Figure 1. Z-stack movies of Golgi. A) Z-stack of live hiPS cells expressing one copy (monoallelic) of mEGFP-tagged sialyltransferase 1 (ST6GAL1) imaged on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. B) Z-stack of live hiPS cells expressing two copies (biallelic) of mEGFP-tagged sialyltransferase 1 (ST6GAL1) imaged on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. Contrast settings for these two movies are identical to highlight the increased signal in the biallelic edited line (right). C) Z-stack of live hiPS cells expressing two copies (biallelic) of mEGFP-tagged sialyltransferase 1 (ST6GAL1; white) stained for DNA (NucBlue Live; cyan) and plasma membrane (CellMask Deep Red; magenta). Movie starts at the bottom of the cells and ends at the top.

Figure 2. Timelapse movies of Golgi. A) Timelapse movie of hiPS cells expressing two copies (biallelic) of mEGFP-tagged sialyltransferase 1 (ST6GAL1). Images were taken in 3D every 3 min for 6 hrs on a spinning-disk confocal microscope. Images are maximum intensity projections with the ‘despeckle’ ImageJ filter applied. Playback speed is 1800x real time. B) Timelapse movie of hiPS cells expressing two copies (biallelic) of mEGFP-tagged sialyltransferase 1 (ST6GAL1). Images were taken in 3D every 5 min for 100 min on a spinning-disk confocal microscope. Images are maximum intensity projections with the ‘despeckle’ ImageJ filter applied. Playback speed is 900x real time.

Figures. Z-stack and timelapse movies of lysosomes. A) Z-stack of live hiPS cells expressing mEGFP tagged LAMP1 imaged on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. Scale bar = 5µm. B–C) Timelapse movie of live hiPS cells expressing mEGFP-tagged LAMP1. Images were taken as a single slice near the top (B) or bottom (C) of the cell every 1 sec for 100 sec on a spinning-disk confocal microscope. Images have had the ‘despeckle’ ImageJ filter applied. Movie sped up 10x real time. Scale bar = 5 µm.
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Observations

• Lysosomes are membrane-bound organelles responsible for breaking down unwanted cellular materials for reuse. They are often considered the “waste disposal” sites within cells. LAMP1 is a transmembrane glycoprotein, which together with LAMP2 is the most abundant protein present in the lysosomal membrane (~50% of membrane proteins), making it a great reporter protein for lysosomes.
• Lysosomes are classically known to form round vesicles of different sizes. However, they can also take on tubular morphologies, something that is less commonly known because these tubules are often lost upon fixation. In hiPS cells, lysosomal tubules are more visible in the bottom half of cells. Smaller vesicles are also predominant near the bottom while larger vesicles are mainly found at the top of the cells.
• Lysosomes are very dynamic. Round vesicular lysosomes move around the cell rapidly, sometimes with a stop and go motion. They also deform, most dramatically when one end is pinched into a tear-drop shape. This can also be associated with the pulling out of tubular lysosomes.
• Tubular lysosomes are also very dynamic, with branches being pulled out and fission and fusion dynamics altering their morphology.
• In recent literature, tubular lysosomes have been implicated as being structures connecting the lysosome to other intracellular compartments (e.g. late endosome) and being involved in the fusion process between these compartments. Tubular lysosomes have also been shown to be a precursor to lysosome formation via budding during cell starvation.

Figure. Centrin and DNA through cell cycle. Single plane images of hiPS cells expressing mTagRFP-T–tagged centrin (green) and labeled with Hoechst dye (DNA; blue) imaged on a spinning-disk confocal microscope. Cells labeled A-F represent different stages of the cell cycle (see diagram). A) G1-phase, B) early S-phase, C) later S-phase, D) G2/M-phase and E–F) M-phase contain centrioles at distinguishable stages of duplication (see zoomed in images and cell cycle diagram).

Observations

• Centrin is a key protein in centrioles, which are small cylindrical structures made primarily of tubulin. Centrioles are important components of centrosomes, the structures that form microtubule organizing centers. Centrioles are also found at the base of cilia and flagella.
• Centrioles localize to the very top of the hiPS cells in interphase, consistent with the presence of primary cilia, which emanate from the centrioles. Centriole duplication and separation is observed throughout the cell cycle consistent with models of cell cycle regulation of centriole behavior. Small daughter centrioles are seen to appear next to their mother centrioles and grow in size.
• See FAQs for reasoning behind on our choice of red-fluorescent protein tagging.