Shining a Light on the Molecular Gateway to the Cell Nucleus
A team of investigators at the University of California - Berkeley Physical Sciences-Oncology Center (UC-Berkeley PS-OC) has traced with unprecedented resolution the paths of cargos moving through the nuclear pore complex (NPC), a selective nanoscale aperture that controls access to the cell's nucleus, and answered several key questions about its function.
Jan Liphardt, principal investigator of the UC Berkeley PS-OC, and colleague Karsten Weis led the team that conducted this study. The investigators published their work in the journal Nature.
The NPC, a large protein assembly shaped like a basketball net fringed with tentacles, is the gateway to the cell nucleus. Several viruses target the NPC to gain entry to the nucleus, and dysfunctional transport between the cytoplasm and the nucleus has been implicated in cancer and multiple other diseases. Each cell nucleus contains roughly 2,000 NPCs embedded in the nuclear envelope; the NPC (which is about 50 nanometers wide) is responsible for all transport into and out of the nucleus. To prevent the contents of the rest of the cell's interior from mixing with that of the nucleus, the NPC discriminates between cargos with great precision.
Scientists have constructed models for the NPC, but how this channel operates and achieves its selectivity has remained a mystery. It is known that to make it through the NPC, large molecules must bind at least a few receptors called importins; whether binding more importins speeds or slows a molecule's passage has been unclear. So, too, has the exact point at which the Ran GTPase protein functions in the transport process.
Previously, scientists had observed the motion of small molecules (a few nanometers in diameter), labeled with fluorescent tags, as they passed through the NPC. But the rapid transit and faint signal of these molecules resulted in sparse, fuzzy data. Drs. Liphardt and Weis's team employed quantum dots, which are about 20 nm in diameter and thus transit through the pore more slowly than small molecules. In addition, quantum dots are far brighter than conventional fluorophores, making it easier for the researchers to track them as they moved through the NPC.
The researchers coated the quantum dots with signals recognized by importins. Using a microscopic technique that allowed them to see a flat, thin visual slice through living cells, they watched hundreds of individual dots entering, jiggling around in, being ejected from, and in some cases admitted through, NPCs. The investigators recorded video data and tracked the motion of 849 quantum dots with nanometer precision.
The spaghetti-like paths of the quantum dots, superimposed on one another, revealed that the particles fell into three classes: "early aborts," which were briefly confined and then bounced out; "late aborts," which wandered in and meandered to the inner end of the pore before exiting the way they came; and "successes," which followed much the same paths as the late aborts but were granted entry.
From the paths' erratic paths, the researchers deduced that the quantum dots were indeed diffusing randomly, rather than being actively transported. And adding more importins to the dots' coating shortened the transit time, suggesting that importins make incoming cargo more soluble within the NPC rather than restricting their movement. The researchers found a particularly interesting result when they withheld the carrier protein Ran from the experiment. Without Ran in the mix, the quantum dots followed exactly the same range of paths as when Ran was present, except that virtually none passed through the NPC.
Considering their path data, the authors drew a model for how the NPC operates. Large cargo is initially captured by the NPC's filament fringe. It then encounters a constriction, through which it can enter a sort of antechamber. Then, in certain cases, Ran exchanges the cargo's GDP for a GTP and it is admitted into the nucleus. Only the final step is irreversible.
One of the main new insights is that the NPC's selectivity seems to result from a cascade of filters, each preferring correct cargos, rather than just one very selective step. This idea helps explain why some things can easily get into the nucleus and other things are excluded. This discovery may have some very practical clinical implications, Liphardt and Weis say. It may enable scientists to develop techniques to efficiently deliver large man-made cargos, such as drug-polymer conjugates and contrast agents, to the nucleus, which contains the genome.
This work, which is detailed in a paper titled, "Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking," was supported by the National Cancer Institute's Physical Sciences in Oncology initiative, a program that aims to foster the development of innovative ideas and new fields of study based on knowledge of the biological and physical laws and principles that define both normal and tumor systems. An abstract of this paper is available at the journal's Web site.