Abstract
The molecular details of transport through nuclear pore complexes (NPCs) have been well characterized, including the key role of Importin Beta (ImpB) receptor. However, the overall system behavior in intact cells is difficult to analyze because of its highly dynamic nature. Contrary to the common single particle tracking (SPT) approach that tracks an isolated particle as it moves through the pore, we set out to track the center of mass of the entire single NPC. While we track the pore, single molecules pass through it, and their location and dynamics are analyzed by fluorescence correlation spectroscopy (FCS). By this unconventional approach we find that ImpB transport is regulated so as to produce a characteristic bump in the autocorrelation function at the NPC. This regulation is spatially restricted to the pore, dependent on ImpB properties, pore structure, and metabolic energy. Combined to simulations our results suggest that ImpB movement within the pore is likely to be directed instead of unbiased, and that the back and forth components of its shuttling are coupled in time. We use our results to discriminate between existing NPC functional models, and identify key features that must be essential for transport in the intact pore.
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