Golgi visualized via sialyltransferase 1 (also known as ß-galactoside α-2,6-sialyltransferase1)

A. Z-stack 1-copy edit

B. Z-stack 2-copy edit

C. Z-stack 2-copy edit

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.

A. Timelapse 6 hours

B. Timelapse 100 minutes

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.

Observations

• Sialyltransferase 1 is a transmembrane protein that resides within the Golgi apparatus. It catalyzes the transfer of sialic acid from an activated donor molecule to galactose.
• In hiPS cells, the Golgi comprises a set of lamellar structures localized near the top of the cell, often partially covering the nucleus (see 3D reconstruction). The structure of the Golgi becomes more tubular as it descends downward into the cell.
• During cell division, the Golgi fragments into numerous small foci that are dispersed throughout the cell, and excluded from the mitotic spindle. These Golgi fragments reassemble in daughter cells after division.
• Golgi morphology is difficult to visualize in immunostained cells as its lamellar structure easily collapses into a tubular morphology in response to fixation.