IFT88 maintains sensory function by localising signalling proteins along Drosophila cilia.

Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the mechanisms underlying their maintenance. Here, we show Drosophila melanogaster IFT88 (DmIFT88/nompB) continues to move along fully formed sensory cilia. We further identify Inactive, a TRPV channel subunit involved in Drosophila hearing and negative-gravitaxis behaviour, and a yet uncharacterised Drosophila Guanylyl Cyclase 2d (DmGucy2d/CG34357) as DmIFT88 cargoes. We also show DmIFT88 binding to the cyclase´s intracellular part, which is evolutionarily conserved and mutated in several degenerative retinal diseases, is important for the ciliary localisation of DmGucy2d. Finally, acute knockdown of both DmIFT88 and DmGucy2d in ciliated neurons of adult flies caused defects in the maintenance of cilium function, impairing hearing and negative-gravitaxis behaviour, but did not significantly affect ciliary ultrastructure. We conclude that the sensory ciliary function underlying hearing in the adult fly requires an active maintenance program which involves DmIFT88 and at least two of its signalling transmembrane cargoes, DmGucy2d and Inactive.

Pericentrin-mediated SAS-6 recruitment promotes centriole assembly.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

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

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 ß-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 ß-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.

The stability of Fbw7α in M-phase requires its phosphorylation by PKC.

Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.

PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space.

Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions.

Polo-like kinases: structural variations lead to multiple functions.

Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. PLKs undergo major changes in abundance, activity, localization and structure at different stages of the cell cycle. They interact with other proteins in a tightly controlled spatiotemporal manner as part of a network that coordinates key cell cycle events. Their essential roles are highlighted by the fact that alterations in PLK function are associated with cancers and other diseases. Recent knowledge gained from PLK crystal structures, evolution and interacting molecules offers important insights into the mechanisms that underlie their regulation and activity, and suggests novel functions unrelated to cell cycle control for this family of kinases.