Purdue researchers Seema Mattoo and Ranjan Sengupta have developed a new electron microscopy technique that allows them to accurately track membrane proteins without damaging the cellular environment.
Membrane proteins play crucial role in many biological processes. According to studies more than 50% of all modern medicinal drugs targets membrane proteins.
Unfortunately for scientists, deducing their structures has been a longstanding challenge because it’s tough to track the protein without causing harm to the cellular membrane using current techniques. Not anymore.
Researchers at Purdue University have created a “cryoAPEX” named new electron microscopy technique that accurately trace membrane proteins in a well-preserved cell. The new electron microscopy method is a hybrid of high-pressure freezing method and chemical fixation method which are commonly used in cell biology.
“We took the best features from each technique and played around with conditions until we found a happy medium where you could stain for your protein while maintaining membrane preservation,” said Seema Mattoo, an assistant professor of biological sciences at Purdue. “We were also able to use this information to develop a 3D image of the protein in the context of the cell.”
In Chemical fixation method antibodies are used to detect proteins, but to do that, the cells need to be “fixed” with alcohol. Alcohol degrades the membrane, essentially resulting holes in it and allowing the signal researchers are looking for to leak out. This produces inaccurate results.
On the other hand, High-pressure freezing preserves the entire cell flawlessly. But this method isn’t compatible with staining techniques, so there’s no way to track the protein of interest.
Membrane proteins play various roles in biological processes, including signalling between a cell’s internal and external environments, transporting molecules and ions across the membrane, and allowing cells to identify and communicate with each other. Ability to track these proteins as they perform their roles in the cell will allow researchers to understand cell signalling mechanisms that regulate protein function.
“Virologists, for instance, will find this technique really useful because now they’ll be able to follow their viruses in the context of a particular viral protein within a cell,” said Mattoo, who is also a member of Purdue’s Center for Cancer Research and Institute of Inflammation, Immunology and Infectious Disease.
Mattoo‘s research focuses on a class of enzymes called Fic proteins. Fic proteins belongs to a family of proteins characterized by the presence of a conserved FIC domain which is involved in the modification of protein substrates by the addition of phosphate-containing compounds. Its human version known as ‘HYPE’, is important in deciding whether cells under stress live or die. Her motive for developing this method was to be able to see where HYPE goes once it enters the endoplasmic reticulum, she said.
During the investigation researchers found that HYPE is strongly drawn to the lumen. This implies that HYPE is tightly modulated, and if it ever does leave the endoplasmic reticulum, it would likely be under disease-specific conditions.
“As we’re trying to figure out how to manipulate HYPE to determine whether it can be used as a therapeutic, we’re going to focus a lot of our energy on the endoplasmic reticulum,” Mattoo said. “But the wider implications of these findings are predominantly in the technique. We developed it so that it can be applied to tissue culture cells and can be used by a wide range of researchers.”
These findings have been published in the Journal of Cell Science.
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