Plk4 triggers autonomous de novo centriole biogenesis and maturation

Abstract

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.

The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

“…However, it is unclear how de novo biogenesis is spatially regulated. Recent studies 48,49 highlight the involvement of pericentriolar material (PCM) in de novo assembly in animals, in agreement with previous observations. 11 Given that many PCM components do not have clear orthologs outside of the Holozoa, 24 other molecules might perform similar functions.…”

“…Despite their complex ultrastructure, 45 centriole assembly is a relatively fast process, lasting around 10-15 min in Caenorhabditis elegans, 46 Naegleria gruberi, 47 and in Drosophila melanogaster. 48 Centriole biogenesis in P. patens might follow a similar temporal dynamics, explaining the low number of cells observed at early bicentriole assembly and centriole separation stages. Still, the observation that both sister centrioles still contained doublet-MTs (Figures 2H, 2I, S2C, and S2D), and had similar lengths (Figures 2G, S2B, and S4), supports the idea that both assembled de novo in a bicentriole conformation.…”

Experimental and Natural Induction of de novo Centriole Formation

“…Similarly, de novo centriole formation induced by the removal of resident centrioles has been observed in cultured cell lines of Drosophila melanogaster . Loss of centrioles by long-term treatment with Plk4/Sak RNAi, followed by the restoration of Plk4/Sak expression, results in the de novo assembly of new centrioles in Drosophila cell lines ( Rodrigues-Martins et al, 2007b ; Dzhindzhev et al, 2010 ; Nabais et al, 2021 ).…”

Centriole growth is not limited by a finite pool of components, but is limited by the Cdk1/Cyclin-dependent phosphorylation of Ana2/STIL

“…Interestingly, a previous study using a similar microscopy set-up but a different mNG-Plk4 knock-in line used FCS to estimate a concentration of ~7-8nM (Nabais et al, 2021). As we could not use FCS, we used Peak-Counting Spectroscopy (PeCoS) to monitor how the cytoplasmic concentration of the transgenically expressed ePlk4-mNG fusion protein varied during nuclear cycle 12 (as the centrioles do not overduplicate in this line).…”