Detecting intracellular signalling in behaving axonal growth cones.

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Neuronal circuits develop with great precision and specificity, starting with the highly accurate guidance of neuronal axons. Over the past two decades much work has been directed towards the identification of axonal guidance cues and their receptors, but how their signalling coordinates the dynamics of growth cone behaviour remains poorly understood. Part of the reason for this is the low spatial and temporal resolution of our understanding of growth cone intracellular signalling pathways. To resolve this, we propose to use a specific class of spinal motor neurons (LMCs), obtained from embryonic chicken explants and a recently-developed mouse embryonic stem cell line that can be easily induced to form spinal motor neurons. Our in vitro analysis indicates that LMCs respond to ephrin-B signals that activate the EphB tyrosine kinase pathway. Recent protein engineering efforts also generated a number of fluorescent indicators of intracellular signalling, such as receptor phosphorylation, signal relay protein conformation changes, or small GTPase activation and cytoskeleton remodelling. However, very few of such indicators have been used in the context of live growth cones, and essentially none in molecularly-defined neurons. We propose to use fluorescent indicators of cellular signalling events to study the live dynamics of axon guidance signalling in spinal motor neurons, in order to link axon guidance receptor signal relay with growth cone dynamics at high spatial and temporal resolution, through the following Aims: Aim 1 Assess EphB signalling dynamics in LMC neurons following treatments with ephrin-B family of proteins. These experiments will first characterise the general response properties of a recently developed EphB sensor, and will be later combined with whole growth cone imaging in order to relate biochemical level events with growth cone morphology changes. Aim 2 Assess cytoskeletal response dynamics in LMCs following ephrin-B treatment. Here, we will take advantage of small GTPase activation biosensors, which will be first characterised in the context of ephrin-B stimulation, and subsequently imaged in the context of whole growth cone dynamics. Aim 3 Link EphB and cytoskeletal signalling events in live growth cones. This will be done by simultaneously imaging EphB signalling, small GTPase activation and cytoskeletal rearrangement.