A Structural Study of Conserved Centriole Duplication Machinery Public Deposited

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  • March 19, 2019
  • Slevin, Lauren K.
    • Affiliation: College of Arts and Sciences, Department of Biology
  • Centrioles are microtubule-based cylindrical structures that act within organelles responsible for nucleating polarized microtubule networks. Centrioles have an inherent nine-fold radial symmetry with species-dependent dimensions. Despite the fact that centrioles have been described by biologists for over a century, the process by which cells license and assemble nascent centrioles has only recently started to come to light. It is now known that centrioles, like DNA, duplicate via a cell cycle-regulated mechanism, and that a core set of five conserved proteins is necessary for centriole duplication. Polo-like Kinase 4 (Plk4) is a highly conserved serine/threonine kinase required for centriole duplication licensing. Plk4, along with all other Plk family members, contains arrayed Polo Box (PB) domains, a motif that undergoes hetero- or homo-dimerization to bind targets, localize to subcellular structures, and regulate kinase activity; however, the mechanism by which Plk4 employs its PBs to license centriole duplication has been unclear. Here, we harness x-ray crystallography, biochemistry, and cell biology to show that Drosophila melanogaster Plk4 contains three complete PB domains with distinct functions. The first two, PB1-PB2, form a homodimer in trans in both crystallographic form and in solution. We use in vitro pulldowns to demonstrate that PB1-PB2 are collectively needed to bind Asterless, a centriole scaffolding protein that localizes FL Plk4 to centrioles. Additionally, the PB1-PB2 homodimer is required for downregulation of the FL molecule, limiting centriole duplication. Finally, we use D.m. S2 cells to show that PB1-PB2 localizes to centrioles via conserved electrostatic interactions. The C-terminal PB domain, PB3, also forms a canonical PB fold, yet it shows species-dependent architecture and oligomerization states, demonstrating that PB3 is a structurally variable domain with species-dependent functions. Further work examines the role of a dynein light chain (LC8) in oligomerizing Anastral-2 (Ana2), a downstream centriole component that is a candidate for Plk4 phosphorylation. LC8-based Ana2 tetramerization has further implications for the role of Ana2 during centriole duplication. Collectively, our work delineates novel domains and interactions in the fundamental centriole licensing and assembly proteins Plk4 and Ana2, and implicates conserved mechanisms in centriole biogenesis.
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  • In Copyright
  • Redinbo, Matthew R.
  • Waters, Marcey
  • Bloom, Kerry
  • Duronio, Robert
  • Slep, Kevin
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2014
Place of publication
  • Chapel Hill, NC
  • This item is restricted from public view for 1 year after publication.

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