TRANSIENT CHOLESTEROL EFFECTS ON NICOTINIC ACETYLCHOLINE RECEPTOR CELL-SURFACE MOBILITY

The raw data set comprises X,Y coordinates and brightness of images corresponding to nicotinic acetylcholine receptor (AChR) particles in live CHO/K1-A5 cells using total internal reflection (TIRF) fluorescence microscopy . Image acquisition for movies was performed with 100 ms exposure in streaming mode using the software SlideBook (Intelligent Imaging Innovations, Boulder, CO) and exported as 16 or 8-bit images. Subsequent analysis was carried out using Image J (NIH, USA, http://rsb.info.nih.gov/ij/) or Matlab (MathWorks, see below) and further exported to Excel (Microsoft) for further data processing. Examples of these Excel spreadsheets corresponding to meta-analysis of some of the physical parameters of the particles, interpreted as supramolecular aggregates (nanoclusters) of AChR macromolecules are also attached. These physical properties were used to characterize the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells. AChR particles tagged with a monovalent ligand, fluorescent α-bungarotoxin (αBTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (~50%), the rest being relatively immobile (~44%). Depletion of membrane cholesterol by methyl-α-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes 20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs10 s to ~20 s. The instantaneous (microscopic) diffusion coefficient D2-4 of the AChR obtained from the MSD analysis diminished from ~0.001 µm2 s-1 to ~0.0001-0.00033 µm2s-1 upon cholesterol depletion, ~30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors.