A neuron has two types of highly polarized cell processes, the single axon and multiple dendrites. One of the fundamental questions of neurobiology is how neurons acquire such specific and polarized morphologies. During neuronal development, various actin-binding proteins regulate dynamics of actin cytoskeleton in the growth cones of developing axons. The regulation of actin cytoskeleton in the growth cones is thought to be involved in axon outgrowth and axon-dendrite specification. However, it is largely unknown which actin-binding proteins are involved in axon-dendrite specification and how they are transported into the developing axons. We have previously reported that collapsin response mediator protein 2 (CRMP-2) plays a critical role in axon outgrowth and axon-dendrite specification (N. Inagaki, K. Chihara, N. Arimura, C. Menager, Y. Kawano, N. Matsuo, T. Nishimura, M. Amano, and K. Kaibuchi, Nat. Neurosci. 4:781-782, 2001). Here, we found that CRMP-2 interacted with the specifically Rac1-associated protein 1 (Sra-1)/WASP family verprolin-homologous protein 1 (WAVE1) complex, which is a regulator of actin cytoskeleton. The knockdown of Sra-1 and WAVE1 by RNA interference canceled CRMP-2-induced axon outgrowth and multiple-axon formation in cultured hippocampal neurons. We also found that CRMP-2 interacted with the light chain of kinesin-1 and linked kinesin-1 to the Sra-1/WAVE1 complex. The knockdown of CRMP-2 and kinesin-1 delocalized Sra-1 and WAVE1 from the growth cones of axons. These results suggest that CRMP-2 transports the Sra-1/WAVE1 complex to axons in a kinesin-1-dependent manner and thereby regulates axon outgrowth and formation.

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.

The transport of tubulin and microtubules in a growing axon is essential for axonal growth and maintenance. However, the molecular mechanism underlying the linkage of tubulin and microtubules to motor proteins is not yet clear. Collapsin response mediator protein‐2 (CRMP‐2) is enriched at the distal part of growing axons in primary hippocampal neurons and plays a critical role in axon differentiation through its interaction with tubulin dimer and Numb. In this study, we show that CRMP‐2 regulates tubulin transport by linking tubulin and Kinesin‐1. The C‐terminal region of CRMP‐2 directly binds to the tetratricopeptide repeat domain of kinesin light chain 1 (KLC1). Soluble tubulin binds to the middle of CRMP‐2 and forms a trimeric complex with CRMP‐2/KLC1. Furthermore, the movement of GFP–tubulin in the photobleached area is weakened by knockdown of KLCs or CRMP‐2. These results indicate that the CRMP‐2/Kinesin‐1 complex regulates soluble tubulin transport to the distal part of the growing axon.

Recent experiments in various cell types such as mammalian neutrophils and Dictyostelium discoideum amoebae point to a key role for the lipid product of PI 3‐kinase, PIP₃, in determining internal polarity. In neurons, as a consequence of the elongation of one neurite, the axon is specified and the cell acquires its polarity. To test the hypothesis that PI 3‐kinase and PIP₃ may play a role in neuronal polarity, and especially in axon specification, we observed localization of PIP₃ visualized by Akt‐PH‐GFP in developing hippocampal neurons. We found that PIP₃ accumulates in the tip of the growing processes. This accumulation is inhibited by addition of PI 3‐kinase inhibitors. Those inhibitors, consistently with a role of PIP₃ in process formation and elongation, delay the transition from stage 1 neurons to stage 3 neurons, and both axon formation and elongation. Moreover, when the immature neurite contacts a bead coated with laminin, a substrate known to induce axon specification, PIP₃ accumulates in its growth cone followed by a rapid elongation of the neurite. In such conditions, the addition of PI 3‐kinase inhibitors inhibits both PIP₃ accumulation and future axon elongation. These results suggest that PIP₃ is involved in axon specification, possibly by stimulating neurite outgrowth. In addition, when a second neurite contacted the beads, this neurite rapidly elongates whereas the elongation of the first laminin‐contacting neurite stops, consistently with the hypothesis of a negative feedback mechanism from the growing future axon to the other neurites.

Disrupted-In-Schizophrenia 1(DISC1) is a candidate gene for susceptibility to schizophrenia. DISC1 is reported to interact with NudE-like (NUDEL), which forms a complex with lissencephaly-1 (LIS1) and 14-3-3ε. 14-3-3ε is involved in the proper localization of NUDEL and LIS1 in axons. Although the functional significance of this complex in neuronal development has been reported, the transport mechanism of the complex into axons and their functions in axon formation remain essentially unknown. Here we report that Kinesin-1, a motor protein of anterograde axonal transport, was identified as a novel DISC1-interacting molecule. DISC1 directly interacted with kinesin heavy chain of Kinesin-1. Kinesin-1 interacted with the NUDEL/LIS1/14-3-3ε complex through DISC1, and these molecules localized mainly at cell bodies and partially in the distal part of the axons. DISC1 partially colocalized with Kinesin family member 5A, NUDEL, LIS1, and 14-3-3ε in the growth cones. The knockdown of DISC1 by RNA interference or the dominant-negative form of DISC1 inhibited the accumulation of NUDEL, LIS1, and 14-3-3ε at the axons and axon elongation. The knockdown or the dominant-negative form of Kinesin-1 inhibited the accumulation of DISC1 at the axons and axon elongation. Furthermore, the knockdown of NUDEL or LIS1 inhibited axon elongation. Together, these results indicate that DISC1 regulates the localization of NUDEL/LIS1/14-3-3ε complex into the axons as a cargo receptor for axon elongation.