Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons

Abstract

The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.

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

“…Given that γ-tubulin, augmin, and TPX2 interact with each other and that MT nucleation from the sides of existing MTs requires all three proteins in Xenopus egg extract (Petry et al, 2013), it is tempting to speculate that RanGTP promotes ncMT nucleation by releasing TPX2 from the inhibitory importin heterodimers which in turn activating the γ-tubulin-augmin-TPX2 complex at the neurite tip. In support of this hypothesis, TPX2 and augmin complex subunits have been observed to localize along neurites (Chen et al, 2017;Cunha-Ferreira et al, 2018). In addition, both importin-α and importin-β molecules have been observed to present throughout the cytoplasm in cultured neurons (Chen et al, 2017;Hanz et al, 2003).…”

“…It has been reported that RanGTP is enriched at the soma and the neurite tip (Chen et al, 2017). To confirm this localization, an antibody which specifically targets the C-terminal tail of Ran GTPase that is only exposed in the GTP-bound state (Richards et al, 1995) was used for immunofluorescence staining to examine 2 days in vitro (2DIV) dissociated hippocampal neurons.…”

“…Interestingly, RanGTP appears to colocalize with the actin-based structure in the growth cone ( Figure 1C). Cytoplasmic RanGTP has also been shown to activate TPX2 and to promote ncMT nucleation in neurons (Chen et al, 2017). To examine whether RanGTP regulates ncMT nucleation, Ran mutants were utilized to alter the level of cytoplasmic RanGTP in neurons.…”

“…This 2-minute time period was selected based on the observation that adding RanQ69L and TPX2 dramatically enhances MT nucleation within 66 seconds in Xenopus egg extract (Petry et al, 2013). To maximize the effect of RanTRAP, photoactivation was carried out in the region of the neurite more than 15 µm from the soma or the neurite tip, where endogenous RanGTP level is low (Chen et al, 2017). EB3-mCherry dynamics was quantified before and after photoactivation ( Figure 3B).…”

“…A recent discovery shows that RanGTP is specially enriched at the tip of the neurite and around the soma (Chen et al, 2017). When a pharmacological perturbation disrupts the interaction between Ran and its downstream effector, ncMT nucleation from the neurite tip is compromised.…”