Category: Nucleus

Histones visualized via Histone H2B type 1-J

8/1/2018

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J 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.

Movie. Time-lapse movie of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J protein. Cells were imaged in 3D on a spinning-disk confocal microscope every 3 min. A single mid-level z-section is shown. Movie plays at 1260x real time. Scale bar, 5 µm.

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J protein. Cell Mask Deep Red Plasma Membrane dye was applied to mark cell boundaries (magenta). NucBlue Live Ready Probe Reagent was used to compare mEGFP-tagged H2B localization (left panel) to a NucBlue Live (Hoechst) DNA stain (left panel). 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.

Observations

Histone H2B type 1-J is a protein that participates in organizing DNA. It and other histone proteins form a core around which DNA wraps. These structures, called nucleosomes, are the fundamental repeating units within chromatin (DNA and histones). The packing of these structures impacts DNA accessibility, affecting functions like transcription, replication, and repair of DNA.
In hiPS cells imaged with spinning-disk light microscopy, mEGFP-tagged Histone H2B type 1-J appears within the nucleus as puncta of varying size and intensity, likely reflecting more densely packed regions of nucleosomes, and in a “haze”, where nucleosomes cannot be resolved.
The localization of mEGFP-tagged Histone H2B type 1-J is similar to the pattern of the DNA-binding dye Hoechst. Hoechst binds the minor grove of DNA (with preference for A-T rich regions), while mEGFP-tagged Histone H2B type 1-J localizes to the nucleosomal cores around which the DNA wraps.
Histone H2B type 1-J pattern of localization and intensity changes through the cell cycle. During interphase, Histone H2B type 1-J localizes throughout the nucleus, with some regions of greater intensity (reflecting a greater density of protein) near the nuclear periphery and the nucleolus. During cell division, Histone H2B type 1-J localizes to chromosomes.

Nuclear pores visualized via Nucleoporin Nup153

8/1/2018

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Nucleoporin Nup153 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.

Movie. Time-lapse movie of live hiPS cell colony expressing mEGFP-tagged Nucleoporin Nup153 protein. Cells were imaged in a 3D volume spanning ~1.5 µm through the middle of the cells on a spinning-disk confocal microscope every 3 min. A single mid-level z-section is shown. Movie plays at 1800x real time. Scale bar, 5 µm.

Observations

Nucleoporin Nup153 is a protein within the nuclear pore complex (NPC). The NPC bridges the double membrane of the nuclear envelope to regulate the exchange of molecules between the nucleus and cytoplasm. Nucleoporin Nup153 is located on the nuclear side of the NPC. It is critically involved in the ability of the NPC to transport molecules between the nucleus and cytoplasm; but it also plays other roles in nuclear properties and functions, including the shape of the NPC and the nucleus, and regulation of transcription.
In hiPS cells, Nucleoporin Nup153 appears as numerous dense puncta along the nuclear envelope, including within invaginations.
During cell division, Nucleoporin Nup153 localization changes with the reorganization of the nuclear envelope. During mitosis, Nuceloporin Nup153 localizes in a dim wavy structure, likely the ER. At the end of mitosis, Nucleoporin Nup153 appears on chromosomes before finally reappearing at the nuclear envelope.

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.

Nucleolus via Fibrillarin

3/16/2017

Z-stack

High magnification timelapse (cell division)

Figure 1. Movies of fibrillarin in Nucleoli. Left: Z-stack of live hiPS cells expressing mEGFP tagged fibrillarin imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Right: Timelapse movie of live hiPS cells expressing mEGFP tagged fibrillarin. Images were collected in 3D every 3 minutes for 1.5 hours on a spinning-disk confocal microscope. Image is a maximum intensity projection. Playback speed is 900x real time.

Figure 2. Time series of cell division. A single cell going through cell division taken from the movie on the right.

Observations

Fibrillarin marks the dense fibrillar component (DFC) of the nucleolus, the nuclear subcompartment where ribosome biogenesis occurs.
During much of interphase the nucleolus exists in 1-2 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. Low levels of fibrillarin are visible on chromosomes during chromosome segregation in mitosis.

Nuclear envelope visualized via lamin B1

3/10/2017

Low magnification timelapse

High magnification timelapse

Figure. Timelapse movies of hiPSC cells expressing mEGFP tagged Lamin B1. Images were collected in 3D every 3 minutes for 12 hours (left) or every 35 seconds for 23 minutes (right) on a spinning-disk confocal microscope. Images are maximum intensity projection (left) or single slices from the middle of the z-stack (right). Playback speed is 1800x (left) and 350x (right) real time.

Observations

Lamin B1 is a member of the lamin family of proteins that make up the nuclear lamina, located just inside the inner nuclear envelope. - In hiPS cells, nuclei in these cells occupy 30-50% of the cell volume making them very prominent. In the center of cells the nuclei occupy almost the entire cytoplasm such that a ‘bird’s eye view’ of the cell monolayer shows nuclei that appear tightly packed together.
As the cells enter mitosis, the lamin B1-containing nuclear envelope is seen to ruffle and take on a wavy morphology as it begins to breakdown. However the nuclear envelope does not completely breakdown during mitosis. As the nuclear envelope reforms, invaginations are seen that can look like spots within the nucleus. These spots decrease and disappear over time suggesting that they may be cell cycle related. These invaginations are also seen with Sec61B, which labels the ER including the peripheral ER surrounding the nucleus (See ER section).

Cell structure observations

Histones visualized via Histone H2B type 1-J

Nuclear pores visualized via Nucleoporin Nup153

Nucleolus visualized via nucleophosmin

Nucleolus via Fibrillarin

Nuclear envelope visualized via lamin B1

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