During growth cone migration, guidance receptors signal via Rac GTPases to reassemble the actin cytoskeleton. Such process enables the growth cone to modulate filopodial and lamellipodial protrusions in order to migrate to its final place. The two redundant Rac GTPases MIG-2 and CED-10 are essential for this process, since the abolishment of Rac activity disrupts virtually every PDE axon. Racs shuffle between an active, GTP-bound state, to an inactive, GDP-bound state. Rac activation via binding to GTP can be aided by Guanine-nucleotide Exchange Factors (GEFs). UNC-73 is a GEF for MIG-2 and CED-10 in axon guidance, but mutations in
unc-73 did not cause defects as severe as the double
mig-2;
ced-10. We then suspected other GEFs may also control Rac activity in parallel to UNC-73. In a screen for other Double Homology (DH) containing molecules, TIAM-1 displayed a genetic interaction with the Racs. Like
unc-73, loss-of-function (lof) mutations in
tiam-1 synergized with lof
mig-2 and
ced-10. Additionally, a gain-of-function (gof) version of
tiam-1 caused ectopic lamellipodia in PDEs that were suppressed by lof
mig-2 and
ced-10. These results suggest TIAM-1 acts upstream of Racs in axon pathfinding. We then investigated the mechanism of action of TIAM-1. We purified the GEF catalytic DH/PH domains of TIAM-1 and performed a mant-GTP based exchange assay. The biochemical results suggest TIAM-1[DH/PH] acts as a GEF specifically for Rac, but not Cdc-42 nor RhoA. A point mutation that is predicted to disrupt TIAM-1 GEF activity abolished the ability of TIAM-1[DH/PH] to exchange mant-GTP in Rac. Next we asked whether TIAM-1 localized to the plasma membrane, where Racs are active. In vivo TIAM-1 expression in neurons, as well as in NIH 3T3 fibroblasts, suggest TIAM-1 localized at the plasma membrane with CED-10. In vitro studies implicated hTiam1 as a downstream effector of hCdc-42. In vivo, lof
tiam-1 and
cdc-42 had no increase of defects in PDE guidance. Plus, gof
cdc-42 displayed ectopic lamellipodia that were suppressed by the lof
tiam-1. These data implicate TIAM-1 as a downstream effector of CDC-42 in vivo. Ventrally-guided neurons are attracted to UNC-6 and repelled by SLT-1. We asked if TIAM-1 acts in either pathway. Lof
tiam-1 did not increase defects seen in
unc-6 animals, but increased in
slt-1. Gof
unc-40 caused ectopic lamellipodia that also required TIAM-1 (but not UNC-73) for its occurrence. Our work revealed the role of the previously unidentified C. elegans TIAM-1 as a GEF for MIG-2 and CED-10, downstream of CDC-42, in the UNC-6/Netrin attractive signaling pathway. Future studies will further dissect the mechanisms of action of TIAM-1 in growth cone morphology and dynamics.