Proteins that are required for efficient nuclear functions are imported into the nucleus through the nuclear pore complexes (NPCs) which are also the export gates for nuclear products that are required in the cytoplasm. These molecules are usually transported by a class of protein vehicles called karyopherins, the best-studied of which is importin . In this study, we investigated in vivo dynamics of such nuclear transport processes, and found that the process that we have known as “import” is not a unidirectional process from cytoplasm to nucleoplasm, but a steady-state established by both influx and efflux between two compartments.
We performed a series of kinetic analyses of importin and its target molecule (cargo) in a living cell by using FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching), as well as in vitro transport analysis and binding assay to extract kinetic parameters of each step of the reaction cycle. These parameters were used to build up a kinetic model of the entire transport cycle. Our model could successfully provide various reaction parameters and information on the cycle, such as intracellular distribution and fluxes (influx and efflux) of the cargo and importin , and a rate limiting step in various conditions. This result revealed a theoretical background of how a steady-state distribution of cellular proteins is established by bidirectional fluxes between cytoplasm and nucleoplasm.
Imagine a kitchen sink filled with a certain amount of water. If you unplug the drain, it will become empty. But, if you open the tap at the same time and keep supplying an appropriate amount of water, the volume of water will be apparently “constant”, which is maintained by the continuous inflow and outflow. This point is referred to as steady-state. This is also the case in living organisms whose cells contain a nucleus connected to the cytoplasm through special drains called nuclear pore complexes (NPCs). Proteins that are required for efficient nuclear functions are imported through the NPCs which are also the export gates for nuclear products that are required in the cytoplasm. These cargoes are usually transported by a class of protein vehicles called karyopherins, the best-studied of which is importin . The dynamic events of the importin -mediated pathway at steady-state are still not well known. In this study, we used fluorescence techniques and kinetic parameters to investigate how the steady-state nuclear transport cycle is maintained.
The cytoplasm of live HeLa cells expressing nuclear-specific cargoes was continuously photobleached and the fluorescence signal of the nucleus was simultaneously monitored in a process called fluorescence loss in photobleaching (FLIP). The entire nucleus was also photobleached and the extent and speed of recovery of fluorescence signals was recorded in a process called fluorescence recovery after photobleaching (FRAP). In addition, in vitro nuclear transport assays and surface plasmon resonance (SPR) were used to obtain kinetic parameters with which a model was established to explain the entire cargo import cycle facilitated by importin .
Our FLIP experiment showed a progressively weaker nuclear fluorescence signal. This indicates that, at steady-state, the imported cargo could also be exported out of the nucleus by importin . The FRAP experiment revealed that the flux (import-export cycles) was inversely proportional to the size of the cargoes. In addition, our kinetic model revealed various reaction parameters and information on the cycle, such as intracellular distribution and fluxes of the cargo and importin The model also revealed that the affinity of importin for its cargo in the cytoplasm determines the rate of the import process. Taken together, our data strongly suggests that shuttling of import and export cargoes may be a general feature of living cells at steady-state.
Our model serves as a basis for understanding a variety of nucleocytoplasmic molecular dynamics at steady-state, and how this balance is disturbed by endogenous/exogenous factors. We will further focus on how the abnormality in this system leads to cellular aberrations and pathological status. Precise control of artificial molecules in cells will also be achieved by predicting steady-state dynamics according to our established system, which will benefit virus suppression and drug delivery techniques.
Lolodi O, Yamazaki H, Otsuka S, Kumeta M, Yoshimura SH. “Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport.” Mol Biol Cell (2015)