Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel

Anastassia L Kantsadi,Georgios N Hatzopoulos,Pierre Gönczy,Ioannis Vakonakis

doi:10.1016/j.str.2022.02.005

Anastassia L Kantsadi, Georgios N Hatzopoulos + Show 2 more

Open Access

https://doi.org/10.1016/j.str.2022.02.005

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Abstract

SummaryCentrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Highlights

In animal cells that have exited the cell cycle, and broadly in unicellular eukaryotes, centrioles dock to the plasma membrane where they serve as basal bodies that template motile cilia and flagella, as well as the sensory primary cilium (Marshall, 2008)
The close stacking of spindle assembly abnormal protein 6 (SAS-6) rings brings to proximity the spokes of the protein, which emanate from the central hub in a radial fashion and correspond to the SAS-6 coiled-coil domain (Figures 1A and 1B)
In vitro reconstituted cartwheels from purified C. reinhardtii SAS-6 showed an $4-nm periodicity at their hub, as well as an indication of spoke merging toward the periphery, suggestive of ring stacking emerging from properties and interactions inherent to SAS-6 (Guichard et al, 2017; Nazarov et al, 2020; Klena et al, 2020)

Summary

Introduction

Centrioles are cylindrical organelles found across the eukaryotic kingdom (Carvalho-Santos et al, 2011; Hodges et al, 2010; Marshall, 2009; Winey and O’Toole, 2014). In animal cells that have exited the cell cycle, and broadly in unicellular eukaryotes, centrioles dock to the plasma membrane where they serve as basal bodies that template motile cilia and flagella, as well as the sensory primary cilium (Marshall, 2008). In this role, centrioles are essential for swimming cellular motility, such as in sperm, cellular signaling, and sensing of the surroundings. The cartwheel recruits and organizes further centriole components, including microtubules, resulting in the formation of a pro-centriole, which matures into a full organelle in the subsequent cell cycle (Azimzadeh and Marshall, 2010; Firat-Karalar and Stearns, 2014; Gonczy and Hatzopoulos, 2019; Jana et al, 2014). Errors in organelle assembly, which lead to the production of either too many or too few centrioles, as well as structurally aberrant ones, can lead to cell- and organism-level pathologies, including genomic instability, developmental abnormalities, and tumor formation (reviewed in Bettencourt-Dias et al, 2011; Chavali et al, 2014; Gonczy, 2015; Thornton and Woods, 2009; Venghateri et al, 2015)

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