Extended Data Fig. 4: Nuclear Accumulation of M9-βGal and Imp α in Permeabilized U-2 OS NUP96-mEGFP Cells.
From: Super-resolved 3D tracking of cargo transport through nuclear pore complexes
a, Nuclear import of M9-βGal(Atto542). The M9-βGal cargo is an ~500 kDa tetramer with four M9 nuclear localization sequences (NLSs) that are recognized by the transportin NTR. Transport reactions were monitored using wide-field fluorescence (λex = 532 nm) in the presence or absence of transportin or RanGTP (RanGDP + GTP) as indicated. The ‘transport mix’ was flowed in at t = 0.5 min. Representative images from four time points are shown (N = 20 cells from 4 independent experiments). [M9-βGal] = 0.25 µM, [transportin] = 0.25 or 1.0 µM, [RanGDP] = 0.5 µM, [NTF2] = 1 µM, [GTP] = 1 mM. b, Kinetics of nuclear import of M9-βGal. Average nuclear fluorescence (±s.d.) was quantified over time for N = 15 (-transportin), 17 (-RanGTP), or 20 (high and low transportin) cells from 4 independent experiments). Background refers to an area far from the cells. c, Cargo-dependent nuclear uptake of Imp α. Robust accumulation of Imp α(Atto542) into the nucleus (λex = 532 nm) occurs in the presence of the NLS-2xBFP cargo (top row) but not in its absence (bottom row). The range of the fluorescence images in the bottom row is 5 times smaller than in the top row, emphasizing the very low nuclear accumulation (N = 18 cells over 4 independent experiments). [Imp α(Atto542)] = 0.5 µM, [Imp β] = 0.5 µM, [NLS-2xBFP] = 0.5 µM, [RanGDP] = 1.5 µM, [NTF2] = 1 µM and [GTP] = 1 mM. Source numerical data are provided in source data.
