Their findings appear in Nature Methods.
Scientists have known for more than a century that proteins, such as histones, aid in packing DNA into the nucleus of a cell. Human cells contain 2 to 3 meters of DNA, which must be kinked and coiled enough to fit into a region 1/10 the width of a human hair.
Despite the use of powerful, high-resolution imaging techniques such as electron microscopy, the mechanism by which this chromatin packing occurs remains a mystery. The densely coiled chromatin fibers are very difficult to visualize, and little is known about how they condense during cell division, or unwind to allow gene expression.
In developing their method, the Illinois team tackled a key difficulty in imaging cells using electron microscopy. Traditional studies “fix” the cells with potent chemicals (called fixatives) to preserve their structure for viewing under a microscope. But standard fixation methods interfere with another step in the imaging process: the use of tagged antibodies to label key components of the cells.
These antibodies, which target and latch on to specific proteins in the cell, can be tagged with fluorescent labels for detection in light microscopy, or with metal particles (gold, in this case) for electron microscopy.
“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.
electron microscopy image
“And if your target is inside the condensed chromatin, the antibodies have no way to penetrate.”
Instead of fixing the cells before staining with antibodies, the researchers first exposed living animal cells to the labeled antibodies. This allowed the antibodies to penetrate more deeply into the chromatin structure, and boosted the number of gold particles adhering to regions of interest. The signal was enhanced by adding a silver solution that precipitated (solidified) upon contact with the gold.
“We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins,” Kireev said.
The researchers had inserted several copies of a bacterial DNA, called the Lac operator, into the chromosomes. A bacterial protein, the Lac repressor, recognizes and binds to the Lac operator in living cells.
The researchers combined a Lac repressor protein with another protein that fluoresces green under blue light. This engineered protein adhered to the chromosomes in regions containing the Lac operator sequences. Under blue light, these regions fluoresced. A gold-tagged antibody targeted against green fluorescent protein (GFP) was then microinjected into the nucleus of a living cell, which added a metallic signal that could be boosted with silver.
“All this combined gives us a much better signal, a much stronger signal, with the very best structural preservation,” Kireev said.
The fluorescing protein helped the researchers find the regions of interest in the cells. These areas were then “immunogold” labeled and targeted for electron microscopy.
In the resulting micrographs the researchers saw enhanced staining of the chromosomes.
“We can now apply this same live-cell labeling 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.
“In trying to understand chromosomes, people have largely been limited to low resolution visualization of specific chromosomal proteins using light microscopy,” Belmost said. “This meant everyone has had to do a lot of guessing of how things are put together, leading in many cases to vague, cartoon models of what are likely to be complicated chromosomal structures carrying out DNA functions such as replication and transcription.”
“Now we hope we can simply look and see the real structure using the more than 10-fold higher resolution of electron microscopy,” Belmont said. “We are really excited to see what we will find using our new method”
Editor’s note: To reach Andrew Belmont, call 217-244-2311; e-mail: email@example.com.
To reach Igor Kireev, call 217-333-8372; e-mail: firstname.lastname@example.org.
Diana Yates | University of Illinois
The balancing act: An enzyme that links endocytosis to membrane recycling
07.12.2016 | National Centre for Biological Sciences
Transforming plant cells from generalists to specialists
07.12.2016 | Duke University
In recent years, lasers with ultrashort pulses (USP) down to the femtosecond range have become established on an industrial scale. They could advance some applications with the much-lauded “cold ablation” – if that meant they would then achieve more throughput. A new generation of process engineering that will address this issue in particular will be discussed at the “4th UKP Workshop – Ultrafast Laser Technology” in April 2017.
Even back in the 1990s, scientists were comparing materials processing with nanosecond, picosecond and femtosesecond pulses. The result was surprising:...
Have you ever wondered how you see the world? Vision is about photons of light, which are packets of energy, interacting with the atoms or molecules in what...
A multi-institutional research collaboration has created a novel approach for fabricating three-dimensional micro-optics through the shape-defined formation of porous silicon (PSi), with broad impacts in integrated optoelectronics, imaging, and photovoltaics.
Working with colleagues at Stanford and The Dow Chemical Company, researchers at the University of Illinois at Urbana-Champaign fabricated 3-D birefringent...
In experiments with magnetic atoms conducted at extremely low temperatures, scientists have demonstrated a unique phase of matter: The atoms form a new type of quantum liquid or quantum droplet state. These so called quantum droplets may preserve their form in absence of external confinement because of quantum effects. The joint team of experimental physicists from Innsbruck and theoretical physicists from Hannover report on their findings in the journal Physical Review X.
“Our Quantum droplets are in the gas phase but they still drop like a rock,” explains experimental physicist Francesca Ferlaino when talking about the...
The Max Planck Institute for Physics (MPP) is opening up a new research field. A workshop from November 21 - 22, 2016 will mark the start of activities for an innovative axion experiment. Axions are still only purely hypothetical particles. Their detection could solve two fundamental problems in particle physics: What dark matter consists of and why it has not yet been possible to directly observe a CP violation for the strong interaction.
The “MADMAX” project is the MPP’s commitment to axion research. Axions are so far only a theoretical prediction and are difficult to detect: on the one hand,...
16.11.2016 | Event News
01.11.2016 | Event News
14.10.2016 | Event News
07.12.2016 | Health and Medicine
07.12.2016 | Life Sciences
07.12.2016 | Health and Medicine