Rotation of Single Cell-Surface Fc Receptors Examined by Quantum Dot Probes

Abstract

Rotational diffusion, which depends linearly on the in-membrane volume of the rotating molecule, is, relative to lateral diffusion, a more sensitive probe of an individual molecule’s size and local environment. Single-particle tracking has provided new perspectives on lateral compartmentalization of membrane proteins. However, little is known at present about rotational motions of single membrane protein molecules. We discuss correlation analysis of fluctuations in fluorescence polarization from proteins labeled by asymmetric quantum dots. Such analysis provides rotational information on single membrane molecules which can be directly related to that from ensemble measurements of cell surface protein rotation. We have used asymmetric quantum dots to examine individual Type I Fce receptors on 2H3-RBL cells and to conduct imaging measurements of receptor rotational diffusion on timescales down to 10 ms per frame. To achieve removal of blinking-based contributions to rotation measurements, we discuss an approach based on the necessary statistical independence of polarization and intensity fluctuations. Imaging results demonstrate rotational correlation times broadly ranging from 50 to 500 ms among individual molecules of the same type. The magnitude of these orientational fluctuations is comparable to the fraction of molecules which appear rotationally immobile when examined on the microsecond timescale by time-resolved phosphorescence anisotropy. These slow motions, not observable previously, appear to be a property of the membrane rather than the receptor state. Our results suggest that individual mesoscale membrane regions may rotate or liberate with respect to the overall cell surface.