Aims:
Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal
development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve
injury.
Background:
Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing
microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated.
Objective:
To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin.
Method:
Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the
transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons.
An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites.
Result:
We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation.
Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for
spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus,
and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the
neurite outgrowth but not branch formation by remodeling microtubules.
Conclusion:
Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.
Cytosolic GαsActs as an Intracellular Messenger to Increase Microtubule Dynamics and Promote Neurite Outgrowth
