Featured Research

from universities, journals, and other organizations

New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells

Date:
October 22, 2012
Source:
Massachusetts Institute of Technology
Summary:
The glowing green molecule known as green fluorescent protein (GFP) has revolutionized molecular biology. When GFP is attached to a particular protein inside a cell, scientists can easily identify and locate it using fluorescence microscopy. However, GFP can't be used with electron microscopy, which offers much higher resolution than fluorescence microscopy. Chemists have now designed a GFP equivalent for electron microscopy -- a tag that allows scientists to label and visualize proteins with unprecedented clarity.

In this electron microscope image of a mitochondrion, the matrix has been stained with APEX, making it appear dark. The lighter projections into the matrix represent the intermembrane space.
Credit: Tom Deerinck and Jeff Martell

The glowing green molecule known as green fluorescent protein (GFP) has revolutionized molecular biology. When GFP is attached to a particular protein inside a cell, scientists can easily identify and locate it using fluorescence microscopy. However, GFP can't be used with electron microscopy, which offers much higher resolution than fluorescence microscopy.

Chemists from MIT have now designed a GFP equivalent for electron microscopy -- a tag that allows scientists to label and visualize proteins with unprecedented clarity.

"With things that may appear only a few pixels across by fluorescence microscopy -- for example, a mitochondrion -- you can't make out any of the internal features. But with electron microscopy it's very easy to discern the intricate internal structures," says Jeff Martell, a graduate student in chemistry at MIT and lead author of a paper describing the new tag in the Oct. 21 online edition of Nature Biotechnology.

The new tag could help scientists pinpoint the locations of many cell proteins, providing new insight into those proteins' functions, according to the researchers.

Improving on nature

Dubbed APEX, the new tag is similar to naturally occurring proteins that have been tried as imaging labels for electron microscopy. Horseradish peroxidase (HRP) is one commonly used tag, but it works only in a few compartments of a cell. Other recently developed tags work throughout a cell but are technically challenging to use because they require light to be shined on the sample and oxygen to be bubbled through it.

To improve on these methods, the researchers started with a protein similar to HRP, called ascorbate peroxidase (APX). APX is more versatile than HRP because it can function within a cell's cytosol, in the main cavity of a cell.

Both HRP and APX belong to a class of enzymes called peroxidases, which remove an electron and a proton from other molecules in a process known as oxidation. Every peroxidase has different targets, and one of HRP's main targets is a molecule called DAB, which when oxidized can be visualized with electron microscopy. The researchers genetically engineered APX so that it would also target DAB.

To use this new APEX tag (for "engineered APX"), the researchers deliver, into a living cell, a small ring of DNA containing the APEX gene joined to the gene for the protein they plan to image. The cell then produces the target protein, bound to the APEX protein.

Next, the researchers need to deliver DAB, which is not normally found in cells. This delivery takes place during the process of "fixing," or stabilizing cells, which must be done before they can be imaged with electron microscopy.

When the APEX protein oxidizes DAB, it generates radicals that rapidly clump together into a tarlike polymer. That polymer can be detected through electron microscopy, allowing the researchers to pinpoint the location of the target protein.

Biological question resolved

To demonstrate the usefulness of their new tag, the researchers set out to resolve an open question regarding the location of a calcium channel protein discovered last year. Two research groups identified the protein and reported that it is located within mitochondria, but they had conflicting theories as to its precise location and orientation. Using the new imaging technique, the MIT-led team labeled the protein and determined that it is embedded in the inner mitochondrial membrane and faces into the innermost part of mitochondria, the mitochondrial matrix.

The team also showed that the new tag can label proteins throughout the cell -- not only within mitochondria but also in the nucleus, the endoplasmic reticulum and the cytosol.

Martell and Alice Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT and senior author of the Nature Biotechnology paper, invented the new technology. Other authors who helped to test the tag and explore biological applications are Mark Ellisman, Thomas Deerinck and Gina Sosinsky of the University of California at San Diego, Yasemin Sancak and Vamsi Mootha of Harvard Medical School, and Thomas Poulos of the University of California at Irvine.

In current studies, the researchers are working on filling entire cells, such as neurons, with their imaging agent. This allows certain neurons in an electron microscope image to stand out, making it easier to trace the connections they make with other neurons. For that project, the MIT researchers are collaborating with Joshua Sanes, a professor of molecular and cellular biology at Harvard University, who says he believes the new labeling technology will be very useful.

"We want to find the exact connections that these cells are making, and APEX is a good way to label cells for electron microscopy. We can label specific types of cells and figure out how they fit into the neural circuitry," Sanes says.

Ting and Martell have filed for a patent on their imaging technology and are now working on making the APEX molecule more stable and better able to bind heme (an iron atom embedded in an organic compound), which is necessary for it to function properly.

The research was funded by the National Institutes of Health.


Story Source:

The above story is based on materials provided by Massachusetts Institute of Technology. The original article was written by Anne Trafton. Note: Materials may be edited for content and length.


Journal Reference:

  1. Jeffrey D Martell, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, Alice Y Ting. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology, 2012; DOI: 10.1038/nbt.2375

Cite This Page:

Massachusetts Institute of Technology. "New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells." ScienceDaily. ScienceDaily, 22 October 2012. <www.sciencedaily.com/releases/2012/10/121022113641.htm>.
Massachusetts Institute of Technology. (2012, October 22). New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells. ScienceDaily. Retrieved July 25, 2014 from www.sciencedaily.com/releases/2012/10/121022113641.htm
Massachusetts Institute of Technology. "New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells." ScienceDaily. www.sciencedaily.com/releases/2012/10/121022113641.htm (accessed July 25, 2014).

Share This




More Matter & Energy News

Friday, July 25, 2014

Featured Research

from universities, journals, and other organizations


Featured Videos

from AP, Reuters, AFP, and other news services

TSA Administrator on Politics and Flight Bans

TSA Administrator on Politics and Flight Bans

AP (July 24, 2014) TSA administrator, John Pistole's took part in the Aspen Security Forum 2014, where he answered questions on lifting of the ban on flights into Israel's Tel Aviv airport and whether politics played a role in lifting the ban. (July 24) Video provided by AP
Powered by NewsLook.com
Creative Makeovers for Ugly Cellphone Towers

Creative Makeovers for Ugly Cellphone Towers

AP (July 24, 2014) Mobile phone companies and communities across the country are going to new lengths to disguise those unsightly cellphone towers. From a church bell tower to a flagpole, even a pencil, some towers are trying to make a point. (July 24) Video provided by AP
Powered by NewsLook.com
Algonquin Power Goes Activist on Its Target Gas Natural

Algonquin Power Goes Activist on Its Target Gas Natural

TheStreet (July 23, 2014) When The Deal's Amanda Levin exclusively reported that Gas Natural had been talking to potential suitors, the Ohio company responded with a flat denial, claiming its board had not talked to anyone about a possible sale. Lo and behold, Canadian utility Algonquin Power and Utilities not only had approached the company, but it did it three times. Its last offer was for $13 per share as Gas Natural's was trading at a 60-day moving average of about $12.50 per share. Now Algonquin, which has a 4.9% stake in Gas Natural, has taken its case to shareholders, calling on them to back its proposals or, possibly, a change in the target's board. Video provided by TheStreet
Powered by NewsLook.com
Robot Parking Valet Creates Stress-Free Travel

Robot Parking Valet Creates Stress-Free Travel

AP (July 23, 2014) 'Ray' the robotic parking valet at Dusseldorf Airport in Germany lets travelers to avoid the hassle of finding a parking spot before heading to the check-in desk. (July 23) Video provided by AP
Powered by NewsLook.com

Search ScienceDaily

Number of stories in archives: 140,361

Find with keyword(s):
Enter a keyword or phrase to search ScienceDaily for related topics and research stories.

Save/Print:
Share:

Breaking News:
from the past week

In Other News

... from NewsDaily.com

Science News

    Health News

      Environment News

        Technology News



          Save/Print:
          Share:

          Free Subscriptions


          Get the latest science news with ScienceDaily's free email newsletters, updated daily and weekly. Or view hourly updated newsfeeds in your RSS reader:

          Get Social & Mobile


          Keep up to date with the latest news from ScienceDaily via social networks and mobile apps:

          Have Feedback?


          Tell us what you think of ScienceDaily -- we welcome both positive and negative comments. Have any problems using the site? Questions?
          Mobile: iPhone Android Web
          Follow: Facebook Twitter Google+
          Subscribe: RSS Feeds Email Newsletters
          Latest Headlines Health & Medicine Mind & Brain Space & Time Matter & Energy Computers & Math Plants & Animals Earth & Climate Fossils & Ruins