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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells
Géza Antalffy1, Ariel J Caride, Katalin Pászty
1Department of Molecular Cell Biology, National Blood Center, Budapest, Hungary.
Abstract:
The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.
Insights
The PMCA2w/b pump localizes to the apical membrane in polarized cells, stabilized by the actin cytoskeleton and NHERF2. Full cell polarization is essential for correct PMCA2w localization.
Area of Science:
- Cell Biology
- Membrane Protein Trafficking
- Epithelial Cell Biology
Background:
- The "w" splice variants of the PMCA2 (Plasma Membrane Calcium ATPase 2) pump are known to localize to specific cellular compartments.
- Previous research suggested PMCA2w/b had incomplete apical targeting in MDCK cells, with broader distribution.
- Overexpression of NHERF2 enhanced apical localization of PMCA2 in epithelial cells.
Purpose of the Study:
- To investigate the localization and function of PMCA2w/b in a stable MDCK cell line.
- To determine the factors influencing PMCA2w/b's precise localization within polarized epithelial cells.
Main Methods:
- Generation of a stable MDCK cell line expressing full-length PMCA2w/b.
- Utilizing reversible surface biotinylation and confocal microscopy in fully polarized cells.
- Investigating the role of the actin cytoskeleton and NHERF2 in pump localization.
Main Results:
- PMCA2w/b exhibits high activity and enhanced apical localization in terminally polarized MDCK cells.
- The pump is stabilized at the apical membrane through interaction with the apical cytoskeleton, NHERF2, and ezrin.
- Disrupting the actin cytoskeleton relocates the pump from apical patches without internalization.
Conclusions:
- Terminal polarization is a prerequisite for the correct apical membrane positioning of PMCA2w variants.
- The apical membrane cytoskeleton and associated proteins like NHERF2 play a crucial role in stabilizing PMCA2w/b at the cell surface.
