Determining Protein Stoichiometry of Cellular Vesicles by Fluorescence Brightness Analysis

Abstract

Fluorescence fluctuation spectroscopy (FFS) has been used to study concentration, transport parameters and interactions of fluorescently labeled molecules. One particularly important FFS parameter is brightness, which determines the stoichiometry of protein complexes inside living cells. Recent studies demonstrated that brightness analysis can be extended to characterize the copy number of viral-like particles that contain hundreds to thousands of proteins. Here we explore extending brightness analysis to study protein-carrying vesicles with significantly lower copy numbers than encountered with viral-like particles. Vesicles fulfill many important functions inside the cell. They store, transport, excrete, and digest cellular products. Characterizing vesicles by FFS is challenging, because the low concentration and brightness of the sample limits the achievable signal-to-noise. We present studies that identify the lowest brightness and concentration limit accessible by FFS using a model system. In addition, we present data on vesicles carrying the kinase PI4KIIbeta, both in vivo and in vitro. The experiments described so far utilize single-color detection, which is sufficient for determining the copy number for a single protein species. We perform dual-color FFS experiments on particles and vesicles to investigate its potential to characterize the copy number of two distinct protein species present on the same vesicle. This work is supported by NIH grants GM064589 and GM075401.