Cryo-EM method limits membrane destruction
Electron tomogram of a cell processed by Cryo-APEX [Mattoo/Purdue]
Researchers at Purdue University have created an electron microscopy technique - cryoAPEX - that accurately tracks membrane proteins in a well-preserved cell.
The new method is a hybrid of two commonly used methods in cell biology; high-pressure freezing and chemical fixation.
“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,” says Professor Seema Mattoo, from Biological Sciences. “We were also able to use this information to develop a 3D image of the protein in the context of the cell.”
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. [Purdue University photo/Rebecca Wilcox]
Membrane proteins play a key role in many biological processes, but determining structures without inflicting cellular damage, has been a long-standing challenge for researchers worldwide.
As Mattoo and colleagues highlights in Journal of Cell Science: “Localization of membrane proteins via electron microscopy at high resolution depends on robust detection technology and on sample preparation methods that confer superior ultrastructural preservation of membranes.”
Indeed, chemically fixing cells can break down the membrane while high-pressure freezing structures isn’t compatible with staining techniques.
Given this, the researchers set out to adapt a recently engineered ascorbate peroxidase enzymatic tag - APEX2 - for cryofixation while preserving subcellular membrane structure.
By coupling chemical fixation and high-pressure freezing of cells with peroxidase tagging, they discovered they could localise membrane proteins within a well-preserved subcellular membrane architecture.
A 3D model of the endoplasmic reticulum (blue) displaying density associated with transiently expressed APEX2-tagged human Fic protein was generated from an electron tomogram of a cell processed by Cryo-APEX. [Purdue University/Seema Mattoo]
“Virologists 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,” highlights Mattoo.
Mattoo’s research focuses on a class of enzymes called Fic proteins; the version present in humans, HYPE, is a critical regulator of whether cells under stress live or die.
Using her latest method, she has been able to show that HYPE is drawn to the lumen once entering the endoplasmic reticulum.
As she points out, these results indicate that HYPE is tightly regulated, and if it leaves the endoplasmic reticulum, it would likely be under disease-specific conditions.
“The wider implications of these findings are predominantly in the technique,” she says. “We developed it so that it can be applied to tissue culture cells and can be used by a wide range of researchers.”