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Effect of monensin on ricin and fluid phase transport in polarized MDCK cells
1Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Abstract:
The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter-grown Madin-Darby canine kidney (MDCK) cells was investigated using 125I-labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 microM) stimulated transcytosis of both markers about 3-fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3-fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane-bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).
Insights
Monensin enhances basolateral to apical transcytosis in Madin-Darby canine kidney (MDCK) cells, while altering apical to basolateral transport and reducing recycling. This impacts cellular membrane transport pathways.
Area of Science:
- Cell Biology
- Membrane Transport
- Biochemistry
Background:
- Madin-Darby canine kidney (MDCK) cells are a widely used model for studying epithelial cell polarity and transport.
- Endocytosis, transcytosis, and recycling are crucial processes for cellular function and membrane protein trafficking.
- Monensin, an ionophore, is known to disrupt intracellular pH and ion gradients, potentially affecting cellular transport.
Purpose of the Study:
- To investigate the effects of monensin on endocytosis, transcytosis, recycling, and Golgi transport in MDCK cells.
- To differentiate the impact of monensin on fluid-phase versus membrane-bound marker transport.
- To elucidate the role of endosomal pH in monensin-mediated transport alterations.
Main Methods:
- Utilized filter-grown MDCK cells to model epithelial polarity.
- Employed 125I-labeled ricin as a membrane transport marker.
- Used horseradish peroxidase (HRP) as a fluid-phase transport marker.
- Quantified endocytosis, transcytosis (bidirectional), recycling, and Golgi apparatus transport in the presence and absence of monensin.
Main Results:
- Monensin (10 µM) significantly stimulated basolateral to apical transcytosis of both ricin and HRP by approximately 3-fold.
- Apical to basolateral transcytosis of HRP was reduced by ~50% with monensin, while ricin transport slightly increased.
- Monensin reduced apical endosome recycling and increased intracellular accumulation of both markers, correlating with increased ricin transport to the Golgi apparatus.
- Basolateral addition of markers showed no change in recycling or accumulation, but a nearly 3-fold increase in ricin transport to the Golgi.
Conclusions:
- Basolateral to apical transcytosis is enhanced by monensin, potentially independent of low endosomal pH.
- Monensin differentially affects apical to basolateral transcytosis of membrane-bound (ricin) versus fluid-phase (HRP) markers.
- The study highlights monensin's complex role in modulating cellular membrane trafficking pathways, impacting transcytosis, recycling, and Golgi delivery.