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It then encounters a constriction, through which it can enter a sort of antechamber. Large cargo is initially captured by the NPC’s filament fringe. 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.Ĭonsidering their path data, the authors drew a model for how the NPC operates. The researchers found a particularly interesting result when they withheld the carrier protein Ran from the experiment. 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 binding to interior walls. 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.įrom the paths’ erratic meanderings, the researchers deduced that the quantum dots were indeed diffusing randomly, rather than being actively transported. The researchers recorded video data and tracked the motion of 849 quantum dots with nanometer precision. 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 researchers coated the quantum dots with signals recognized by importins. employed “quantum dots”, which are about 20 nm in diameter-and hence slower than smaller molecules-and much brighter than conventional fluorophores. But the rapid transit and faint signal of these molecules resulted in sparse, fuzzy data.
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Previously, scientists had observed the motion of small molecules (a few nm in diameter), labeled with fluorescent tags, through the NPC. (Weis and Liphardt are members of QB3.) The research was published September 1 st in the journal Nature, in a paper on which Berkeley post-doc Alan Lowe and graduate student Jake Siegel were joint first authors. Karsten Weis, a UC Berkeley professor of molecular and cell biology, Jan Liphardt, a UC Berkeley professor of physics, and colleagues conducted advanced imaging experiments that resolved these issues. So, too, has the exact point at which a carrier protein called “Ran” plays a crucial part, substituting one molecule of GTP (a cellular fuel, an analog of the better-known ATP) for one of GDP that the large molecule brings with it when it enters the NPC. 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. Scientists have constructed models for the NPC, but how this channel operates and achieves its selectivity has remained a mystery. Several viruses target the NPC to gain entry to the nucleus, and dysfunctional transport between the cytoplasm and the nucleus has been implicated in multiple diseases including cancer. (b) A quantum dot cargo moves through an NPC. (a) Larger cargos (red) require a transport receptor (green) to pass through the gate. The nuclear pore complex (NPC) gates the traffic of all molecules between the cytoplasm and the nucleus of eukaryotic cells. 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. The NPC (which is about 50 nanometers wide) is responsible for all transport into and out of the nucleus. Each cell nucleus contains roughly 2,000 NPCs, embedded in the nuclear envelope. The NPC, a large protein assembly shaped like a basketball net fringed with tentacles, is the gateway to the cell nucleus, where genetic information is stored. QB3 biophysicists have 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. Berkeley Research Infrastructure Commons (RIC)