amino peg functionalised qds , with an emission peak of 605 nm , were functionalised with sulfosuccinimidyl-4-(n - maleimidomethyl)cyclohexane-1-carboxylate , followed by reaction of the maleimide group with the free sulfhydryl group of the purified ibb / z - domain protein .
hela cells were permeabilised using digitonin and the qd - ibb cargos ( 50 pm ) were introduced in a transport buffer ( 20 mm hepes , 110 mm koac , 5 mm naoac , 2 mm mgoac , 2 mm dtt , ph 7.3 ) containing 0.5 mm importin - b , 4 mm rangdp , 1 mm ntf2 , and 1 mm gtp .
single - molecule data were acquired with 25 ms time resolution using a custom - built near - tirf microscope , incorporating a 100 1.65 na objective , 100mw 532 nm continuous wave laser and an andor ixon+ camera .
 the nuclear pore complex ( npc ) mediates all exchange between the cytoplasm and the nucleus . 
 small molecules can passively diffuse through the npc , while larger cargos require transport receptors to translocate1 . 
 how the npc facilitates the translocation of transport receptor / cargo complexes remains unclear . 
 here , we track single protein - functionalized quantum dot ( qd ) cargos as they translocate the npc . 
 import proceeds by successive sub - steps comprising cargo capture , filtering and translocation , and release into the nucleus . 
 the majority of qds are rejected at one of these steps and return to the cytoplasm including very large cargos that abort at a size - selective barrier . 
 cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely . without ran , cargos still explore the entire npc , but have a markedly reduced probability of exit into the nucleus , suggesting that npc entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos . 
 the overall selectivity of the npc appears to arise from the cumulative action of multiple reversible sub - steps and a final irreversible exit step . 
Methods summary
Supplementary Material