Schematic of the NPC mimic, including a single pore, the device that supports the membrane and the experimental setup on a microscope. FG-nup, FG-nucleoporin. Reprinted from Nature.

The nuclear pore complex (NPC) is a heterogeneous scaffold of proteins that selectively filters biomolecules moving between the nucleus and the cytoplasm. The NPC is an ancient yet highly conserved gate decorated with phenylalanine-glycine (FG)-nucleoporins, natively disordered protein domains that interact directly with the transporters that ferry biological cargo through the pore.

In an effort to determine whether an artificial pore lined with FG-nucleoporins is sufficient to recapitulate NPC selectivity, Brian Chait and Mike Rout, in conjunction with their colleagues at Rockefeller University, engineered a nanosorter that mimics the NPC. “We worked for many years on a detailed description of the nuclear pore complex,” says Chait, and “what we didn't find was any sort of motor molecules or ATPases that would make this thing move in a concerted way.” Rout adds, “We knew that the NPC was a gate, but when we think of a gate, we think of something that opens and shuts.” The absence of conventional molecular motors and moving parts means that the NPC is an unusual gate. Chait says, “At that point, we realized that this machine would have to work by diffusion.”

The initial impression that the NPC was a hole surrounded by FG-nucleoporins did not change as more details emerged. “This is a virtual gating machine; it is always open to the right kind of things, but always closed to the wrong things,” says Chait. To verify this hypothesis, they decided to try and build one. They engineered a porous polycarbonate membrane that was functionalized with FG-repeat domains conjugated to the membrane through a thin layer of gold. The NPC mimic, or nanosorter, incorporated several key features of the NPC, among them that the nanopores had appropriate diameters, FG-nucleoporins were present in appropriate orientations and densities, and the business end of the membrane itself was thin. The researchers tested their NPC mimic by measuring the flux of fluorescently labeled proteins from one chamber to another on the opposite side of the membrane. Amazingly, their relatively simple nanosorter recapitulated many fundamental properties of the NPC, including selectivity.

Looking forward, this nanosorter should prove highly useful to scientists interested either in understanding how the NPC works or in its practical applications as a selective filter. In this first application of the nanosorter, an unanticipated role emerged for transporters that carry cargo through the pores. As Rout explains, “Rather than just being carriers of cargo back and forth across the NPC, [the transporters] seem to help exclude things that are not supposed to be there.” Thus, the transporters are an unexpected but possibly major component of the gate between nuclear and cytoplasmic compartments. Further experiments of this nature, with more sophisticated nanosorters, should provide greater mechanistic insight into NPC selectivity and function.

Another application that Chait and Rout envision for improved versions of this nanosorter is molecular sorting. According to Rout, “Currently, molecular sorting is based on chromatography, and that's great, but it has its limitations. It would be nice to have alternative techniques in your pocket. Biological membranes are selective filters. Once we fully understand the principles of how they work, we could design our own.” Chait adds, “Ultimately, we may be able to sort all kinds of different molecules from very messy milieus.”