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Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy
R N Ghosh1, D L Gelman, F R Maxfield
1Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Journal of Cell Science
|August 1, 1994
Summary
Endosomes in HEp2 cells have distinct sorting and recycling compartments for low-density lipoprotein (LDL) and transferrin (Tf). This study clarifies endosomal pathways, reconciling previous findings in CHO and HEp2 cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Endocytosis and Intracellular Trafficking
Background:
- Endosomes in Chinese Hamster Ovary (CHO) cells are known to comprise distinct vesicular and tubulovesicular compartments.
- Previous studies reported extensive tubular reticulum in HEp2 cell endosomes, contrasting with findings in CHO cells.
- Understanding endosomal sorting and transport is crucial for cellular function and various physiological processes.
Purpose of the Study:
- To investigate and reconcile the differing observations on endosomal structure and function in HEp2 cells compared to CHO cells.
- To elucidate the sorting and intracellular transport pathways of endocytosed macromolecules in HEp2 cells.
- To characterize the roles of specific endosomal compartments in the trafficking of low-density lipoprotein (LDL) and transferrin (Tf).
Main Methods:
- Utilized HEp2 human carcinoma cells for studying endosomal transport.
- Employed fluorescently labeled low-density lipoprotein (LDL) and transferrin (Tf) as probes for lysosomal and recycling pathways, respectively.
- Applied laser scanning confocal microscopy for simultaneous visualization of probe sorting and post-sorting behavior.
- Quantified 3-dimensional cellular distributions and trafficking rates using pulse-chase experiments.
Main Results:
- Both LDL and Tf initially co-localized in punctate sorting endosomes.
- Transferrin (Tf) rapidly exited sorting endosomes into a distinct post-sorting recycling compartment, with a half-life of approximately 2.5 minutes.
- Low-density lipoprotein (LDL) remained in sorting endosomes, which subsequently matured into late endosomes, with a recycling half-life of approximately 7 minutes.
- Morphological and kinetic data supported the existence of separate sorting and recycling compartments in HEp2 cells.
Conclusions:
- HEp2 cells possess distinct short-lived sorting endosomes and long-lived post-sorting recycling compartments.
- The observed tubular endosomes in HEp2 cells likely represent tubular portions of sorting and recycling compartments.
- Endosomal trafficking and sorting of Tf and LDL in HEp2 cells follow a similar scheme to that observed in CHO cells.