Figure 7. FCS analysis of nuclear transport proteins in NPC.

Importin β, importin α, NTF2, Ran wt, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into the cytoplasm of B104 cells. Cells were incubated for 30–60 minutes after injection and analyzed by FCS with observation volume positioned over the nuclear envelope. For each protein at least 20 cells were analyzed. To resolve protein populations specific to NPC and nuclear envelope, contributions from adjacent nucleoplasm and cytoplasm were subtracted by a global fitting procedure. Number of molecules per NPC for each protein component was calculated based on number of pores encompassed by FCS observation volume. Distributions of each protein between populations with different mobilities 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 component compared with wt Ran is shown. The table shows autocorrelation decay times (τ_D), concentrations, numbers of molecules per NPC, or fractions for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.