Abstract
Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.
Highlights
Netrin-1 protein secreted by floor plate cells functions as a chemoattractant that directs commissural axon extension to the ventral midline of the embryonic vertebrate neural tube [1,2,3,4,5]
We investigated the intracellular signaling mechanism involved, demonstrating that netrin-1 rapidly recruits Deleted in colorectal cancer (DCC) to the leading edge of commissural neuron growth cones and that src family tyrosine kinase (SFK) activation is required for netrin-1 activation of Cdc42 in neuronal growth cones
To address the temporal and spatial activation of signal transduction mechanisms activated by netrin-1 downstream of DCC, we utilized a CFP/YFP FRET biosensor for the Rho GTPase Cdc42 [16,17]
Summary
Netrin-1 protein secreted by floor plate cells functions as a chemoattractant that directs commissural axon extension to the ventral midline of the embryonic vertebrate neural tube [1,2,3,4,5]. While netrin-1 is essential for normal neural development, and many downstream components of netrin-1 signaling have been identified, how the activation of these proteins directs growth cone motility remains incompletely understood. A member of the Rho family of small GTPases, exhibits a highly conserved capacity to co-ordinate the intracellular signaling mechanisms that underlie directional migration [6]. Biochemical studies of spinal commissural neurons revealed that netrin-1 signals through the transmembrane receptor Deleted in colorectal cancer (DCC) to activate Cdc in embryonic. FLIM FRET Imaging of Cdc Activated by Netrin
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