Investigating the role of EML2 and EML4 alternative splicing in microtubule remodelling and neuronal cell differentiation

Neurons are highly complex differentiated cells that lie at the heart of the nervous system. Generation of elongated axons and dendrites during neuronal cell differentiation is dependent on organization and dynamics of the microtubule network. Yet our understanding of how microtubules are remodelled during this process is far from complete. EMLs are a family of microtubule-associated proteins highly expressed in neuronal cells. Humans express six EML proteins, with EML1 to EML4 being structurally similar, containing an N-terminal region that binds to polymerized microtubules and a C-terminal region that binds to soluble tubulin heterodimers. Contrasting evidence suggests that some EML proteins stabilize microtubules, whereas others destabilize them. Moreover, mutations in EML1 are responsible for neuronal heterotopia, a defect in neuronal cell migration.

Previous studies in our lab have shown that EML4 recruits the NEK9 and NEK7 kinases to microtubules, while NEK6 and NEK7 phosphorylate the N-terminus of EML4 resulting in its detachment from microtubules. Database analysis revealed that EMLs are subject to alternative splicing in their N-terminal regions, leading to the production of one long (L) variant that contains a complete N-terminus as well as the C-terminus, and possibly several short (S) variants that differ in the amount of N-terminus. In this project, we explored the expression and localization of the long and one of the short splice variants of EML4 and EML2, as well as NEK6 and NEK7, during differentiation of SH-SY5Y neuronal cells. We also tested the consequences of overexpression and depletion of these EML proteins on neurite outgrowth and branching before and after differentiation.

We found that differentiation led to decreased expression of the EML4-L variant, whereas the EML4-S variant remained relatively constant. In contrast, the EML2-L variant increased in expression, while EML2-S decreased in expression. Overexpression of EML2-L and EML2-S led to increased and decreased neurite lengths, respectively. Interestingly, neurite branching increased in EML2-S transfected cells. Using localization approaches, we found that EML2-L but not EML2-S localized strongly to interphase microtubules. However, the EML4-S variant stabilized microtubules less effectively than EML4-L. Depletion of both EML4 and EML2 led to neurite outgrowth. We propose that the presence of the N-terminal microtubule-binding region present in the long variants of EML2 and EML4 promotes stabilization of microtubules and neurite extension. However, the short variants might promote neurite branching. Together, these data suggest that regulated expression and localization of different splice variants of EML proteins are crucial to the remodelling of microtubules that occurs during neuronal differentiation.