October 18, 1999
Volume 52, No. 8
A day in a lab's life: Of Xenopus and DNA
When Maureen Powers looks in a mirror, tracing the ridge of her brow, moving down along her nose and back up to her eyes, she sees an expression of the genes that made her face.
Powers, assistant professor of cell biology, knows that gene expression occurs at the cellular level when regulatory proteins cross into the nucleus (home to a cell's DNA) and switch genes off and on. Turned on, a gene begins transcribing its genetic message into messenger RNA that leaves the nucleus to be translated into proteins.
"One of the biggest advances in cells as they evolved from bacteria to higher-ordered cells is the development of the nucleus," Powers said. "This packages up the genes into a compartment, allowing for better control of what the DNA encodes by controlling access of regulatory proteins."
But passage in and out of the nucleus is not random; it is a tightly controlled process. Anything entering or leaving the nucleus must pass through the nuclear pore complex (NPC). Scattered throughout the nuclear membrane, these gateways determine which materials contain the correct signal for passage. In short, the nuclear pore complex acts as a significant regulator of genetic expression and cellular function.
Powers studies the NPC, and it is almost impossible to relate a typical day in her lab. Scientific research is by nature a process of constant change involving continual reaction to interesting results and ever-evolving ideas. That being noted, a day in her lab might happen like this:
In the morning, a researcher collects eggs laid by Xenopus frogs. This South African amphibian lays a large number of eggs, providing enough material for multiple experiments. Although it appears small, a Xenopus egg is actually a very large single cell, quite visible to the naked eye. And like the microscopic cells making up a human body, the egg contains components for making a nucleus.
Traditional model species like the fruit fly or the worm have the advantages of powerful genetics. Xenopus is a unique model system because materials prepared from its eggs can be used to build a working nucleus in the test tube. "It's not very cute," Powers said of her aesthetically challenged subject, "but it's reasonably adaptable to the lab."
The frogs had been injected the previous day with a hormone to induce egg laying, and the result is hundreds of eggs. After collection, the eggs are spun down in a centrifuge. By varying the time and force of the spin, the researcher can pellet larger components of the cell while smaller cellular components remain in solution. In Powers' case, the cell membranes are removed as pellet, while all of the stored-up parts of the disassembled nucleus remain in solution.
"There are between 50 and 100 proteins that make up the NPC," Powers said. "This is one of the biggest machines in the cell."
Next, her lab passes the cell solution over an antibody column. The antibodies are specific "handles" for one protein of the NPC, binding to that particular piece and removing it from the solution. When the NPC is later reassembled, any lack of function can be attributed to the missing protein removed by the antibody.
To build a nucleus, the solution is mixed with some of the membrane that was pelleted earlier in the centrifuge. After mixing, DNA extracted from sperm is also added, and the membrane spontaneously begins to form a nucleus by enclosing the DNA. As the nucleus reforms, the NPC also reassembles, but lacking the single protein removed by the antibody.
At this point, Powers can finally study the NPC's function and how it was changed by removal of one of its protein parts. She might label a protein with a fluorescent tail and then mix this protein with the newly formed nuclei. Under a fluorescent microscope, she can follow the progression of the protein into the nucleus. Likewise, she can tag RNA, inject it into the nucleus and determine if it is no longer transported out. All of this transport into and out of the nucleus will be mediated by the newly modified NPC, the nuclear doorman.
Yet the function of the NPC is more complex and dynamic than in the simple examples just described. "Some things that were thought to be regulated at the point of going into the nucleus are actually regulated by when they go out," Powers said. "They are going in and out all the time; this is the steady state."
Although there is constant traffic through the NPC, it is not understood how this immense structure actually works. By studying the function of individual parts of the NPC, Powers' lab hopes to develop a blueprint of how the parts work together to form the machine that conducts substrates into and out of the nucleus.
"It is important that we understand how this very basic and critical process works," she said. "Not only is nuclear transport essential for the life of the cell, but infecting viruses also take advantage of this process in their life cycle. HIV, for example, makes several proteins that effect transport through the NPC. By understanding the basic mechanism of transport, we increase our chance of designing strategies to combat viral replication."