Endosomes visualized via Ras-Related Protein Rab-5A

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.