A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

Abstract

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-offunction experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter

“…3A). As previously reported, Stx and Munc18 are also present in growth cones (22,23), where they extensively colocalize as expected from their tight association ( Fig. 3 B and C).…”

“…This observation is significant given previous reports that correct plasma membrane trafficking of Stx strongly depends on the presence of Munc18 (8)(9)(10)(11)(12)(13)22). Noteworthy, Stx is able to bind FEZ1 in the absence of Munc18.…”

Calcium signaling in axon guidance

“…These results suggest that it is the resulting Ca 2+ concentration that is critical rather than the method of calcium entry. [50]. Notably, these responses are not merely the same response with the sign reversed; attraction to the left of a growth cone invokes a distinctly different response to repulsion from the right.…”

“…In addition to VAMP2-mediated exocytosis, syntaxin-1 (Sytx1) and tetanus neurotoxin-insensitive (TI)-VAMP mediate netrin-1 signals; this complex associates with the netrin-1 receptor in growth cones, deleted in colorectal cancer (DCC) [50]. Noticeably Sytx1 coassociates TI-VAMP, rather than its more common partner VAMP2, in response to netrin-1 signals.…”

“…This suggests a unique mechanism of exocytosis in netrin-1-induced attraction. Sytx1 and TI-VAMP are required for in vitro attraction in response to netrin-1 whereas VAMP2 inhibition (via TeNT) does not affect attraction [50]. Further, TI-VAMP and Sytx1 are both necessary for in vivo commissural axon pathfinding (an event known to be mediated by netrin-1) [50,52].…”