Seeing Signaling: Tools for Dynamic Imaging of Kinase Activity in the Nervous System

Matthew B. Dalva, Ph.D.

University of Pennsylvania

Department of Neuroscience
Funded in June, 2009: $200000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY .

LAY SUMMARY

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Using Molecular Imaging to Identify Signaling that Influences Synaptic Development and Plasticity

Researchers will examine the dynamics of intracellular signaling that give rise to the formation of synapses, junctions connecting one brain cell to another to facilitate communication and plasticity.

One key component in the development of synapses between two brain cells is activation (phosphorylation) of amino acids called “tyrosine kinases.” Overall, several families of tyrosine kinases are involved in synaptic events. These events include formation of “dendritic spines” (tiny mushroom-like protrusions that receive neurotransmitters from across the synapse), synaptic maturation, and brain plasticity. Precise activation of tyrosine kinase receptors located on cells helps to guide development of synaptic connections, while their over-activation is implicated in developmental disorders and in diseases such as cancers. The investigators hypothesize that the spatial and temporal dynamics of tyrosine kinase signaling, and regulation of these, help control the outcomes of these synaptic functions.

They will test this hypothesis using molecular imaging with fluorescent phosphorylation reporters, which enable them to visualize and quantify increases and decreases in tyrosine kinase signaling within laboratory animal neurons in tissue cultures. First, they will image this signaling as cells’ dendritic spines explore their environment and generate synaptic connections. Then, the investigators will image events involved in neuronal plasticity, to see how signaling flows between or along pathways. These experiments are anticipated to define the signaling events that underlie cells’ dynamic establishment and loss of synaptic connections.

ABSTRACT

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Seeing Signaling: Tools for Dynamic Imaging of Kinase Activity in the Nervous System

During the past decade, stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons.

To begin, we have focused on visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. Activation of tyrosine kinases transmits extracellular cues to signal transduction cascades that result in a diverse set of adaptive events, while misregulation of tyrosine kinase signaling is prominent in many diseases. We hypothesize that the spatial and temporal dynamics of tyrosine kinase activity is fundamental to determining its diverse downstream effects. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation.

We now propose two specific aims to use tools we have developed and generate additional molecules to visualize signaling during neuronal development.  We propose to: (1) determine the local tyrosine kinase activity during filopodial motility dependent synapse formation; and (2) determine the kinetics of different kinases during signaling within neurons. Using our indicators, we will elucidate the dynamics of signals that underlie synapse maturation. Moreover, because abnormal tyrosine kinase activity underlies many diseases, our tools will be of broad use and enable novel insights into essential mechanisms that underlie human disease.

INVESTIGATOR BIOGRAPHIES

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Matthew B. Dalva, Ph.D.

Dr. Matthew B. Dalva received his undergraduate degree from Stanford University. He earned a Ph.D. in neurobiology from Duke University working with Dr. Lawrence Katz to develop scanning laser photostimulation. Dr. Dalva then moved to Harvard University/Children’s Hospital for a postdoctoral fellowship in the laboratory of Dr. Mike Greenberg. There, Dr. Dalva embarked on a successful hunt for proteins that direct maturation of synapses. After showing that a specific family of tyrosine kinases known as EphBs regulated NMDAR clustering, function and synapse development, he joined the faculty and started his own laboratory at the University of Pennsylvania in 2003.

Dr. Dalva is currently an assistant professor in the Department of Neuroscience and serves as Co-Director of the Center for Dynamic Imaging of the Nervous System. Dr. Dalva’s laboratory investigates how excitatory spine synapses are formed and lost, and what impact the normal morphology and numbers of these structures have on brain function. In 2008 his laboratory demonstrated that a particular receptor tyrosine kinase (EphB) is required for filopodial motility dependent excitatory synapse formation.

In addition to the 2009 award from the Dana Foundation’s Program in Brain and Immuno-imaging, Dr. Dalva has received awards from the McCabe Foundation (2003), the Mallinckrodt Foundation (2003), the Philadelphia Foundation (2005), and the Whitehall Foundation (2005).