Characterizing the passive permeability barrier of yeast NPCs. (A) Expected decay in passive permeability rates with molecular mass in a rigid versus soft barrier to passive transport. (B) Permeability of yeast NPCs determined in vivo using a FRAP approach. From left to right: At time t = 0 s, the cytoplasmic pool of reporter macromolecules is photobleached with a spot-bleaching laser (cyan crosshair), and the nuclear fluorescence in a single cell is plotted over time, as the nuclear pool of reporters equilibrates with the cytoplasm through passive diffusion. Bars, 1 µm. Note, at steady state, the cytoplasm can appear ∼20% less bright than the nucleus because of vesicles and other structures below imaging resolution that do not contain GFP. We have not adjusted for this small deviance from the ideal N/C of 1, as the effect is small and consistent between strains. The mean transport time τ is the time constant of the exponential decay in nuclear fluorescence; the nuclear volume, cytoplasmic volumes, and number of NPCs are then quantified based on 3D reconstruction of the cell, using a complete image series through the z axis acquired after the time course. These data are integrated to compute the permeability coefficient in a single cell. The population mean of several such measurements from many cells is computed from multiple independent measurements, where each dot in the violin plot indicates a permeability coefficient from a single cell. AU, arbitrary units.