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, 29 (9), 1565-72

Procentriole Assembly Revealed by Cryo-Electron Tomography

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Procentriole Assembly Revealed by Cryo-Electron Tomography

Paul Guichard et al. EMBO J.

Abstract

Centrosomes are cellular organelles that have a major role in the spatial organisation of the microtubule network. The centrosome is comprised of two centrioles that duplicate only once during the cell cycle, generating a procentriole from each mature centriole. Despite the essential roles of centrosomes, the detailed structural mechanisms involved in centriole duplication remain largely unknown. Here, we describe human procentriole assembly using cryo-electron tomography. In centrosomes, isolated from human lymphoblasts, we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A-microtubule is capped by a conical structure and the B- and C-microtubules elongate bidirectionally from its wall. These observations suggest that the gamma tubulin ring complex (gamma-TuRC) has a fundamental role in procentriole formation by nucleating the A-microtubule that acts as a template for B-microtubule elongation that, in turn, supports C-microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Cryo-electron tomography of purified human centrosomes. Z slices (20 nm spacing) from a three-dimensional reconstruction (140 slices) of a centrosome, showing longitudinal sections of a centriole and its procentriole surrounded by the pericentriolar material (*: PCM). The procentriole (P) is perpendicular to the proximal part of the mature centriole (C). Arrows point towards microtubule blades. Ring-shaped objects (open arrowheads) are visible in the PCM. The central structure of the cartwheel (ctw) is distinguished in the procentriole (arrowhead in slice 60). The distal extremity of the mature centriole is filled with electron dense material (open arrow in slice 80). Scale bar, 250 nm.
Figure 2
Figure 2
Visualisation of the initial procentriole structures. (A) Z-section of a tomogram showing the central structure of the cartwheel and the connecting stalk (boxed in yellow). (B) Magnified view of the boxed region in (A), showing the central tube of the cartwheel (purple arrowhead) and the connecting stalk (red arrowhead). (C, E) Projection images of 23 Z-sections from two cryo-tomograms containing the central cartwheel structure. (D, F) Profile plots obtained from corresponding images (dotted lines) in (C, E). Maxima appear each 11.75 nm (with a s.d. of 2.5 and 1.16 nm, respectively). Scale bars: 100 nm (A) and 20 nm (B, C, E).
Figure 3
Figure 3
Procentriole microtubule triplet growth. (A) Microtubule singlet of a procentriole. The proximal part of the microtubule is capped by a conical structure. The right panel is a 3 × magnification of the boxed region. (B) Two Z sections of an A-microtubule (singlet) spaced by 5 nm. The distal part of the microtubule forms an outwardly curved extension (open arrow). (CE) Microtubule doublets from three different tomograms. Under each panel, a schematic representation of microtubule organisation is shown: A-microtubule is in purple, B-microtubule is in red (C). The B-microtubule is 35 nm from the tip of the A-microtubule cap. (D) The B-microtubule starts at 18 nm from the tip of the A-microtubule cap and shows an outwardly curved extension at its proximal extremity (open arrow). (E) The B-microtubule is at the level of the tip of the A-microtubule cap. (F) Microtubule triplet of a procentriole. The C-microtubule is attached to the side of the B-microtubule at a distance of 46 nm from the distal tip of the cap. The distal and proximal extremities of the C-microtubule display curved extensions (open arrows). (G) Microtubule triplet of a mature centriole. The microtubules are opened at their distal and proximal extremities. (F, G) Next to the panel, a schematic representation of microtubule organisation is shown: A-microtubule is in purple, B-microtubule is in red, and C-microtubule is in green. Scale bar: 20 nm.
Figure 4
Figure 4
The nine microtubule blades of the centriolar barrel. (A) Microtubule singlets present in a procentriole. Note that the length of each microtubule differs and is not arranged in order of size around the centriolar wall. Next to the panel, a schematic representation of the microtubule organisation in the centriolar wall is shown. A-microtubules are represented by a purple circle and numbered. (B) Procentriole displaying singlet or doublet microtubules. Note that three microtubule blades are not yet formed (2, 3, 7), whereas the blade 4 already shows a doublet microtubule. Next to the panel, a schematic representation of the microtubule organisation in the centriolar wall is shown. Present A-microtubules are represented by a purple circle and numbered, absent A-microtubules are in light purple, and B-microtubule is in red. Scale bar: 20 nm. (C, D) The length of the A-microtubule is measured from the proximal tip to its distal end. (C) Procentriole with nine blades of singlet (blade 5 and 6) or doublet microtubules. Note that the length of the A-microtubule is variable (in purple). The B-microtubule (in red) already reaches a length of 180 nm in blade 9, whereas the others are only 60 nm long (blade 1 or 2) or have not yet assembled (blades 5 and 6). (D) Procentriole with nine blades of doublet or triplet microtubules. Note that the C-microtubule (in green) in blade 8 is absent.
Figure 5
Figure 5
Model of human procentriole assembly. (A) Mature centriole. (B, C) The first step of procentriole assembly involves the formation of a stalk and the central hub of the cartwheel at the proximal end of the mature centriole (A). (C) The cartwheel assembles and organises the procentriolar wall, in which some A-microtubules start growing from the γ-TuRCs. (D) Enlargment of the procentriole shown in (C). (E) Before completion of the nine A-microtubule round, some B-microtubules start growing from the wall of A-microtubules. (F) B-microtubules grow bidirectionally, and C-microtubules start growing from the wall of the B-microtubules. (G) C-microtubules grow bidirectionally until the B- and C-microtubules reach the proximal end of the A-microtubule. (H) Growth continues at the distal end until completion of the microtubule triplet blades.

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