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Ran GTPase regulates non-centrosomal microtubule nucleation and is transported by actin waves towards the neurite tip

Yung-An Huang, Chih-Hsuan Hsu, Ho-Chieh Chiu, Chris T. Ho, Wei-Lun Lo, Eric Hwang

Preprint posted on June 28, 2019 https://www.biorxiv.org/content/10.1101/684720v1

On the Ran: the small GTPase Ran promotes non-centrosomal microtubule nucleation in neurons and is transported to the neurite tip by actin waves

Selected by Ramona Jühlen

Background

In neurons microtubules are nucleated from the centrosome, but as neurons mature the centrosome loses its ability to act as a microtubule-organizing center (MTOC). Several cellular components in neurons have been identified to serve as non-centrosomal MTOC (ncMTOC): the Golgi, the augmin complex and the protein TPX2.

The Ras-related nuclear protein (Ran) is a small GTPase predominately regulating nucleocytoplasmic transport, but also mitotic spindle formation. Ran regulates TPX2 activity and thereby, TPX2-mediated ncMT nucleation in mitotic neurons. The question of whether and how Ran plays a role in MT nucleation in post-mitotic neurons remains open. However, there is evidence that Ran regulates neuronal morphogenesis and is enriched at the tip of the neurite.

By using an optogenetic tool the authors shed light on the ability of Ran to nucleate ncMT and discover how Ran is localized to the neurite tip.

Key findings

First, the authors show that specifically activated Ran (RanGTP) is able to regulate MT formation at the neurite tip. For this purpose they used dissociated hippocampal neurons (2 days in vitro) expressing Ran mutants: the constitutively active Ran (RanQ69L; mimicking activated RanGTP) and the dominant negative Ran (RanT24N; reducing RanGTP levels).

In order to verify that RanGTP is able to nucleate MT at other sites in the neuron than the neurite tip, the authors used an optogenetic tool (called RanTRAP; a variation of the original LOVTRAP system (1)) to spatially control RanGTP levels at any location in the neuron. By releasing RanGTP at the photoactivated region, they could verify that RanGTP is indeed able to enhance nucleation of MTs along the neurite.

The authors showed earlier in the preprint that RanGTP is enriched at the neurite tip, but since Ran is potentially produced in the soma of the neuron, they were curious about how Ran is localized or transported to the neurite tip. There has been some evidence that actin-based structures are involved in that process: firstly, RanGTP co-localizes with actin filaments in the growth cone of the neuron and secondly, it has been observed that wave-like dynamics of actin filaments, transporting a variety of protein cargoes in neurons, proceed MT nucleation. By depolymerizing actin filaments the authors present that RanGTP is no longer enriched at the neurite tip and that the frequency of MT nucleation is reduced (leaving MT polymerization velocity unchanged). Finally, they show in live cell imaging that the RanGTP-mimic mutant co-migrates with actin waves, whereas the dominant negative Ran-mutant does not.

Overall, the authors show that Ran plays a crucial role in regulating ncMT nucleation along the neurite and they speculate that RanGTP promotes ncMT nucleation by releasing TPX2 from the inhibitory importin heterodimers. TPX2 can then interact with γ-tubulin and the augmin complex in order to initiate MT nucleation (Figure 1).

Figure 1. Scheme explaining the optogenetic tool RanTRAP and the hypothetical mechanism of RanGTP regulating non-centrosomal microtubule nucleation. The LOV2 domain is fused to a mitochondrial targeting sequence (TOM20) and the RanGTP-mimic RanQ69L is fused to the ZDK domain. In absence of light the activity of RanQ69L is sequestered to the mitochondria (LOV2 binds to ZDK). Upon light irradiation, RanQ69L is released from the mitochondria (LOV2 and ZDK dissociate due to a conformational change in LOV2) and activates TPX2 activity by binding to the inhibitory importin-α/β heterodimer. TPX2 can initiate microtubule nucleation.

What I like about this work

I am fascinated about this kind of protein moonlighting: in my research, so far, I was only concerned about the crucial role of RanGTPase cycling for nucleocytoplasmic import and export. But the obvious LINC between the nucleus and the cytoskeleton, composed of SUN-proteins and nesprins, gets more and more competition by nuclear pore components and associated proteins.

Moreover, this work provides one more and novel indirect connection between the actin and microtubule cytoskeleton.

Open questions

It has been recently shown that CRM1 (exportin 1) can serve as a docking station for ncMT nucleation in yeast (2). How is the current work about Ran and the work by Bao et al. about CRM1 ncMT nucleation possibly linked?

Additional references

1. H. Wang et al., Nat Methods. 13, 755–758 (2016).

2. X. X. Bao et al., eLife. 7, doi:10.7554/eLife.33465.

Tags: actin, lovtrap, non-centrosomal microtubules, rangtpase

Posted on: 16th July 2019

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