Science Literature

All traffic between a cell's nucleus and its cytoplasm has to pass through a nuclear pore complex (NPC) which selects which macromolecules will pass and which will not. Research into the structure of the NPC, previously discussed here and here, is being supplemented by research into the mechanisms that turn a pore into a gateway. A major step forward has been the construction of an artificial nanopore that mimics the action of the NPC.

Artificial transportation. A schematic representation of the genuine (top) and artificial (bottom) nuclear pore complexes. By experimenting with a nuclear pore complex "mimic", researchers have shown how transport factors (red), which help proteins move through the complex, are assisted by proteins called FG-nucleoporins (twisting lines).(Source here)

In the nuclear envelope, the pores are about 30 nm in diameter. Previous work revealed that, "at its simplest, the pore's organization consists of an anemone-like configuration, with folded proteins forming the hole itself and unfolded, tentacle-like proteins called FG-nucleoporins around the opening." This formed the basis of work to mimic the structure.

"[T]he researchers are looking at the functionality of the complex at its most basic configuration: a membrane pockmarked with FG-nucleoporin-coated holes. "We wondered whether we could create a simple artificial mimic, made of just a tiny hole and some of these tentacle proteins," Chait says. "So we built one to see if it really works." To do so, postdoctoral associate Tijana Jovanovic-Talisman started with simple polycarbonate membranes strewn with little holes and coated with a thin layer of gold, and then attached a type of FG-nucleoporin to the membrane."

The nuclear envelope is represented by a polycarbonate sheet perforated with nanopores about 30 nm in diameter. One face of the sheet was coated in gold, to which active FG-nucleoporins were attached, separated by low complexity hydrophilic spacers. The nucleoporins were not synthesised, but obtained from budding yeast. They were arranged in approximately the same density as found in the NPC (about 70 molecules per pore).

"Using confocal microscopy, [Jovanovic-Talisman] tested how efficiently proteins crossed this artificial membrane. Then she added transport factors, which bind to the FG-nucleoporins and selectively ferry cargo across the membrane barrier. The researchers saw that transport factors crossed the artificial membrane much faster than proteins alone, just as occurs in the natural nuclear pore complex. Without the transport factors, that selectivity largely disappeared. The research gave the scientists and their collaborators [. . .] a new perspective on the cell's nuclear transport mechanism. "We're beginning to think of the transport factors in a different way," Jovanovic-Talisman says, "as if they're a mobile part of the nuclear pore complex machine."

This role for the transport factors is a significant addition to knowledge. The artificial nanopore permits experimentation to establish critical factors. For example, as predicted, "the larger the pore size, the weaker the selectivity of the membrane". Other experiments showed the potential for "future carefully tuned alterations in the design of our device could significantly enhance its selectivity".

"The artificial pore could be used to test the importance of pore shape and size. And it has potential biomedical applications, too. Because it can separate particular proteins out of very complex mixtures, the device could have enormous implications for biopharmaceuticals. "It's a device that mimics what nature does and has some beautiful properties, in that it decides what passes through a hole in a very complex mixture," Chait says."

From a design perspective, these findings are tremendously important. Instead of finding simplicity as the black box of the NPC is opened, we discover hidden depths of complexity. There are numerous factors that have to be in place before there is functionality. The characteristics of irreducible complexity are becoming clearer with time. What this means is that incremental development and billions of years cannot be involved in any explanation of the origin of the NPC. It must be formed abruptly if it is to work at all. But note this:

"The team is now working toward making the synthetic pore as selective and efficient as the natural one. "Our machine doesn't work as well as the nuclear pore complex. We've had only three years, while nature's had billions of years to do this," Chait says. "We've got lots of work to do." "

Unfortunately for Chait, geology will not allow him billions of years either. There is evidence for eukaryotes just before the Great Oxidation Event (back to 2.6 Ga) and the distinctive structure of eukaryotes appears to have been very stable since then. Abrupt appearance followed by stasis is the norm in the fossil record, and it does strain gradualist thinking beyond breaking point. It is suggested that a research methodology based on Design will yield productive outcomes quicker than any other approach.

Artificial nanopores that mimic the transport selectivity of the nuclear pore complex
Tijana Jovanovic-Talisman, Jaclyn Tetenbaum-Novatt, Anna Sophia McKenney, Anton Zilman, Reiner Peters, Michael P. Rout & Brian T. Chait
Nature 457, 1023-1027 (19 February 2009) | doi:10.1038/nature07600

Nuclear pore complexes (NPCs) act as effective and robust gateways between the nucleus and the cytoplasm, selecting for the passage of particular macromolecules across the nuclear envelope. NPCs comprise an elaborate scaffold that defines a ~30 nm diameter passageway connecting the nucleus and the cytoplasm. This scaffold anchors proteins termed 'phenylalanine-glycine' (FG)-nucleoporins, the natively disordered domains of which line the passageway and extend into its lumen. [. . .] To test whether a simple passageway and a lining of transport-factor-binding FG-nucleoporins are sufficient for selective transport, we designed a functionalized membrane that incorporates just these two elements. Here we demonstrate that this membrane functions as a nanoselective filter, efficiently passing transport factors and transport-factor-cargo complexes that specifically bind FG-nucleoporins, while significantly inhibiting the passage of proteins that do not. This inhibition is greatly enhanced when transport factor is present. Determinants of selectivity include the passageway diameter, the length of the nanopore region coated with FG-nucleoporins, the binding strength to FG-nucleoporins, and the antagonistic effect of transport factors on the passage of proteins that do not specifically bind FG-nucleoporins. We show that this artificial system faithfully reproduces key features of trafficking through the NPC, including transport-factor-mediated cargo import.

See also:

Researchers construct a device that mimics one of nature's key transport machines, PhysOrg.com, January 6th, 2009