Brightness Characterization of Internal Endomembrane Proteins by Z-Scan Fluorescence Fluctuation Spectroscopy

Abstract

Traditionally, fluorescence fluctuation spectroscopy (FFS) has been used to quantify the stoichiometry of soluble proteins in the nucleus and the cytoplasm of mammalian cells by brightness analysis. The development of z-scan FFS broadened the capability of brightness analysis to include proteins distributed across stratified layers, such as the cytoplasm and the plasma membrane. In this work, we further extend z-scan FFS to study proteins that reside on or within internal endomembranes, including the endoplasmic reticulum (ER) membrane/lumen and the nuclear envelope/perinuclear space. Experimentally, we place a 20 amino acid ER signal sequence in front of EGFP (SS-EGFP); the complex is translated into the lumen of the ER where of the signal sequence is cleaved, leaving EGFP to diffuse within the ER and nuclear envelope. The brightness of SS-EGFP is determined by performing z-scan FFS measurements where corrections are applied for both the thin layer geometry and coexcitation of adjacent layers. Additionally, we create and test a tandem dimeric EGFP protein (SS-EGPF2) to establish a model for calibrating brightness and stoichiometry. Finally, we test the limits of our technique and apply z-scan FFS to characterize the brightness of other proteins found within the endomembrane system. This research was supported by grants from the National Institutes of Health (R01GM064589) and the National Science Foundation (PHY-0346782).