PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

Abstract

The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo . How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011 ; Zitouni et al., 2016 ). Surprisingly, we observe that in vitro , PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo . The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.

Centriole assembly at a glance

“…Whereas PLK4 and hsSAS-6 are essential in both cases, the higher order oligomerization of hsSAS-6 dimers necessary for centriole assembly in the canonical pathway is dispensable during de novo formation (Wang et al, 2015), perhaps because peripheral elements, such as the A-C linker or MT, are particularly crucial in this case. βand γ-tubulins, thereby generating microtubule organizing centers (MTOCs) (Montenegro Gouveia et al, 2018). Although analysis by electron microscopy (EM) indicates that these condensates are not centrioles, this observation can lead one to imagine that PLK4 concentrates other components than just STIL during organelle biogenesis.…”

Plk4 triggers autonomous de novo centriole biogenesis and maturation

“…Therefore, Plk4 auto-phosphorylation and interaction with Stil may provide spatial and temporal regulation of Plk4 kinase activity in cells, activating Plk4 preferentially at the centrosome where it is concentrated. Moreover, Plk4 ability to form large order oligomers (“condensates”), may be important for the onset of centriole biogenesis (Montenegro Gouveia et al, 2018; Leda et al, 2018; Shohei and Kitagawa, 2018; Park et al, 2019). We hypothesised that, in the case Plk4-driven centriole biogenesis is based on a positive feedback mechanism, the initiation of biogenesis is concentration-dependent and relies on overcoming a critical, local threshold in kinase activity (Fig.…”

“…For example, Sas6 self-assembly into homodimers is at the heart of the universal 9-fold symmetry (Nakazawa et al, 2007; Breugel et al, 2011; Kitagawa et al, 2011; Guichard et al, 2017). At high concentration, Xenopus Plk4 forms supramolecular scaffolds that bind other centrosomal proteins and nucleate MTs (Montenegro Gouveia et al, 2018). In human cells, the association of Plk4 into condensates was shown to be mediated by disordered regions within Plk4 (Yamamoto and Kitagawa, 2019) and regulated by autophosphorylation (Montenegro Gouveia et al, 2018; Yamamoto and Kitagawa, 2019; Park et al, 2019).…”

“…The PCM may generate protein scaffolds in the cytoplasm where centriolar proteins bind with higher affinity, therefore locally concentrating these molecules and forming stable seeds for centriole biogenesis. Moreover, Gamma-tubulin promotes MT nucleation, which may attract more components via motor-based transport or through entrapment of proteins with MT-binding capacity, such as Plk4 (Montenegro Gouveia et al, 2018). These manifold properties of the PCM may promote centriole biogenesis within biochemically-confined environments in the cytoplasm.…”

“…It is still not fully understood how Plk4 acquires basal catalytic activity, but it is likely that other centrosomal proteins regulate this process, such as its substrates Ana2 and Asl (Klebba et al, 2015b; a; Moyer et al, 2015; Zitouni et al, 2016; Mclamarrah et al, 2018; Boese et al, 2018; Aydogan et al, 2019). Moreover, at high concentration, Plk4 self-assembles into nanoscale condensates in Xenopus extracts and in human cultured cells, which may be important for centriole assembly (Montenegro Gouveia et al, 2018; Yamamoto and Kitagawa, 2019; Park et al, 2019).…”