Self-assembling SAS-6 Multimer Is a Core Centriole Building Block

Paul Guichard,Sergio Marco,Jayachandran Gopalakrishnan,Tomer Avidor-Reiss,Heinz Schwarz,Andrew H. Smith,David A. Agard

doi:10.1074/jbc.m109.092627

Abstract

Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a "cartwheel" with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with approximately 6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.

Highlights

SAS-6 Is a Component of the Centriole Central Tubule—To gain structural insights into the centriole central tubule, we biochemically isolated centrosomes from Drosophila embryo extract and processed them for thin-section electron microscopy (EM) as previously described [26]
This is similar in size to the central tubules of most of the model organisms with the exception of the hexapod Acerentomon microrhinus, where the diameter of the central tubule varied according to the centriole diameter [27]
We examined whether SAS-6 is a component of the central tubule in Drosophila

Summary

Introduction

The procentriole has an internal cartwheel structure that is required for the stability of the centriole and the 9-fold symmetry of cilia [11]. Based on the structure of the cartwheel, it is expected that the central tubule would itself display 9-fold symmetry [12] (supplemental Fig. S1A). It has been proposed that the central tubule generates the 9-fold symmetry of the centriole [13, 14]. Null mutants of SAS-6 in Chlamydomonas [11] and Drosophila [11, 19] lack the cartwheel. The SAS-6 null mutants of Chlamydomonas and Drosophila are defective in establishing the 9-fold symmetry of centrioles. In Caenorhabditis elegans, RNAi studies show that SAS-6 is required for the formation of a centriole central tube. To investigate the mechanism of SAS-6 function, we employed a Drosophila model that allows for biochemical isolation

Methods

Results

Conclusion