Characterizing Protein Interactions In Different Cellular Compartments By Axial Scan Fluorescence Fluctuation Spectroscopy

Abstract

Fluorescence fluctuation spectroscopy (FFS) is a noninvasive tool for measuring protein interactions, concentrations and transport directly in living cells. Brightness analysis of FFS experiments focuses on the photon counts rate of protein complexes and provides a unique approach to quantify homo- and hetero- interactions between proteins. However, FFS theory assumes that the fluorescent particles are uniformly distributed within the optical observation volume. This assumption is violated when measuring thin cytoplasmic sections of cells, because fluorophores only occupy part of the observation volume. Another problem for conventional FFS analysis is the situation where more than one cellular compartment is enclosed by the observation volume. If the concentrations or the interactions of the proteins differ in adjacent compartments, the brightness determined from conventional FFS theory can be strongly biased. We present examples that highlight these biases and introduce a model based on axial scan FFS that avoids these potential pitfalls. The performance of the axial scan FFS and the modified FFS theory is characterized with simple model systems. We apply axial scan FFS to study the protein interactions of the NTF2 protein in the cytoplasm and the nucleus of cells. This work is supported by the National Science Foundation (PHY-0346782) and NIH grant R01GM064589.