Control of daughter centriole formation by the pericentriolar material

Abstract

Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.

Figure 1

Laser ablation of daughter centrioles induces reduplication of the remaining mothers in S-phase arrested HeLa cells. (A) Daughter centriole in one of the two diplosomes was ablated (arrow in 00:00). The mother remained single for >1 hr , and then developed a “shadow” (arrow in 02:07, also see Fig.S4), indicating formation of a new daughter. This cell was fixed for EM at 02:08. (B) Selected 80-nm EM sections from the complete series of the cell presented in (A). Notice that the new daughter centriole (arrow) is significantly smaller than the daughter in the other, non-irradiated diplosome (arrowhead). (C) Repetitive ablations of daughter centrioles. After ablation of the first daughter (arrow in 00:00), the mother (arrowheads) developed a new daughter in ∼4 hr (arrow in 04:07). This daughter was subsequently ablated (arrow in 09:04) and ∼10 hr later another daughter developed (arrow in 20:43). Notice that the centrioles in the non-irradiated diplosome remained engaged throughout the experiment. EM analysis confirmed that the shadow seen in 20:43 was in fact a daughter centriole. (D) Mother centriole in one of the two diplosomes was ablated (arrowhead in 00:00) and the remaining daughter (arrows) did not duplicate. All images in A, C, and D are maximal intensity projections of complete Z-series through the centrosome. Due to the significant differences in fluorescence intensity between the mature and newly formed centrioles, it is impossible to reproduce the two types simultaneously through use of a linear grey-scale look-up table (LUT). Instead, a pseudo-colour intensity LUT (shown to the right of panel B) has been applied to the images. The first image in each series is also presented in contrast-enhanced grey-scale (non-linear Υ factor). Scale bars in A, C, and D represent 1 μm. Time stamps in panels A, C, and D are in hours : minutes.

Figure 2

Examples of centriolar configurations in S-phase arrested CHO cells. (A) Centrosomes in a typical CHO cell after 38 hr in 2-mM hydroxyurea (also see Fig. S4), as visualized by immunostaining. This centrosome contained two brighter mother centrioles (M1 and M2) and two dimmer daughter centrioles formed in the first round duplication (D1 and D2). At the time of fixation, both mothers were reduplicating. Centrioles M1 and D2 were associated with single daughters thus forming classical diplosomes, while centriole M2 was associated with two daughters, thus forming a triplosome. The other daughter centriole (D1) remained single during the second round of replication. Scale bar = 1 μm. (B – D) Centriole ultrastructure in a typical cell fixed after 37 hr in HU. (B) single centriole, (C) diplosome, and (D) triplosome. The first image in each panel presents a maximal-intensity projections of the centrin-GFP fluorescence recorded immediately after fixation. These images are pseudocolored with the same LUT as in Fig.1. The remaining images in each panel depict selected consecutive 80-nm EM sections from the complete series through the cell. Proximal ends of mother centrioles are marked ‘p’. In contrast to the behaviour observed in HeLa cells (Figs. 1B, S1), daughter centrioles in S-phase arrested CHO cells develop to full length (∼400 nm). Notice that daughter centrioles are orthogonal to their mothers in both the diplosome (C) and triplosome (D) One daughter in the triplosome (arrow in D) is closer to the proximal end of the mother than is the other daughter (arrowhead in D). Also note that the triplosome in this cell was formed by the oldest mother centriole, which contained the greatest amount of centrin-GFP, and was the only centriole in the cell to carry distal appendages (marked ‘da’). Scale bars in C-E = 0.5 μm.

Figure 3

Ablation of all daughter centrioles within a centrosome induces reduplication of the mother in S-phase arrested CHO cells. (A) In this cell, daughter centrioles in both diplosomes were ablated (arrows in 00:00). Both mother centrioles (arrowheads) remained single for more than 1 hr. Then, one of the mothers developed a new daughter (arrow in 02:30), while the second mother remained single. Approximately 9 hr after ablation, the second mother also developed a new daughter centriole (arrow in 09:00). (B) An example of triplosome formation after ablation of the original daughter centriole. Both daughter centrioles were ablated, as in (A). However, one of the mothers in this cell developed two daughter centrioles (arrows in 03:00, 04:30, and 06:30). Formation of the two daughters was not simultaneous. However, at later time points the two daughters appeared to be of the same size (not shown). The second mother developed a single daughter centriole (arrowhead in 06:30). (C) In this cell the mother centriole in one of the diplosomes was ablated (arrowhead). Because the daughter and mother centrioles can undergo natural disengagement during the course of >20-hr long experiments, which complicates the analysis, the second diplosome was also completely ablated. As the result, the cell was left with just one daughter centriole (arrows). This centriole remained single for more than 11 hr but ultimately developed a daughter (arrowhead in 21:00). (D) Both daughters within a triplosome (arrows in 00:00) were ablated. The mother remained a single centriole for ∼9 hrs and then developed a new daughter (arrows in 09:54 and 10:42). (E) Just one of the two daughters in a triplosome was ablated (arrows in 00:00) converting this triplosome into a diplosome. The other daughter centriole (arrowhead) remained engaged with the mother for more than 24 hrs, and the ablated daughter did not regenerate. Time stamps in hours : minutes. Scale bars = 1 μm. Same LUT as in Fig.1.

Figure 4

Effects of PCM exaggeration on the number of daughter centrioles. (A) Overexpression of pericentrin in S-phase arrested CHO cells results in the formation of a large cloud of pericentriolar material that contains numerous centrin-GFP aggregates cloud. overexpressing pericentrin. (A) Lower-magnification view of the centrosome in a cell expressing pericentrin (visualized with an anti-HA antibody). Inset in the Υ-tubulin frame depicts the size of the Υ-tubulin cloud of normal centrosomes in CHO cells shown at the same magnification. (B) Higher-magnification view of the region boxed in “(A) Merged”. (C) Some but not all centrin aggregates in pericentrin-overexpressing cells are centrioles. Centrin-GFP distribution in two cells that were analyzed by serial-section EM (see Fig. S7 for EM data). In the cell fixed 25 hr after transfection (left frame) centrin-GFP distribution is generally diffused with two brighter and several dimmer discrete spots. The brighter spots (M1 and M2) correspond to full-length mother centrioles while five of the dimmer spots (D1-D5) correspond to short randomly-oriented daughter centrioles (see Fig. S7A). In the cell fixed 45 hrs after transfection (right frame) discrete centrin spots are more prominent. Two brightest spots (M1 and M2) correspond to mother centrioles and twelve of the dimmer spots (D1-D12) correspond to daughter centrioles (see Fig. S7B). (D) 3-D organization of centrosomes with multiple daughter centrioles. Notice that centrin spots corresponding to the distal ends of centrioles may reside within the Υ-tubulin cloud or protrude outside. In contrast, the proximal ends of daughter centrioles (marked by SAS-6) consistently reside within the PCM although they are not always in the proximity of the mother centriole . See Videos 9-11 for surface-rendered models of the centrosomes presented here.