Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

FRET-FLIM optimization shows activity of two signaling molecules in single dendritic spine

01.11.2016

Researchers at Max Planck Florida Institute for Neuroscience and Stanford University teamed up to develop a new molecular tag which allowed visualization of two signaling proteins' activity in a single dendritic spine in real time.

An ongoing challenge for scientists working to understand the brain is being able to see all its parts. Researchers have spent centuries developing better imaging techniques to see beyond the abilities of our naked eyes. They've built microscopes that gather information down to the electron level. They've engineered fluorescent tags that make cells and structures of interest more visible.


These are representative fluorescence lifetime images of RhoA and CaMKII activation in a dendritic segment of a hippocampal neuron. Structural plasticity was induced in a single spine using 2p glutamate uncaging.

Credit: Max Planck Florida Institute for Neuroscience

One of the most effective imaging techniques for neuroscientists has been the combination of FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy). This duo gives scientists the power to view biochemical dynamics of proteins with high spatial and temporal accuracy, while also allowing them to calculate the minuscule distances between molecules in real time.

At the Max Planck Florida Institute for Neuroscience (MPFI), researchers in Ryohei Yasuda's laboratory, such as postdoctoral researcher, Tal Laviv, Ph.D., have been working to understand the cellular and molecular mechanisms of learning and memory.

The team has been using FRET-FLIM to study the activity of proteins in dendritic spines, protrusions that form off of neuronal branches, and make synaptic connections and communicate with other neurons. Dendritic spines are known to emerge, change shape, and even disappear over a lifetime, and these changes are considered the cellular basis for learning and memory. These imaging techniques were a key factor in helping the team elucidate some of the molecular mechanisms behind this type of plasticity.

Nonetheless, there is an important limitation in using these techniques to understand how multiple types of proteins and molecules interact in living samples. There is only one FRET donor tag (GFP) that will work within a FLIM protocol, so if researchers want to study two proteins, they'll investigate "Protein A" in one set of experiments, following an additional set of experiments to examine "Protein B" activity.

Following these experiments, the researchers will need to draw conclusions about how these two proteins interact based on these two sets of experiments. This not only increases the amount of time it takes to study multiple proteins, but also makes it more difficult to analyze how they interact in space and time. "For those types of systems," explained Dr. Laviv, "it's crucial to look at as many proteins as possible at the same time to correlate their activities."

Researchers at Stanford University, from the research team of Dr. Michael Lin, which specializes in building protein based tools for molecular imaging, reached out to the Yasuda Lab at MPFI after they identified a new set of red fluorescent proteins, or RFPs, named CyRFPs. They suggested this new set of RFPs could be used in combination with GFP for simultaneous imaging.

To determine if this could work, the scientists tested the ability to visualize dendritic spine structure and function using CyRFP and a GFP-based calcium bio-sensor. Using this combination, they were able to monitor the structure and function of spines in real time, even in the brains of living animals. Finally, the team tweaked a variant of CyRFP, which now could be used as a fluorescent FRET donor a part of a FRET pair, named monomeric cyan-excitable red fluorescent protein (mCyRFP1). Scientists in the Yasuda Lab conducted a series of experiments to test the newly proposed FRET pair alongside a GFP bionsensor. The technique allowed them to view, for the first time, the activities of two signaling molecules within a single dendritic spine as the spine was undergoing synaptic plasticity. A description of this new technique was published on October 31, 2016 in Nature Methods.

Dr. Laviv explains that the new technique will increase both accuracy and efficiency of FRET-FLIM imaging experiments and could potentially increase our understanding of how learning and memory ultimately alters the structure and function of dendritic spines.

###

This work was supported by grants from Human Frontiers Science Program, NSF Graduate Fellowship, a Siebel Scholar Award, National Institute of Health grants R01MH080047, 1DP1NS096787, 1U01NS090600 and P50GM107615, a Burroughs Wellcome Foundation Career Award for Medical Scientists, a Rita Allen Foundation Scholar Award and the Max Planck Florida Institute for Neuroscience.

About Max Planck Florida Institute for Neuroscience

The Max Planck Florida Institute for Neuroscience (Jupiter, Florida, USA) specializes in the development and application of novel technologies for probing the structure, function, and development of neural circuits. It is the first research institute of the Max Planck Society in the United States.

Jennifer Gutierrez | EurekAlert!

More articles from Life Sciences:

nachricht Could this protein protect people against coronary artery disease?
17.11.2017 | University of North Carolina Health Care

nachricht Microbial resident enables beetles to feed on a leafy diet
17.11.2017 | Max-Planck-Institut für chemische Ökologie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: A “cosmic snake” reveals the structure of remote galaxies

The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.

Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...

Im Focus: Visual intelligence is not the same as IQ

Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.

That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...

Im Focus: Novel Nano-CT device creates high-resolution 3D-X-rays of tiny velvet worm legs

Computer Tomography (CT) is a standard procedure in hospitals, but so far, the technology has not been suitable for imaging extremely small objects. In PNAS, a team from the Technical University of Munich (TUM) describes a Nano-CT device that creates three-dimensional x-ray images at resolutions up to 100 nanometers. The first test application: Together with colleagues from the University of Kassel and Helmholtz-Zentrum Geesthacht the researchers analyzed the locomotory system of a velvet worm.

During a CT analysis, the object under investigation is x-rayed and a detector measures the respective amount of radiation absorbed from various angles....

Im Focus: Researchers Develop Data Bus for Quantum Computer

The quantum world is fragile; error correction codes are needed to protect the information stored in a quantum object from the deteriorating effects of noise. Quantum physicists in Innsbruck have developed a protocol to pass quantum information between differently encoded building blocks of a future quantum computer, such as processors and memories. Scientists may use this protocol in the future to build a data bus for quantum computers. The researchers have published their work in the journal Nature Communications.

Future quantum computers will be able to solve problems where conventional computers fail today. We are still far away from any large-scale implementation,...

Im Focus: Wrinkles give heat a jolt in pillared graphene

Rice University researchers test 3-D carbon nanostructures' thermal transport abilities

Pillared graphene would transfer heat better if the theoretical material had a few asymmetric junctions that caused wrinkles, according to Rice University...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Ecology Across Borders: International conference brings together 1,500 ecologists

15.11.2017 | Event News

Road into laboratory: Users discuss biaxial fatigue-testing for car and truck wheel

15.11.2017 | Event News

#Berlin5GWeek: The right network for Industry 4.0

30.10.2017 | Event News

 
Latest News

NASA detects solar flare pulses at Sun and Earth

17.11.2017 | Physics and Astronomy

NIST scientists discover how to switch liver cancer cell growth from 2-D to 3-D structures

17.11.2017 | Health and Medicine

The importance of biodiversity in forests could increase due to climate change

17.11.2017 | Studies and Analyses

VideoLinks
B2B-VideoLinks
More VideoLinks >>>