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.