FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

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.

Tuning cell-surface affinity to direct cell specific responses to patterned proteins.

Interactions with local extracellular cues direct cell migration. A versatile method to study cell response to a protein consists of patterning the protein cue on a substrate and quantifying the distribution of cells between patterned and non-patterned areas. Here, we define the concepts of (i) cell-surface affinity to describe cell choices, and of (ii) reference surface (RS) to clarify that the choice is made relative to a reference.

Netrins: versatile extracellular cues with diverse functions.

Netrins are secreted proteins that were first identified as guidance cues, directing cell and axon migration during neural development. Subsequent findings have demonstrated that netrins can influence the formation of multiple tissues, including the vasculature, lung, pancreas, muscle and mammary gland, by mediating cell migration, cell-cell interactions and cell-extracellular matrix adhesion. Recent evidence also implicates the ongoing expression of netrins and netrin receptors in the maintenance of cell-cell organisation in mature tissues.

Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP.

The study of cellular responses to changes in the spatial distribution of molecules in development, immunology and cancer, requires reliable methods to reproduce in vitro the precise distributions of proteins found in vivo. Here we present a straightforward method for generating substrate-bound protein patterns which has the simplicity required to be implemented in typical life science laboratories. The method exploits photobleaching of fluorescently tagged molecules to generate patterns and concentration gradients of protein with sub-micron spatial resolution.

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear.