Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.
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.
Colarusso
Pina
P
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603, USA. colarusp@nhlbi.nih.gov
Spring
Kenneth R
KR
D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D005456:Fluorescent Dyes / Q000494:pharmacology; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D010477:Perfusion; D047392:beta-Cyclodextrins
C035005:3,3'-dihexadecylindocarbocyanine; D002232:Carbocyanines; D003505:Cyclodextrins; D005456:Fluorescent Dyes; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D015638:Cytochalasin D; D002784:Cholesterol