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.
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!
Rochester scientists discover gene controlling genetic recombination rates
23.04.2018 | University of Rochester
One step closer to reality
20.04.2018 | Max-Planck-Institut für Entwicklungsbiologie
Physicists at the Laboratory for Attosecond Physics, which is jointly run by Ludwig-Maximilians-Universität and the Max Planck Institute of Quantum Optics, have developed a high-power laser system that generates ultrashort pulses of light covering a large share of the mid-infrared spectrum. The researchers envisage a wide range of applications for the technology – in the early diagnosis of cancer, for instance.
Molecules are the building blocks of life. Like all other organisms, we are made of them. They control our biorhythm, and they can also reflect our state of...
University of Connecticut researchers have created a biodegradable composite made of silk fibers that can be used to repair broken load-bearing bones without the complications sometimes presented by other materials.
Repairing major load-bearing bones such as those in the leg can be a long and uncomfortable process.
Study published in the journal ACS Applied Materials & Interfaces is the outcome of an international effort that included teams from Dresden and Berlin in Germany, and the US.
Scientists at the Helmholtz-Zentrum Dresden-Rossendorf (HZDR) together with colleagues from the Helmholtz-Zentrum Berlin (HZB) and the University of Virginia...
Novel highly efficient and brilliant gamma-ray source: Based on model calculations, physicists of the Max PIanck Institute for Nuclear Physics in Heidelberg propose a novel method for an efficient high-brilliance gamma-ray source. A giant collimated gamma-ray pulse is generated from the interaction of a dense ultra-relativistic electron beam with a thin solid conductor. Energetic gamma-rays are copiously produced as the electron beam splits into filaments while propagating across the conductor. The resulting gamma-ray energy and flux enable novel experiments in nuclear and fundamental physics.
The typical wavelength of light interacting with an object of the microcosm scales with the size of this object. For atoms, this ranges from visible light to...
Stable joint cartilage can be produced from adult stem cells originating from bone marrow. This is made possible by inducing specific molecular processes occurring during embryonic cartilage formation, as researchers from the University and University Hospital of Basel report in the scientific journal PNAS.
Certain mesenchymal stem/stromal cells from the bone marrow of adults are considered extremely promising for skeletal tissue regeneration. These adult stem...
13.04.2018 | Event News
12.04.2018 | Event News
09.04.2018 | Event News
23.04.2018 | Physics and Astronomy
23.04.2018 | Physics and Astronomy
23.04.2018 | Trade Fair News