Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

Abstract

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Once and only once: mechanisms of centriole duplication and their deregulation in disease

“…In vitro , SAS-6 can oligomerize into structures closely resembling the cartwheel hub, suggesting that SAS-6 may impart the typical nine-fold symmetry to centrioles 21–23 . However, the assembly of stable cartwheels in vivo likely requires additional proteins, interactions with the microtubule wall and/or preexisting centrioles 24,25 .…”

“…The conserved centriole duplication factor STIL (Ana2 in Drosophila ), interacts with SAS-6 and plays a central role in promoting SAS-6 recruitment and/or assembly 26–32 . In Chlamydomonas , cartwheel formation requires the protein Bld10p 19,33 , which interacts with SAS-6 to relieve an inhibitory action of the SAS-6 C-terminus on cartwheel assembly 23 . In human cells, the putative Bld10p homolog CEP135, also interacts with hSAS-6 34 , but most CEP135 localizes to the parent centriole and not the procentriole 35,36 , suggesting additional roles in centriole biogenesis and PCM assembly.…”

“…(b) Micrograph shows lattice of in vitro reconstituted cartwheel hub and spoke structures visualized by cryo-electron microscopy. Adapted with permission from 23 . (c) Image derived from cryotomogram sections of Chlamydomonas procentriole emphasizes cartwheel and triplet microtubules.…”

The Rise of the Cartwheel: Seeding the Centriole Organelle

“…How distinct cartwheel elements and associated structures are organized during organelle assembly remains largely an open question. However, some clues came from a recent analysis using cryo‐ET of isolated Chlamydomonas procentrioles, which could be divided into three regions. First, a most proximal region that harbors only a cartwheel and no pinhead or microtubules; such a core cartwheel is characterized by two densities termed D1 and D2 (Figure ).…”

Coupling Form and Function: How the Oligomerisation Symmetry of the SAS-6 Protein Contributes to the Architecture of Centriole Organelles

“…[46,47] and C. reinhardtii [48] centrioles revealed that the spokes of successive SAS-6 rings merge as they radiate outwards, leading to stacking of SAS-6 rings with a periodicity of approximately 8.5 nm at the central hub and 17 nm at the end of spokes ( Figure 6A). SAS-6 stacking periodicity aligns with the axial periodicity of microtubules at the outer centriole wall.…”