An intriguing new method which allows for more detailed electron microscopy images is set to shed light on the complex chromosomal structures that carry out DNA functions such as replication and transcription.
The technique has created excitement amongst the research community, particularly geneticists and drug researchers, who believe more intimate knowledge of the cell division and gene expression process will aid greatly in their research.
Scientists from the University of Illinois adopted a method that exposes living cells to labelled antibodies. These antibodies, which aim and fasten on to certain cell proteins, were labelled with gold particles for detection under electron microscopy.
The method developed also deals with a common problem in imaging cells particularly with electron microscopy. In "fixing" cells with chemicals to preserve cell structure, standard fixation methods interfere with the use of labelled antibodies to tag key structures of the cells.
Instead the researchers first exposed living animal cells to the antibodies, allowing the antibodies to penetrate more intensely into the chromatin structure. The antibodies also allowed more gold particles to be attracted to regions of interest.
As the gold particles migrated to the various chromatin structures, researchers added silver solution that solidified upon contact with the gold, strengthening the signal in the process.
"If you fix the cells first, you have a dramatic drop in the efficiency of these immunochemical reactions," said Igor Kireev, a visiting scientist in the department of cell and developmental biology and lead author of the paper.
"And if your target is inside the condensed chromatin, the antibodies have no way to penetrate. We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins," added Kireev.
Scientists have known for more than a hundred years that histone proteins assist in stuffing DNA into the cell nucleus. Human cells normally contain 2-3 metres of DNA, which must be tightly wound to fit into a space 1/10 the width of a human hair.
Although high-resolution imaging techniques such as electron microscopy has greatly advanced the understanding of this process, pressing questions have cropped up leading to a slowdown in research as scientists attempt to make progress despite big holes in cell replication knowledge.
Key to this information glut is the densely coiled chromatin fibres, which are considered very difficult to visualise. Because of this little is known about how they condense during cell division, or unwind to allow gene expression.
To further strengthen the signal, the scientists placed a number of copies of a Lac operator (bacterial DNA), into the chromosomes. Bacterial protein, known as a Lac repressor, identifies and attaches to the Lac operator in living cells.
The Lac repressor protein was paired with another protein that fluoresces green under blue light. When inserted into the cell the protein migrated towards the chromosomes in regions containing the Lac operator sequences. These regions fluoresced under blue light.
As a finishing touch an antibody labelled with gold and targeted against green fluorescent protein (GFP) was injected into the cell's nucleus. This added a metallic signal that was boosted with silver.
The resulting micrographs allowed the researchers to observe enhanced chromosome staining.
"We can now apply this same live-cell labelling method to study at high resolution many different GFP-tagged proteins in the cell cytoplasm or nucleus," said Andrew Belmont, a professor of cell and developmental biology and senior author of the paper.
"Now we hope we can simply look and see the real structure using the more than 10-fold higher resolution of electron microscopy," Belmont added. "We are really excited to see what we will find using our new method."
In trying to understand chromosomes, scientists have largely been limited to low resolution visualisation of specific chromosomal proteins using light microscopy. This meant they have had to make a lot of assumptions as to how things are put together.
This has lead to vague, crude models of what are likely to be complicated chromosomal structures carrying out DNA functions such as replication and transcription.