We are now ENABLED: An elegant technique for advancing molecular knowledge

About the School of Medicine Paper of the Month

The OHSU School of Medicine spotlights a recently published faculty research paper each month. The goals are to describe to the public the exceptional research happening at OHSU as well as inform our faculty of the innovative work underway across the school’s departments, institutes and disciplines. The monthly paper is selected by Assistant Dean for Basic Research Mary Heinricher, Ph.D.

We are now ENABLED: An elegant technique for advancing molecular knowledge

January 30, 2015

January’s featured paper is called “Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins,” published in The Journal of Neuroscience. The paper is published by a team from the Mao and Zhong Labs.

Paper of the Month, January 2015

It’s a paradox – how can you observe a system without disturbing the system?  Molecular biologists have struggled with this problem for decades, and the field tried to solve it by using a variety of fluorescent probes to observe protein activity in cells.

Although many techniques have been developed and refined over the years, their use has been shown to disrupt normal cellular function. So the question remains:  How can scientists visualize individual proteins in live cells for high-contrast and high-sensitivity imaging without affecting cellular function?

Why is this question so important to science? Understanding how a protein is organized and how it moves in living cells is the primary approach to understanding the function of the protein and the cell.  Despite all the advances in modern microscopy, one critical component in the imaging process remains unsolved: how to label the otherwise invisible protein in living cells so that it becomes visible under a microscope.

Imperfect solutions

Currently, the primary method to visualize protein activity in living cells is to link a target protein with GFP or similar fluorescent proteins in their DNA form, and then introduce the DNA into cells using a variety of methods.

The drawback, though, is the protein is expressed in addition to the normal protein levels (also called the endogenous levels), and therefore is “overexpressed.” Protein overexpression is known to be associated with many undesirable side effects. Even the very basic properties of cell-cell communication and regulation can be altered by overexpression.

Over the years, scientists have attempted different solutions to bypass the need for overexpression in live cell imaging. One solution is to “knock-in” the fluorescent protein tag into the genomic localization of the target protein. In this case, all of the endogenous proteins are replaced by the labeled protein without overexpression complications.

However, this approach is rarely employed because it would label the protein in all cells that normally express this protein. In tissue – the native habitat of cells – the imaging contrast resulted from a knock-in approach would be very low because each cell becomes the background of adjacent cells. The key to successful imaging studies is high cellular contrast that results from selective labeling of a very sparse subset of cells that are well isolated from each other.

Now, an elegant technique

This month’s featured paper was chosen by Mary Heinricher, Ph.D., assistant dean for basic research, because Drs. Tianyi Mao and Haining Zhong have managed to create an elegant technique that allows researchers to label individual proteins using live-cell imaging in a manner that minimally disrupts normal cellular function but still allows for high contrast and high sensitivity imaging. 

“We developed a novel strategy to label fluorescence protein that we call ENABLED (endogenous labeling via axonal duplication),” said Dr. Zhong, an assistant scientist at the Vollum Institute. “This strategy involves genetic manipulation of genomic DNAs in a way that the DNA can be edited in a specific manner if the cell also expresses another protein called Cre recombinase.”

He continued, “In the labeled cells, the targeted protein is expressed under native expression control at endogenous expression levels.”

Tianyi Mao, Ph.D., an assistant scientist at the Vollum Institute and collaborator with Dr. Zhong, elaborates. “The important feature of our strategy is that, in the absence of Cre recombinase (i.e., before editing), the cell is not labeled, and wild type, unlabeled protein is expressed,” she said. “By controlling how Cre recombinase is introduced, we can achieve labeling either in all cells, in a sparse subset of cells or in a selected subtype of cells.”  

Huge advance for the field

Drs. Mao and Zhong were able to demonstrate that their labeling strategy, at least for their protein of interest PSD-95, did not interfere with cellular function. This represents a huge advance in the field, as conventional approaches to examine PSD-95 disrupted organization and dynamics of the protein.

“Our study provides, for the first time, a generalizable strategy to label a large number of protein species in living cells without overexpression,” said Dr. Zhong. “This will give the scientific community the previously unattainable ability to examine the function of these proteins and their contribution to cellular function under physiological conditions. Indeed, many research laboratories throughout the world have contacted us to request the PSD-95-ENABLED mouse for their own studies.”

“This is a capability that was literally unimaginable just a few years ago,” commented Dr. Heinricher.

Further improvements, future experiments

Drs. Mao and Zhong are already looking to future projects.  “Like all first-generation techniques, the ENABLED strategy can be further improved,” said Dr. Mao. “The current ENABLED strategy is only compatible by tagging a protein from the end of a protein. Although a large portion of proteins can be labeled this way, some proteins can only be tagged from the beginning of the protein or in the middle of the protein. We will continue to expand the capability of the ENABLED strategy to label these other proteins.”

“Furthermore,” added Dr. Mao, “the PSD-95-ENALBED mouse that we generated allows one to visualize the type of synapses in the brain that are implicated in many neurological disorders but are otherwise difficult to identify. This ability will open up a new field in neuroscience research regarding the function of these synapses. Our team, including Dr. Dale Fortin who is the first author of the current paper, Joshua Melander and Maozhen Qin are taking advantage of this unique ability to examine several important functional aspects of this type of synapse.” 

“I'm excited to see not just what they do with it, but where it leads the field,” concluded Dr. Heinricher.

•    Read the paper
•    Read about the Mao Lab
•    Read about the Zhong Lab

Pictured above from left to right: Maozhen Qin, Haining Zhong, Tianyi Mao, Joshua Melander. Front row: Dale Fortin.


Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

Dale A. Fortin, Shane E. Tillo, Guang Yang,  Jong-Cheol Rah, Joshua B. Melander, Suxia Bai, Omar Soler-Cedeño, Maozhen Qin, Boris V. Zemelman, Caiying Guo, Tianyi Mao and Haining Zhong 

J Neurosci. 2014 Dec 10;34(50):16698-712. doi: 10.1523/JNEUROSCI.3888-14.2014