Eukaryotic cells contain two major functional compartments, the nucleus and the cytoplasm. The former contains the genome, the latter comprises the cytosol and organelles for cellular functions such as protein and ATP synthesis. The nuclear envelope (NE) constitutes the physical boundary between the nucleus and the cytoplasm. Molecules pass across the NE in both directions through the nuclear pore complexes, with small molecules (R < 2.5 nm), such as ions and nucleotides, freely diffusing, and larger molecules, such as proteins, requiring a complex transport process. Large molecules are actively shuttled in/out the cell nucleus by nuclear transport receptors (importins/exportins), depending if the cargo carries a nuclear localization signal or nuclear export signal (NLS/NES). While the molecular players are known, the spatio-temporal dynamics of the nucleo-cytoplasmic transport remains elusive. In this work, we study the size-dependent dynamics of the nucleo-cytoplasmic transport in space and time. To this end, we designed a series of fluorescently labeled molecular cargo using tandem-mCherry with localization signals, ranging in size 29–135 kDa. Using spinning disk confocal microscopy, we monitor size-dependent nucleo-cytoplasmic transport of the fluorescent cargos in live human cells. In particular, we focus on decoupling the effects of passive and active transport by inhibiting active import/export using biochemical perturbations. In addition, we implement a photo-activatable cargo system, which allows us to control the directionality of its active transport and thus isolate the contributions of active and passive transport. This work holds promise to provide fundamental insights into biophysical mechanism(s) underlying the nucleo-cytoplasmic transport. Such knowledge will aid our understanding of the nucleus in both health and disease. This work was supported by the National Institutes of Health grants R00-GM104152 and R01-GM145924 and by the National Science Foundation grants CAREER PHY-1554880, CMMI-1762506, and PHY-2210541.
Sahu et al. (Sun,) studied this question.