Imaging Ras Signaling in Neurons

Ryohei Yasuda, Ph.D.

Duke University, School of Medicine

Funded in December, 2005: $200000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

ABSTRACT

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Imaging Ras Signaling in Neurons

The modification of synaptic connections between neurons is thought to underlie the ability to form memories and learn new behaviors. In the central nervous system, most of excitatory synapses terminate on dendritic spines, tiny (~0.5 µm in diameter) protrusions emanating from the dendritic surface. Calcium influx into spines activates various signaling pathways that underlie synaptic plasticity. The small GTPase protein Ras couples calcium influx to many forms of synaptic plasticity, such as rapid synaptic potentiation and new synapse formation. Ras activation can also trigger protein synthesis and gene transcription important for the long-term maintenance of synaptic plasticity and for many other neuronal responses, including cell survival, death, and differentiation. Consistent with many essential roles of Ras signaling in neuronal plasticity, mutations in the Ras signaling pathway are associated with diseases causing cognitive impairments and learning deficits such as autism, X-linked mental retardation and neurofibromatosis 1.

The extensive branching of the Ras pathway raises a question of how Ras can control such diverse downstream effects, including local synapse-specific plasticity and global neuron-wide responses. Our hypothesis is that particular spatial and temporal patterns of calcium elevation result in specific patterns of Ras activation, which subsequently turn on distinct sets of downstream proteins, leading to specific effects. However, most of our knowledge of Ras signaling comes from classical biochemical experiments that lack spatial resolution. For a deeper understanding of molecular mechanisms of synaptic plasticity, and ultimately learning and memory, measurements of Ras activity at the level of individual spines are crucial.

To study Ras signaling in spines and dendrites, we have been developing a Ras activity sensor based on Fluorescence Resonance Energy Transfer (FRET). Since FRET occurs only when the two fluorescent molecules are within a few nanometers of each other, it can be used as a sensitive detector of interactions between proteins labeled with fluorophores. We use fluorescence lifetime, which is defined as the time elapsed between fluorophore excitation and photon emission, as a robust and quantitative measure of FRET.

Although these optical techniques have been implemented in other systems, there are obstacles to applying them to the study of signaling in individual spines. Since often only a few copies of each protein exist in each spine, single molecule sensitivity is required. In addition, strong light-scattering of the brain tissue makes high resolution imaging impossible with conventional microscopy. Because 2-photon laser scanning microscopy (2pLSM) allows robust signal detection necessary for single molecule sensitivity and sufficient spatial resolution, we combine 2pLSM with fluorescence lifetime measurements (2-photon fluorescence lifetime imaging microscopy, or 2pFLIM). This microscopy technique enables us to image Ras activity by measuring interactions between Ras fused with green fluorescence protein and Ras binding domain of Raf, which binds selectively to active Ras, fused with red fluorescence protein.

Using this Ras imaging technique, we will measure the spatial and temporal dynamics of Ras activity in response to physiological stimuli causing different calcium localization, and examine how the Ras pathway processes the dynamics of calcium elevation spatially and temporally. Specifically, we will ask to what extent Ras signaling is restricted in spines in response to spine specific calcium elevation, and what kind of stimulation causes more global Ras activation. Through these experiments, this project is expected to reveal how Ras couples particular patterns of calcium signaling to specific cellular responses.

A deep understanding of the Ras pathway will provide immediate insights into treatments for Ras related diseases. Furthermore, our method will open new possibilities to analyze how mutations associated with mental diseases affect normal Ras activity and neuronal plasticity at the single synapse level. This will significantly impact the development of therapeutics for mental diseases.

HYPOTHESIS

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Hypothesis:
A signaling protein Ras relays calcium elevations to signaling cascades leading to many forms of neuronal plasticity. We hypothesize that the spatiotemporal pattern of Ras activity is important in determining its diverse downstream effects.

Goals:
1. Establish methods to visualize Ras signaling in neurons with high spatiotemporal resolution.

2. Elucidate mechanisms of spatiotemporal regulation of Ras in response to synaptic stimulations.

Methods:
We will visualize Ras activity by measuring binding between Ras and the Ras binding domain of Raf (RBD), reporter molecules which bind selectively to active proteins, using fluorescence resonance energy transfer (FRET). To image FRET with high resolution in light-scattering brain tissue, we will combine fluorescence lifetime measurement and 2-photon laser scanning microscopy (2-photon fluorescence lifetime microscopy).

FINDINGS

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Lay Results:
Changes in synaptic connection are believed to underlie memory and learning. A signaling protein Ras is important for the regulation of synaptic connection. In this project, we have developed a new technique to image the activity of Ras protein in single synapses in the moment synapse connection. Using this technique, we discovered that activated Ras travels in neurons for about ten microns to activate surrounding synapses.

Scientific Results:
We have developed a technique to image Ras signaling in single dendritic spines using FRET based Ras sensor and 2-photon fluorescence lifetime imaging microscopy. Using this technique, we imaged Ras following the induction of long-term potentiation in individual spines. We found that Ras activation increased in the stimulated spine, and spread along the parent dendrites for more than 10 microns. Due to this spreading, surrounding spines were activated similarly to the stimulated spine. This spreading of Ras activity suggests that short segment of dendrite may act as a functional unit. 

SELECTED PUBLICATIONS

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Yasuda R., Harvey C.D., Zhong H., Sobczyk A., van Aelst L., and Svoboda K. Super-sensitive Ras activation in dendrites and spines revealed by 2-photon fluorescence lifetime imaging.  Nat Neurosci. 2006 Feb;9(2):283-91. Erratum in: Nat Neurosci. 2006 Mar;9(3):453.

Yasuda R., Sabatini B.L., and Svoboda K. Plasticity of calcium channels in dendritic spines. Nat Neurosci. 2003 Sep;6(9):948-55.