Polarized fluorescence photobleaching for measuring fast rotational motion of cell surface receptors.

Abstract

Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules. Compared with other existing techniques (such as phosphorescence depolarization), this technique has two major advantages: (a) it does not require sample deoxygenation; (b) the characteristic time of the rotational motion of the biomolecules under study can vary over a wide range. This project has significantly improved the PFRAP technique by (a) shortening the time resolution to $\sim$1 $\mu$s by use of faster electronics and subnanosecond bleaching pulses; (b) increasing the amplitude of the initial bleaching anisotropy; and (c) speeding data acquisition by automating the microscope stage motion. A theoretical molecular state model is postulated, which semiquantitatively accounts for the experimentally observed dependence of reversible and irreversible bleaching on subnanosecond bleaching pulse intensity. The improved PFRAP system has been used to measure the rotational mobility of the two distinct populations of acetylcholine receptors (AChRs) (clustered and nonclustered) on embryonic rat myotubes in cultures. It is found that clustered AChRs are essentially immobile, and that nonclustered AChRs display a mixture of different rotational diffusion rates, some of which are faster than any previously measured on this system. The fastest rotational component for nonclustered AChRs has a rotational diffusion coefficient D $\sim$ 10,000 $\rm sec\sp{-1}$ with a characteristic time $\tau$ $\sim$ 25 $\mu$s, and accounts for $\sim$40% of the AChR population in nonclustered areas. According to a hydrodynamic model, this component probably corresponds to an aggregate consisting of only a few AChR monomers at most. Rat embryonic brain extract (EBX) applied to the cell cultures is known to disperse the endogenous clusters at the cell-substrate contact regions and to induce new clusters on the top surface of myotubes. The PFRAP experimental results here show that there is no significant change in the bleaching anisotropy, the rotational diffusion rates and the fractions of the rotational components of AChR in nonclustered areas after the addition of EBX, and that both the endogenous clusters and the EBX-induced clusters are essentially immobile.