SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture

Abstract

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five-to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight-or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six-to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight-and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.The formation of centrosomes in animal cells, as well as that of cilia and flagella in eukaryotic cells, critically depends on the evolutionarily conserved microtubule-based cylindrical centriole. In most species, newly formed centrioles are organized around a nine-fold radially symmetric 'cartwheel' structure. This cartwheel comprises a central hub ∼25 nm in diameter from which nine spokes emanate and connect through a pinhead structure to nine microtubule triplets, which are linked together and constitute a peripheral 'microtubule wall' (reviewed in refs 1,2). A generally acknowledged model posits that the cartwheel acts as a molecular scaffold for the formation of the nine-fold symmetric architecture of centrioles (reviewed in refs 1-7). In its strictest sense, this 'scaffold model' of centriole formation postulates that the nine-fold symmetric cartwheel is formed first and then dictates the assembly of a nine-fold symmetric microtubule wall.A key prediction of the scaffold model is that changes in cartwheel symmetry should dictate likewise changes in the architecture of the centriole. A central component of the cartwheel is SAS-6, a protein that is essential for cartwheel formation across eukaryotes (reviewed in refs 1,2). In Chlamydomonas, however, despite the absence of cartwheels, ∼20% of cells bearing a deletion of the sole SAS-6 gene nevertheless assemble centrioles that comprise a circular microtubule wall with a nine-fold symmetry in ∼70% of cases, but with seven-, eight-, ten-or eleven-fold symmetries in the remaining cases 8 . These observations suggest that the cartwheel is critical for proper assembly of the microtubule wall in Chlamydomonas, but also that in this species the centriolar microtubules have an inherent ability to assemble into near-nine-fold symmetrical structures. In contrast, knockdown of SAS-6 proteins in other systems, including human cells 9,10 , results in a failure...

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

“…The resulting SAS-6 oligomers show Heterogeneity in symmetry was also observed in in vitro experiments where SAS-6 oligomers were reconstituted and their symmetry directly examined. For example, rotary metal shadowing electron microscopy (EM) of CrSAS-6 oligomers revealed the formation of rings with variable diameters corresponding to 7.8 to 9.4-fold radial symmetry [31], a conclusion also supported by atomic force microscopy (AFM) studies [37,38] of the same oligomers ( Figure 4A). These experiments argued that symmetry differences inferred from the SAS-6 crystallographic structures did not represent artefacts, but rather genuine protein flexibility at the SAS-6 NN and CC interaction interfaces which drive oligomerisation.…”

“…These experiments argued that symmetry differences inferred from the SAS-6 crystallographic structures did not represent artefacts, but rather genuine protein flexibility at the SAS-6 NN and CC interaction interfaces which drive oligomerisation. Atomistic molecular dynamics (MD) simulations of CrSAS-6 performed previously [37], and similar work on LmSAS-6 presented here (described in Appendix A), showcase this conformational flexibility. In this type of analysis, SAS-6 oligomers are built using a fixed model of one interface from crystallography and variable models of the second interface derived from snapshots of the interface MD trajectory.…”

“…Indeed, direct visualization of CeSAS-6 spiral oligomers by negative stain EM demonstrated an even broader symmetry distribution from 7-to 11-fold; it is therefore possible that the structural variation seen in crystallographic structures underestimates the true degree of interface flexibility. MD simulations of the CeSAS-6 CC interface resulted in spiral SAS-6 oligomers of 7-to 16-fold symmetry ( Figure 3E; [37]); more strikingly, when combining simulations of the NN and CC interfaces, CeSAS-6 oligomer symmetries ranged from 6.4-to 16-fold, with a mean symmetry of 8.8-fold ( Figure 3F). This indicates that, despite the steric clash at the hinge region imposed on CeSAS-6, the protein remains flexile and able to accommodate a range of oligomer symmetries.…”

“…Symmetry 2017, 9, 74 5 of 10 substantial fluctuations in symmetry; for example, CrSAS-6 oligomers built using a simulated NN interface range from 7-to 13-fold symmetry [37], while we noted 6-to 23-fold symmetries for LmSAS-6 oligomers built from a simulated CC interface ( Figure 3C). Furthermore, we derived 10,000 distinct oligomer models of LmSAS-6 by combining independent MD simulations of the NN and CC dimerization interfaces ( Figure 3D), and noted that a substantial fraction (24.5 ± 2.7%) of LmSAS-6 oligomers acquire 12-fold or larger symmetries, with a mean oligomer symmetry at approximately 10-fold.…”

“…Furthermore, disruption of SAS-6 oligomerisation abrogates formation of the central centriole scaffold and strongly hinders organelle assembly [29,31]. Thus, SAS-6 oligomers are thought to comprise the centriole cartwheels, thereby influencing the radial symmetry of this organelle [26,37].…”