Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons

Abstract

Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function. This article has an associated First Person interview with the first author of the paper.

Endogenously expressed Ranbp2 is not at the axon initial segment

“…We immunostained cultured hippocampal neurons at 10 days in vitro (DIV). Consistent with previous reports (Hamdan et al, 2020;Khalaf et al, 2019), the anti-Ranbp2 antibody (A301-796A) labeled nuclei, but also colocalized with AnkG at the AIS (Fig. 1A).…”

“…Given the potential link of Ranbp2 to the microtubule-based cytoskeleton, and the number of microtubule-associated proteins that regulate AIS function (Leterrier, 2018), we sought to determine the function of Ranbp2 at the AIS. However, we found that the anti-Ranbp2 antibody used in both previous studies, and that labels AIS (Hamdan et al, 2020;Khalaf et al, 2019), is not specific to Ranbp2. Instead, it cross-reacts with neurofascin (Nfasc).…”

“…These molecular weights are similar to neurofascin splice variants, including a 140 kDa variant (NF140) expressed during early development (Zhang et al, 2015), a 155 kDa variant (NF155) found at paranodal junctions of myelinated axons (Tait et al, 2000), and a 186 kDa variant (NF186) found at the AIS and nodes of Ranvier (Davis et al, 1996). Similar to Khalaf et al (2019), we found that immunostaining of myelinated axons using antibody A301-796A labeled nodes of Ranvier and paranodal junctions (Fig. 1D), the same locations at which NF186 and NF155 are clustered in high density.…”

“…An exogenously expressed N-terminal fragment of Ranbp2 localizes at the AIS The results presented thus far suggest that immunostaining of the AIS using antibody A301-796A reflects cross-reactivity between Ranbp2 and NF186. However, Khalaf et al (2019) showed that an exogenously overexpressed N-terminal fragment of Ranbp2 (amino acids 1-900) can also be clustered at the AIS. Because of this surprising result, we generated constructs expressing a Flag-tagged N-terminal fragment of Ranbp2 that is followed by GFP and selfcleaving P2A peptide sequence to visualize neuronal morphology.…”

“…Immunostaining for Ranbp2 showed AIS localization. In a separate study, Khalaf et al (2019) also reported Ranbp2 at the AIS and suggested that its AIS localization requires AnkG. Nuclear Ranbp2 is thought to be linked to the microtubule cytoskeleton, in which it may contribute to cell cycle control, nuclear transport and neuronal cytoarchitecture (Goldberg, 2017).…”