Abstract
EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.
Highlights
Dynamic instability is an essential property of microtubules that allows them to play diverse roles in intracellular trafficking, organelle positioning, cell migration and cell division [1]
This phosphorylation is catalysed by the NIMA-related kinase-6 (NEK6) and NEK7 kinases and perturbs electrostatic binding of the EML4 protein with the tubulin C-terminal tails that extend from the surface of the microtubule lattice (Fig. 8A)
Our data suggest that this interaction most likely occurs through electrostatic interaction of the basic N-terminal domain of EML4 with the acidic tubulin C-terminal tails that are exposed on the surface of the microtubule
Summary
Dynamic instability is an essential property of microtubules that allows them to play diverse roles in intracellular trafficking, organelle positioning, cell migration and cell division [1]. EMLs are highly conserved and have been described in a variety of organisms, including flies, worms and humans [8,9,10]. Their association with microtubules has been shown in different systems, their function in regulating microtubule dynamics remains unclear with studies to date suggesting that different family members might contribute to stabilization and/or destabilization of microtubules [11,12,13]. A delay in mitotic chromosome congression observed upon depletion of EML3 or EML4 in human cells confirms their importance for cell division and supports roles for members of the EML family in spindle organization [14, 15]
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