Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

摘要

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 raftsanodomains (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 raftsanodomains (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.