were imaged in 3D on a spinning-disk confocal microscope every 5 min. Panels show the same colony at different z-positions. Left) The bottom z-section of the cells. Middle) A maximum intensity projection through the middle 5 z-sections of the cells. Right) A maximum intensity projection through the top 12 z-sections of the cells. Frames in each panel were histogram matched to adjust for photobleaching. Movie plays at 1500x real time. Scale bar = 5 µm.

Figure 2. Timelapse movie of plasma membrane. Timelapse movie of a live hiPS cells in an epithelial sheet containing both unedited cells (which remain unseen in black), and cells expressing the CAAX domain of K-Ras tagged with mTagRFP-T. Cells were imaged in 3D on a spinning-disk confocal microscope every 3 min; the top and bottom z-sections are shown pseudocolored in green and magenta, respectively. Movie plays at 1800x real time. Scale bar = 10 µm.

Observations

The plasma membrane of the cell forms a barrier between the cytoplasm and the extracellular space and neighboring cells. We labeled CAAX, the membrane-targeting domain of K-Ras with mTagRFP-T to label the plasma membrane.
In hiPS cells the plasma membrane exhibits differing behavior depending on the location in the cell. At the bottom of the cell, the membrane surrounds dynamic lamellar protrusions, which overlap with neighboring cells. At the middle region of cells, the plasma membrane clearly outlines the boundaries between cells. At the very top of cells, the membrane surrounds dynamic microvilli, seen as small textured puncta.
In addition, bright puncta are present throughout the cell, with a greater number near the top of the cell. These are likely endosomes created by plasma membrane taken into the cell.
By mixing the cells expressing this FP-tagged CAAX domain with unedited cells, the behavior of individual cells within a colony can be observed more clearly. Pseudocoloring a frame from the bottom of a z-stack in a different color from a frame from the top of the same stack permits visualization of the different behavior of the cell at each location. The bottom of the cell changes shape and explores more space than the top of the cell. The tops of cells appear anchored in space, consistent with the localization of cell junctional proteins to apical cell-cell boundaries.

Endosomes visualized via Ras-Related Protein Rab-5A

4/24/2018

A. Z-stack

B. Timelapse

Figures. Z-stack and timelapse movies of endosomes. A) Z-stack of a live hiPS cell colony expressing two copies (biallelic) of mEGFP-tagged Ras-related protein Rab-5A 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. B) Timelapse movie of a live hiPS cell colony expressing two copies (biallelic) of mEGFP-tagged Ras-related protein Rab-5A. Cells were imaged on a spinning-disk confocal microscope. A single, mid-level plane was acquired every second. A zoom-in of the area boxed in the left panel is shown on the right. Movie sped up 10x real time. Scale bar = 5 µm.

Observations

Ras-related protein Rab-5A is a member of the Rabs, small Ras family GTPases that regulate intracellular membrane trafficking. Rab-5A localizes to endosomes; it plays a role in the fusion of endocytic vesicles with the early endosome, endosome maturation and vesicular transport. We thus visualized a subset of endosomes in cells via Rab5A.
In hiPS cells Rab5A-labeled endosomes are seen as small round puncta throughout the cytoplasm but they also sometimes exhibit a tubular morphology. Their size ranges from ~250nm to 2 µm in diameter with smaller endosomes predominantly at the bottom of cells and larger endosomes at the top.
Two different types of movement are seen, small random movement within a confined area, and directed longer-range movement, likely along microtubules. Tubular structures emanating from round endosomes were occasionally observed during their transport.
Endosome dynamics also included fission and fusion events.

Peroxisomes visualized via peroxisomal membrane protein PMP34

4/24/2018

A. Z-stack

B. Timelapse

Figures. Z-stack and timelapse movies of peroxisomes. A) Z-stack of a live hiPS cell colony expressing mEGFP-tagged peroxisomal membrane protein PMP34 imaged in 3D on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. Scale bar = 5 µm. B) Timelapse movie of a live hiPS cell colony expressing mEGFP-tagged peroxisomal membrane protein PMP34. Cells were imaged on a spinning-disk confocal microscope every 1s. Movie sped up 10x real time. Images are shown with the despeckle ImageJ filter applied. Scale bar = 5 µm.

Observations

Peroxisomal membrane protein PMP34 localizes to peroxisomes, which are membrane-bound organelles that play a role in metabolism and in detoxifying hydrogen peroxide and other harmful substances in the cell.
In hiPS cells, peroxisomes are located throughout the cytoplasm in small puncta less than 100 nm in diameter. Two different types of movement are seen, small random movement within a confined area, and longer-range directed movement, likely along microtubules.
Peroxisomes do not noticeably change their morphology and distribution during cell division.

Cell–cell contacts: Gap junctions visualized via connexin-43

4/24/2018

A. Z-stack

B. Timelapse

Figures. Z-stack and timelapse movies of gap junctions. A) Z-stack of a live hiPS cell colony expressing mEGFP-tagged connexin-43. 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 = 5 µm. B) Timelapse movie of a live hiPS cell colony expressing mEGFP-tagged connexin-43. Cells were imaged on a spinning-disk confocal microscope every 3.25 min. Image is a maximum intensity projection of 20 z-slices acquired at the top of the cells. Contrast is enhanced to show localization of connexin-43 to internal structures. Frames were histogram matched to adjust for photobleaching. Movie plays at 975x real time. Scale bar = 5 µm.

Observations

Connexin-43 is a key component of gap junctions, which are channels between cells that allow for small molecules and ions to pass directly between the cytoplasm of adjacent cells. Gap junctions often cluster in larger gap junction plaques.
In hiPS cells, connexin-43 localizes most prominently to many puncta (presumably clusters of individual channels) at apical cell-cell junctions near the very top of the cells. Some puncta are also seen near the bottom and middle of the cell although in smaller numbers.
Connexin-43 is also localized to the plasma membrane and in internal structures that resemble the Golgi. This localization likely reflects the assembly and trafficking of connexin into gap junctions.
As hiPS cells progress though mitosis, the apical puncta at cell-cell junctions remain intact, but the connexin-43 associated with internal organelles, e.g. the Golgi, becomes diffuse throughout the cell.

Cell Structure Observations

Sarcomere visualized via Troponin I, slow skeletal muscle

Nucleolus visualized via nucleophosmin

Plasma Membrane visualized via CAAX domain of K-Ras

Endosomes visualized via Ras-Related Protein Rab-5A

Peroxisomes visualized via peroxisomal membrane protein PMP34

Cell–cell contacts: Gap junctions visualized via connexin-43

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