Abstract
There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking.
Highlights
When EGF receptor (EGFR)-expressing cells are exposed to fluorescent Epidermal growth factor (EGF) (FL-EGF), the majority of the fluorescence will first be visible at the plasma membrane (~0–3 min) and within internalized endocytic and recycling endosomes and within lysosomes
We found that fluorescent EGF (FL-EGF) produced very strong intracellular fluorescence when taken up by endocytosis into Huh-7 cells
This report investigates whether removal of specific solutes from extracellular medium can affect the trafficking of organelles
Summary
Epidermal growth factor (EGF) is a peptide growth factor that binds to the cell surface EGF receptor (EGFR) and induces receptor dimerization and activation of the EGFR cytosolic tyrosine kinase. This stimulates a host of cellular activities including DNA synthesis, cell proliferation, and migration. Receptor binding stimulates endocytosis of EGF-EGFR, resulting in the formation of cytosolic vesicles and tubules containing EGF-EGFR within their lumen. These undergo endocytic processing and maturation through early endosomes, the recycling compartment, and culminating in lysosomes where EGF-EGFR is degraded and its cellular signaling is quenched [8]. Beyond 30 min of endocytic processing, internalized EGF is typically found in lysosomes [9], but this may be affected by the experimental manipulations presented below, and images and data containing FL-EGF may represent lysosomes as well as other organelles along this pathway
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