Accurate Fluorescence Fluctuation Analysis of Diffusing Proteins in Living Cells with Time-shifted Segmented Q Analysis

Characterizing the oligomeric state and mobility of proteins in the nuclear envelope (NE) of living cells by traditional fluorescence fluctuation methods suffers from artifacts introduced by local volume fluctuations that result from slow undulations of the two nuclear membranes. To address these shortcomings we introduced the time-shifted mean segmented Q (tsMSQ) function and demonstrated that tsMSQ successfully removed the bias from data analysis by simultaneously describing the fluctuations from diffusing proteins and from the volume fluctuations. To improve the tsMSQ technique we extended the temporal range of the analyzed data segments, which led to the discovery of a mismatch between theory and data at short segment lengths. This deviation is particularly evident for slow diffusing membrane proteins at the NE. Further investigations suggested that the phenomenon responsible for the observed deviations at short segment times is caused by a kinetic and not a diffusive process. We developed a theory that integrates a kinetic rate process into the tsMSQ function and demonstrate that the experimental data are described within error by including a fluorophore flickering rate. Next, we applied the extended tsMSQ theory to analyze fluorescence fluctuation data of proteins diffusing at the plasma membrane. Comparing the results obtained with tsMSQ and autocorrelation analysis revealed that tsMSQ is more sensitive to identifying the presence of a blinking process and its parameters. We examine the cause for this difference between the two analysis methods and comment on the relative strengths and weaknesses of each technique. We further demonstrate the importance of including the blinking process to recover unbiased stoichiometry and mobility data. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).