Centriole assembly in Caenorhabditis elegans

Abstract

Centrioles are necessary for flagella and cilia formation1,2, cytokinesis3,4, cell-cycle control5 and centrosome organization/spindle assembly6. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

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Figure 1: Centriole proteins are recruited in two steps.
Figure 2: SPD-2 and ZYG-1 are required for the recruitment of SAS proteins to the site of centriole assembly.
Figure 3: Centriole assembly in Caenorhabditis elegans is a multi-step process.
Figure 4: The SAS proteins are required at different steps during centriole assembly.

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Acknowledgements

We thank A. Dammermann and K. Oegema for their gift of the GFP–ZYG-1 strain and for sharing unpublished results; F. Barr for anti-GFP antibodies; M. Tipsword, M. Ruer and D. Drechsel for generating the ZYG-1 antibodies; A. Pozniakovsky for generating the GFP–SAS-5 and GFP–SAS-6 constructs; O. Molodtsova for biolistic bombardment; J. Mäntler and Q. DeRobillard for help with electron microscopy; C. Lorenz for secretarial assistance; K. McDonald, P. Verkade and I. Baines for help in developing the single-embryo high-pressure freezing approach; C. Eckmann for providing the FOG-1 feeding construct; and F. Friedrich for help with graphical artwork. We are grateful to G. Warren, W. Zacheriae, R. Kittler and F. Quittnat Pelletier for their comments on the manuscript, and Hyman laboratory members for stimulating discussions. The Boulder Laboratory for 3D Electron Microscopy of Cells is supported by a grant from the National Institutes of Health to J. R. McIntosh. L.P. is supported by a postdoctoral fellowship from the HFSP.

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Correspondence to Laurence Pelletier.

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Supplementary information

Supplementary Methods

This file contains additional details of the methods used in this study. (DOC 152 kb)

Supplementary Movie

This file contains text to accompany the below Supplementary Movies. (DOC 30 kb)

Supplementary Figure Legends

This file contains text to accompany the below Supplementary Figures. (DOC 30 kb)

Supplementary Figure 1

In glutharaldehyde-fixed C. elegans embryos daughter centrioles are first observed at pronuclear migration. (JPG 1431 kb)

Supplementary Figure 2

Mating assays can be used to monitor the recruitment of centriole proteins to the site of daughter centriole assembly. (JPG 58 kb)

Supplementary Figure 3

ZYG-1::GFP is recruited during meiosis. (PDF 8162 kb)

Supplementary Figure s4

Electron tomography of staged one-cell embryos. (JPG 40 kb)

Supplementary Figure s5

Quantification of 3D centriole models (PDF 3133 kb)

Supplementary Figure s6

The use of the slicer tool to display centriole structure. (JPG 195 kb)

Supplementary Figure s7

Modelling centriole structures in 3dmod. (JPG 150 kb)

Movie Figure 3A1.

Complete tomographic volume and three-dimensional model of centrioles at pronuclear appearance. (MOV 12527 kb)

Movie Figure 3A2.

Complete tomographic volume and three-dimensional model of centrioles at pronuclear appearance. (MOV 5058 kb)

Movie Figure 3B1

Complete tomographic volume and three-dimensional model of centrioles during pronuclear migration. (MOV 5982 kb)

Movie Figure 3B2

Complete tomographic volume and three-dimensional model of centrioles during pronuclear migration. (MOV 8137 kb)

Movie Figure 3C1

Complete tomographic volume and three-dimensional model of centrioles during the rotation of the pronuclei/centrosome complex. (MOV 3718 kb)

Movie Figure 3C2

Complete tomographic volume and three-dimensional model of centrioles during the rotation of the pronuclei/centrosome complex. (MOV 6824 kb)

Movie Figure 4A

Complete tomographic volume and three-dimensional model of a zyg-1(RNAi) embryo during the rotation of the pronuclei/centrosome complex. (MOV 6467 kb)

Movie Figure 4B

Complete tomographic volume and three-dimensional model of a sas-6(RNAi) embryo during rotation of the pronuclei/centrosome complex (MOV 3422 kb)

Movie Figure 4C

Complete tomographic volume and three-dimensional model of a sas-4(RNAi) embryo at pronuclear appearance. (MOV 8180 kb)

Movie Slicer Tool 1

Movie through serial tomographic slices showing a mother centriole in cross section and a daughter central tube during pronuclear migration. (MOV 6256 kb)

Movie Slicer Tool 2

Movie through serial tomographic slices of the data set in “Movie Slicer Tool 1” rotated to display the mother centriole along the longitudinal axis. (MOV 4011 kb)

Movie SAS-5(RNAi).

Complete tomographic volume and three-dimensional model of a sas-5(RNAi) embryo during rotation of the pronuclei/centrosome complex. (MOV 14434 kb)

Movie SAS-4(RNAi).

Complete tomographic volume and three-dimensional model of a sas-4(RNAi) embryo during rotation of the pronuclei/centrosome complex. (MOV 23365 kb)

Supplementary Table 1

Quantitative analysis of centriole assembly in one-cell wild-type and RNAi embryos. (DOC 88 kb)

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Pelletier, L., O’Toole, E., Schwager, A. et al. Centriole assembly in Caenorhabditis elegans. Nature 444, 619–623 (2006). https://doi.org/10.1038/nature05318

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