Mouse monoclonal antibodies against EEA1 and CD63 were from BD Biosiences (Franklin Lakes, NJ) and from Ancell (Bayport, MN), respectively. Jahn (Max Planck Institute, Goettingen, Germany). Mouse mAb against Syb2 (clone 69.1) was a generous gift from R. The polyclonal anti-green fluorescent protein (GFP) antibody we used was described previously ( Martinez-Arca et al., 2001). Mouse monoclonal antibody (mAb) to TI-VAMP (Cl 158.2) was described previously ( Muzerelle et al., 2003). In this article, we set out to test the possible coordination of TI-VAMP-mediated trafficking with actin in neuronal growth cones as a way to integrate membrane traffic and cytoskeleton dynamics in neurite elongation and polarization. However, the precise mechanism by which TI-VAMP-mediated fusion leads to growth and how it is regulated are not known. We have shown that a TeNT-insensitive vesicle-associated membrane protein (TI-VAMP also called VAMP-7), which is also a member of a subfamily of vesicular SNAREs ( Filippini et al., 2001 Rossi et al., 2004), plays a major role in neurite outgrowth (Martinez-Arca et al., 2000, 2001 Alberts et al., 2003). In contrast, a specific membrane fusion machinery may be involved in neuronal development because a mutant of the exocyst component Sec5 shows impaired neurite outgrowth but normal release of neurotransmitter in Drosophila melanogaster ( Murthy et al., 2003). Accordingly, the brains of Syb2 null mice develop normally up to birth ( Schoch et al., 2001).

The membrane fusion mechanism involved in neurite outgrowth differs from the вЂњclassicalвЂќ synaptic core machinery inasmuch as it is unaffected by treatment with tetanus neurotoxin (TeNT), which cleaves the classical synaptic vesicle SNARE synaptobrevin 2 (Syb2) and so blocks neurotransmitter release ( Osen-Sand et al., 1996 Grosse et al., 1999). Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) constitute the core machinery for membrane fusion and are involved in all of the fusion events along the biosynthetic and endocytic pathways (for review, see Rothman, 2002). Although the role of cytoskeletal dynamics in the navigation of growth cones is well established ( Song and Poo, 2001 da Silva and Dotti, 2002), no link has yet been found between the cytoskeleton and the membrane trafficking machinery that delivers membrane vesicles for growth. Leading the movement at the tips of the developing neurites, the growth cones extend by fusing membrane vesicles with their plasma membrane and by the constant growth and reorganization of the cytoskeleton below this plasma membrane that pushes the leading edge forward. Axons and dendrites elongate through specialized, highly motile growth cones. One of the most striking features of neurons is their extremely elongate form, with their axons and dendrites extending frequently over tens of centimeters.

Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Its accumulation correlates with and depends upon the presence of F-actin.

Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture.

Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis.

X v-amp patch editor