To study this process, the Salk researchers created a cell-free system based on frog's eggs (oocytes) that was able to recapitulate the insertion of the nuclear pore complex into the nuclear membrane. Using advanced real-time imaging tools the scientists watched as a nuclear membrane ?pores and all ?formed within an hour.
"We were able to visualize single nuclear pore complexes," says graduate student and co-first author Daniel Anderson. "This allowed us not only to watch as single pores formed but also to demonstrate that they formed from scratch without the help of already existing pores."
In another experiment, the group used four-dimensional confocal microscopy to follow the formation in cultured human cells of a single pore that had been labeled with a fluorescent tag. If the nuclear pore had split to give rise to two daughter pores, two bright dots would have emerged from one; however, the researchers tracked movement of only one dot, confirming their previous finding that pores formed from scratch.
Additional research demonstrated that nuclear pore assemblies are added in a stepwise, coordinated process requiring components on both sides of the nuclear membrane. "This has important consequences for the next big issue ?the question of how these structures all fuse together," Hetzer says.