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Summary Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development. [ABSTRACT FROM AUTHOR]

Summary: Microtubules are cytoskeletal polymers with two structurally and functionally distinct ends, the plus- and the minus-end. Here, we focus on the mechanisms underlying the regulation of microtubule minus-ends by the CAMSAP/Nezha/Patronin protein family. We show that CAMSAP2 is required for the proper organization and stabilization of interphase microtubules and directional cell migration. By combining live-cell imaging and in vitro reconstitution of microtubule assembly from purified components with laser microsurgery, we demonstrate that CAMSAPs regulate microtubule minus-end growth and are specifically deposited on the lattice formed by microtubule minus-end polymerization. This process leads to the formation of CAMSAP-decorated microtubule stretches, which are stabilized from both ends and serve as sites of noncentrosomal microtubule outgrowth. The length of the stretches is regulated by the microtubule-severing protein katanin, which interacts with CAMSAPs. Our data thus indicate that microtubule minus-end assembly drives the stabilization of noncentrosomal microtubules and that katanin regulates this process. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46-positive microtubules to the plasma membrane. Using in vitro reconstitution and cellular assays, we demonstrate that TRIM46 forms parallel microtubule bundles and stabilizes them by acting as a rescue factor. TRIM46-labeled microtubules drive retrograde transport of Neurofascin-186 to the proximal axon, where Ankyrin-G prevents its endocytosis, resulting in stable accumulation of Neurofascin-186 at the AIS. Neurofascin-186 enrichment in turn reinforces membrane anchoring of Ankyrin-G and subsequent recruitment of TRIM46-decorated microtubules. Our study reveals feedback-based mechanisms driving AIS assembly. • Ankyrin-G in complex with EBs recruits microtubule bundles to the plasma membrane • TRIM46 is a rescue factor that forms stable parallel microtubule bundles • TRIM46-bound microtubules direct Neurofascin-186 trafficking to the proximal axon • Ankyrin-G controls Neurofascin-186 retention in the axon initial segment Fréal et al. report the molecular mechanisms involved in axon initial segment (AIS) assembly. This study describes in detail how feedback-driven coupling between AIS membrane proteins and axonal microtubules allows for the formation and maintenance of a functional AIS. [ABSTRACT FROM AUTHOR]