The GTPase proteins Rho, Rac, and CDC42 have each been implicated in the formation of different cytoskeletal structures in migrating cells. Thus, these proteins act as a signaling intermediaries between extracellular signals such as growth factors or migration guidance cues and the cytoskeletal rearrangements and cell movements that these signals cause. They function not only in growth cones, but also in most migrating cells, including not only cells that migrate as part of their developmental program, but also metastatic cancer cells. The C. elegans VD motor neuron growth cone can be specifically labeled with green fluorescent protein and visualized with time- lapse confocal microscopy. The VD growth cone responds to specific substrates with reproducible rounded, lamellar, and extended spike morphologies.
Aim 1 A: I will construct transgenic worms expressing dominant negative and dominant active forms of the C. elegans Rho family GTPase genes in the VD neuron.
Aim 1 B: I will examine the VD growth cone behavior in these worms and in worms carrying mutations that affect the function of Rho GTPases. I expect to discover whether any or all of these GTPase genes affects specific aspects of the growth cone cytoskeleton.
Aim 2 A: I will carry out a screen using both chemical and transposon mediated mutagenesis to find mutations in genes affecting growth cone dynamics. I intend to concentrate my efforts on mutations that affect the growth cone in ways that resemble the defects caused by mutations in the small GTPase signaling pathways.
Aim 2 B: Mutations will be cloned by conventional positional cloning strategies or by using the technique of transposon display. For transposon mutagenesis, I intend to take advantage of transgenic control of Tc3 transposition, an emerging technology under development in Dr. Jorgensen's lab.
