Cytoskeleton dynamics are critical phenomena that underpin many fundamental cellular processes. Collapsin response mediator proteins (CRMPs) are highly expressed in the developing nervous system, mediating growth cone guidance, neuronal polarity, and axonal elongation. However, whether and how CRMPs associate with microtubules and actin coordinated cytoskeletal dynamics remain unknown. In this study, we demonstrated that CRMP2 and CRMP4 interacted with tubulin and actin in vitro and colocalized with the cytoskeleton in the transition-zone in developing growth cones. CRMP2 and CRMP4 also interacted with one another coordinately to promote growth cone development and axonal elongation. Genetic silencing of CRMP2 enhanced, whereas overexpression of CRMP2 suppressed, the inhibitory effects of CRMP4 knockdown on axonal development. In addition, knockdown of CRMP2 or overexpression of truncated CRMP2 reversed the promoting effect of CRMP4. With the overexpression of truncated CRMP2 or CRMP4 lacking the cytoskeleton interaction domain, the promoting effect of CRMP was suppressed. These data suggest a model in which CRMP2 and CRMP4 form complexes to bridge microtubules and actin and thus work cooperatively to regulate growth cone development and axonal elongation.

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Changing the microtubule dynamics is sufficient to alter the axon and dendrite specification and development. Spastin participates in the growth and regeneration of neurites by severing microtubules into small segments, and collapsin response mediator protein 5 (CRMP5) provides structural support and serves as a track for cargo transport by promoting microtubule polymerization. Nevertheless, how spastin and CRMP5 cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. In our present study, spastin interacted with CRMP5 in vitro and in vivo. The binding domains for the spastin and CRMP5 interaction were the N‐terminal fragment of spastin (residues 270–328) and the C‐terminal fragment of CRMP5 (residues 472–564). Spastin and its truncation mutants, including the microtubule‐binding domain (MTBD) and ATPases associated with diverse cellular activities (AAA) domain, were necessary for the severing of microtubules. Furthermore, we demonstrated that microtubule polymerization of CRMP5 interfered with the microtubule‐severing function of spastin. Knocking down either spastin or CRMP5 inhibited neurite outgrowth in hippocampal neurons. However, co‐transfected spastin and CRMP5 promoted the outgrowth of neurites including dendrites and axons. Taken together, our data support a model in which the spastin interaction with CRMP5 promotes neurite outgrowth by controlling the microtubule dynamics.

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