Rotation of Single Cell Surface Molecules Examined via Polarized FCS Measurements using Quantum Dot Probes

Rotational motions of cell surface molecules are of particular interest given the sensitivity of such motions to molecular size and aggregation state. The time-autocorrelation function (TACF) of fluctuations in fluorescence polarization from asymmetric quantum dots (QD) such as Molecular Probes’ Qdot655 labeling cell surface molecules indicates the rotational correlation times (RCT) of the QD-containing complexes. Type I Fce receptors (FceRI) and insulin receptors (IR) on 2H3 cells, subjected to various treatments potentially affecting receptor rotation, including polyvalent DNP-BSA, methyl-β-cyclodextrin, cytochalasin D or paraformaldehyde, were labeled by Qdot655s. Over 700 individual QD were examined. Side-by-side vertically(v)- and horizontally(h)-polarized fluorescence images of QD-labeled cells were obtained simultaneously as image stacks at 10 ms per frame by means of a Princeton Instrument Dual-View. To minimize apparent polarization TACF arising from QD lateral diffusion, v- and h-substacks were aligned to sub-pixel accuracy. From fluorescence in identical regions around each QD in each stack, intensity and polarization fluctuation TACF were calculated. Given the blinking of QD, experimental parameters such as g-factor, camera background, etc. were carefully optimized for each QD to minimize cross-correlation between polarization and intensity fluctuations. This could otherwise appear as intensity fluctuation TACF contributing to the apparent polarization fluctuation TACF. Using these techniques, the initial polarization fluctuation TACF for FceRI averages about 0.0015 and the geometrically-averaged RCT is about 55-85 ms, both independent of cell treatment. More limited results on IR appear similar. Absence of treatment effects on magnitudes or decay rates of polarization TACF is puzzling. Previous time-resolved phosphorescence anisotropy studies on FceRI suggest limited presence of orientational relaxations slower than 1 ms. However, the absence of treatment effects here suggests such slow reorientation may be a property of the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale membrane regions. Supported by NSF grant MCB-1024668 and NIH grant CA175937 to BGB.