Abstract
Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 +/- 49.8 nm ranging 84.5-365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 +/- 63.8 nm ranging 42-360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.
Highlights
Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported
Canine kidney Madin-Darby canine kidney cells (MDCK) epithelial cells [NBL-2; American Type Culture Collection (ATCC)] were cultured in the medium of ATCC-formulated Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS) in the 37°C, 5% CO2 incubator
The reagents used in the study were as follows: biotin for negative control, biotinylated Cholera toxin subunit B (CTB) and fluorescein isothiocyanate (FITC)-conjugated CTB were purchased from Sigma; mouse IgM for isotype control and mouse anti-GM3 IgM mAb (GMR6) were purchased from BD Biosciences (San Jose, CA) and Seikagaku America (Falmouth, MA), respectively
Summary
Canine kidney MDCK epithelial cells [NBL-2; American Type Culture Collection (ATCC)] were cultured in the medium of ATCC-formulated Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS) in the 37°C, 5% CO2 incubator. The reagents used in the study were as follows: biotin for negative control, biotinylated Cholera toxin subunit B (CTB) and fluorescein isothiocyanate (FITC)-conjugated CTB were purchased from Sigma; mouse IgM (kappa-chain) for isotype control and mouse anti-GM3 IgM mAb (GMR6) were purchased from BD Biosciences (San Jose, CA) and Seikagaku America (Falmouth, MA), respectively. Biotinylated goat anti-mouse IgM (m chain) IgG mAb and streptavidin-conjugated QD655 were purchased from Invitrogen. All negative control experiments showed negative results (data shown). To remove QD aggregates in commercial QD solution, the following procedures were followed to prepare QD dyes for use in each experiment, as mentioned previously [4]. The resultant supernatant was ready for use
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