Fig. 7.

Co-depletion of Poc1 proteins leads to loss of centriole integrity. (A–D) HeLa cells transfected with siRNAs against luciferase (siGL2) or Poc1A+Poc1B were fixed after 72 hours. Cells were stained with acetylated-tubulin (red) and centrin-2 (green) antibodies. Interphase cells are shown in A and mitotic cells in C. (B) The percentage of interphase cells with 2 strong, 1 weak and 1 strong or only 1 acetylated-tubulin-positive centrosomes is indicated with respect to the number of Centrin-2 dots. Data are means ± s.d. from two separate experiments (n = 157 for siGL2 and n = 92 for siPoc1A+siPoc1B). (D) The percentage of mitotic cells with 2 centrin dots at each pole (2/2), 2 centrin dots at one pole and one at the other (2/1), 2 centrin dots at one pole and none at the other (2/0), 1 centrin dot at each pole (1/1) or 1 centrin dot at one pole and none at the other (1/0). Data are from a single experiment (n = 176 for siGL2 and n = 179 for siPoc1A+siPoc1B). (E) HeLa cells transfected with siRNAs against luciferase (siGL2) or Poc1A+Poc1B for 72 hours were stained with antibodies against γ-tubulin (red) and centrobin (green). DNA was stained with Hoechst 33258 (blue). (F) A schematic model showing the consequences of depleting Poc1 protein (green) on centriole stability and duplication, together with the predicted localization of centrobin under these conditions (pink). Upon siRNA depletion, Poc1 remains stably associated with the parental centrioles but is not incorporated into the nascent procentrioles, preventing their stabilization. When this cell enters mitosis, it will produce daughter cells that have a stable mother centriole but an unstable daughter centriole. When this cell then enters S-phase, the stable mother centriole will initiate duplication but produce another unstable centriole, which is incapable of duplication. When this cell arrives at the next mitosis, the cell contains one spindle pole (pole no. 1) containing the old mother centriole and an unstable procentriole, and one pole (pole no. 2) containing a single unstable daughter centriole or no centriole at all. Such a cell may generate an unequal bipolar or monopolar spindle.