One phenomenon that intrigues neuronal cell biologists is that of axonal differentiation — that is, how do immature neurites develop into an axon and mature dendrites? Given the elaborate shapes of neurons and the lengths that some axons can reach, the molecular mechanisms by which axonal differentiation occurs must be highly regulated. In a previous report, Kaibuchi and colleagues showed that collapsin response-mediator protein-2 (CRMP-2) was required for axonal growth and axon–dendrite fate, and the group now reports that CRMP-2 regulates axonal growth and branching by binding to tubulin heterodimers and promoting microtubule assembly.

They began by searching for CRMP-2-binding proteins, and identified α- and β-tubulin as proteins from developing rat brain extracts that bound to immobilized CRMP-2. Both proteins also co-immunoprecipitated with CRMP-2. Under conditions that favoured tubulin-dimer formation, purified tubulin and purified CRMP-2 interacted directly, a result that was confirmed by sedimentation profiling using sucrose density gradient centrifugation. Further analysis showed that the CRMP-2–tubulin complex comprises one CRMP-2 molecule and one tubulin α/β-heterodimer.

To find out what influence this might have on microtubule dynamics, Kaibuchi and co-workers incubated purified tubulin with wild-type or deletion mutants of CRMP-2. Wild-type CRMP-2 and the carboxy-terminal half (residues 323–572) efficiently assembled microtubules, but the amino-terminal half (1–322) did not. Subsequent studies found that residues 323–381 effectively mediated microtubule assembly. The wild-type construct even increased the growth rate of plus ends of pre-formed microtubules. Surprisingly, though, CRMP-2 bound to pre-formed microtubules about ten times less efficiently than to tubulin heterodimers. The association of CRMP-2 with microtubules led the authors to investigate whether this is coupled to tubulin polymerization and, indeed, they found that CRMP-2 seems to co-polymerize with tubulin dimers into microtubules.

So what happens in a cellular context? In transfected fibroblasts, CRMP-2 associates with cellular microtubules. When it is overexpressed in a neuronal cell line, CRMP-2 induces the cells to differentiate and extend neurites. Deletion of residues 323–381 (CRMP-2-Δ323–381), however, inhibited the neurite-inducing activity. So although this 59-amino-acid fragment could induce microtubule assembly, it couldn't induce neurite formation, which indicates that the microtubule-assembly activity of CRMP-2 is required, but is not sufficient, for neurite outgrowth. Similarly, expression of residues 323–381 had no effect on the axonal growth of hippocampal neurons, whereas CRMP-2-Δ323–381 inhibited endogenous microtubule assembly. In addition to inducing axonal growth, CRMP-2 is also required for axonal branching.

The authors propose that CRMP-2 promotes microtubule assembly by binding and delivering tubulin heterodimers, with which it co-polymerizes, to the growing (plus) ends of microtubules. So, microtubule assembly might then affect microtubule-polymer length during axonal growth. But although CRMP-2 is a strong candidate for promoting microtubule assembly, this function alone cannot explain the role(s) of CRMP-2 in axonal differentiation and growth, as the 59-residue microtubule-assembly-promoting fragment couldn't mediate axonal growth on its own. It remains to be seen, then, what CRMP-2's other talents are.