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Published on: March 16, 2017
Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography
Pina Colarusso1, Kenneth R Spring
1Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603, USA. colarusp@nhlbi.nih.gov
Abstract:
High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane.
Insights
Confocal microscopy revealed a reticulated fluorescence pattern on MDCK cell apical membranes. This pattern, caused by microvilli and microridges, is due to increased optical path lengths, not lipid differences.
Area of Science:
- Cell Biology
- Membrane Biophysics
- Microscopy
Background:
- The apical membrane of epithelial cells exhibits complex structures like microvilli.
- Understanding the biophysical properties of these structures is crucial for cell function.
- Lipid probes are used to investigate membrane organization and dynamics.
Purpose of the Study:
- To investigate the spatial distribution and biophysical properties of lipids on the apical membrane of Madin-Darby canine kidney (MDCK) cells.
- To determine the cause of the observed reticulated fluorescence pattern.
Main Methods:
- High spatial resolution confocal microscopy using the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC(16)).
- Differential interference contrast (DIC) microscopy to visualize cell surface structures.
- Multi-spectral and fluorescence anisotropy microscopy.
- Fluorescence recovery after photobleaching (FRAP).
- Treatment with Cytochalasin D and methyl-beta-cyclodextrin for cholesterol depletion.
Main Results:
- A distinct reticulated fluorescence pattern was observed on the apical membrane of MDCK cells stained with DiIC(16).
- Bright fluorescence regions coincided with microvilli and microridges observed by DIC microscopy.
- FRAP experiments showed similar diffusion coefficients for both bright and dim regions, indicating no significant lipid domain differences.
- The pattern was abolished by cholesterol depletion but unaffected by Cytochalasin D.
Conclusions:
- The reticulated fluorescence pattern is attributed to increased optical path lengths through apical microvilli and microridges, rather than distinct lipid domains.
- Cholesterol plays a role in maintaining the structural integrity or optical properties of these apical membrane features.
- Confocal microscopy with lipophilic probes can reveal topographical features of cell membranes.
