TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment

Abstract

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.

Feedback-Driven Assembly of the Axon Initial Segment

“…To correlatively image COS-7 cells with fluorescence and electron microscopy we used a recently developed approach (Harterink et al., 2019). COS-7 cells were grown on needle engraved Aclar pieces (Electron Microscopy Sciences; 50426–10 (Jiménez et al., 2010))…”

“…TRIM46 is an early axonal marker because it localizes to the future axon before neuronal polarization and AIS assembly. Interestingly, expression of TRIM46 in heterologous cells induces the formation of bundles of closely spaced MTs linked by electron-dense cross-bridges, which closely resemble the axon-specific MT fascicles (Harterink et al., 2019, van Beuningen and Hoogenraad, 2016, van Beuningen et al., 2015). Defining the interplay between AnkG and TRIM46-induced MT fascicles may thus provide a molecular pathway for AIS assembly.…”

The model of local axon homeostasis - explaining the role and regulation of microtubule bundles in axon maintenance and pathology

“…However, due to its limited expression in the cerebellum, MTCL1 cannot explain similar sheet-like arrangements in the AISs of other neuron types. A more likely candidate is TRIM-46 (tripartite motif-containing protein 46) which is expressed in AISs in many nervous tissues, contains only one central MT-binding domain, can induce sheet-like arrangements when expressed in non-neuronal cells, localises to the cross-bridges, and its knock-down in cultured neurons causes reduced cross-linkage [335, 336].…”

Cytoskeletal organization of axons in vertebrates and invertebrates

“…The electron-dense lateral bridges contain and require TRIM46 ( Fig. 3 B ), which is widely expressed in the nervous system ( Harterink et al, 2019 ; van Beuningen et al, 2015 ). The MT–MT cross-linker MTCL1 (microtubule cross-linking factor 1) was shown to promote MT fascicle alignment; however, its role is restricted to the AIS of Purkinje cells ( Satake et al, 2017 ), leaving open how MTCL1 functionally relates to TRIM46, which is likewise present in this cell type ( van Beuningen et al, 2015 ).…”