Localization of hSas-6 and C-Nap1 differentiates MTOC-competent from noncompetent centrioles. (A) RPE1 cells going through cell division and exiting mitosis were recorded using time-lapse microscopy. The daughter cells (marked by arrows and arrowheads in phase images) were located and analyzed for γ-tubulin recruitment at the two inherited centrioles (arrows) that were previously mother and daughter centrioles. Note that all centrioles recruited similar amounts of γ-tubulin (left), indicating that daughter centrioles had converted to motherlike centrioles that were active in recruiting PCM. Centrioles in these early G1 cells were also examined for hSas-6 and C-Nap1 localization (right), which negatively and positively correlate, respectively, with modified centrioles that recruit PCM. (B) De novo centrioles (arrows) and centriole rosettes (arrowheads) induced in RPE1 cells as described in Fig. 2 were analyzed for hSas-6 and C-Nap1 localization. All unmodified centrioles, freestanding or engaged, were labeled with hSas-6 but lacked C-Nap1, a reverse pattern to that of modified centrioles shown (top). (A and B) Insets show a higher magnification of centrosomes. (C) RPE1 cells induced to form de novo centrioles and centriole rosettes during interphase were traced by time-lapse microscopy and allowed to pass through mitosis. After division, centrioles in one of the daughter cells (arrows) were analyzed for hSas-6 and C-Nap1 localization. Note that all centrioles displayed a pattern for modified centrioles (strong C-Nap1 and no hSas-6).