Abstract:

The kinetics of proteins passing through individual nuclear pore complexes (NPCs) of the nuclear envelope (NE) was studied using near-field scanning optical microscopy (NSOM) in combination with fluorescence correlation spectroscopy (FCS). The NSOM probe was placed over a single pore in an unsupported native NE to observe fluorescence-labeled NTF2 moving in the transport channel. A correlation analysis of the arising fluorescence fluctuations enabled us to characterize the translocation as driven by Brownian motion and to determine the related kinetic constants. Though trapped in the pore, NTF2 turned out to be highly mobile within a large axial extension. Our findings support the idea that molecules in transit interact with NPC proteins containing phenylalanine-glycine-repeat domains at the periphery of the channel. NSOM-FCS may help to understand the facilitated translocation in more detail and offers a new way to study single molecule mobility on a nanoscale.