Figure 4. FCS analysis of nuclear transport proteins in nucleus.

Fluorescently labeled importin β, importin α, NTF2, Ran, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into cytoplasm of B104 cells. After injected protein achieved steady state nucleocytoplasmic distribution, FCS measurements were performed with confocal volume positioned inside nucleus. For each protein at least 30 cells were analyzed. Autocorrelation data from all cells were globally fitted to resolve protein populations with different mobilities. Immobile fraction for each protein was determined from photobleaching amplitudes. Concentrations for each population were calculated based on total concentration and nucleocytoplasmic distribution of endogenous protein. Concentrations of each component are shown on bar graphs and in the table. Fast, medium, slow and immobile populations are labeled with red, green, blue, and black colors respectively. For mutant Ran proteins, the fraction of each population is shown compared with wt Ran. The table shows autocorrelation decay times (τ_D), fractions, and concentrations for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.