Figure 3.
Spatio-temporal kinetics of de novo centriole biogenesis. (A) Schematic representation of the experimental data analysis for distances. The first four centrosomes formed de novo in the explants were tracked in 3D using the intensity signal from the Jupiter (MT reporter) channel (first tracking round) and Spd2 (centrosomal reporter) channel (second tracking round) combined. For each of the de novo birth events, an XYZT coordinate matrix was retrieved, from which the inter-event distances were calculated. Experimental n = 68 explants/eggs. (B) Scatter plot of observed inter-event distances for all pairwise combinations of the first four de novo biogenesis events. Horizontal lines and error bars represent median and interquartile distance, respectively. (C) Cumulative distribution functions (CDF) of inter-event distance. Distributions were not significantly (ns) different (Kruskal–Wallis mean rank test, P = 0.467). (D) In silico simulations were performed to test if the observed experimental data deviates from a theoretical scenario in which all four birth events occurred at independent and identically distributed random positions with a uniform probability density distribution, within explants with similar geometry as in the experiments. Four random events were obtained in 100 simulations of 68 explants. The graph depicts the median CDF of all experimentally observed (obs, solid line) and all simulated (sim, dashed line) inter-events distances, while the gray envelope indicates the 95% confidence interval (from quantile 0.025 to 0.975) for the simulated data. The experimental observations do not deviate from random simulations. (E) Schematic representation of the experimental data analysis for time. For each of the four de novo birth events, an XYZT coordinate matrix was retrieved, from which the inter-event time were calculated. Experimental n = 68 explants/eggs. (F) Scatter plot of observed inter-event time between the first four de novo biogenesis events. Horizontal lines and error bars represent median and interquartile range, respectively. The first event time is significantly different (**) from subsequent inter-event times (Kruskal–Wallis mean rank test, P = 0.0047). Note that this first event time exhibits high (systematic) variability due to an ill-defined time reference (see Materials and methods). (G) Cumulative distribution functions of observed (continuous) and in silico obtained inter-event time (dashed). Simulations were performed to test if the observed experimental data deviates from a theoretical scenario where all four birth events occurred independently at a constant rate within an explant with similar geometry as in the experiments. Four random events were obtained in 100 simulations of 68 explants. In the simulation, the first event rate of birth was approximated to the inter-event time between the first and second events. The graph depicts the median CDF of the experimentally observed (obs, continuous line) and simulated (sim, dashed line) waiting times between the first and second, second and third, and third and fourth events, while the gray envelope indicates the 95% confidence interval (from quantile 0.025 to 0.975) for the simulations. The observed and simulated waiting time distributions do not overlap, and differ more as centriole number increases, suggesting that the rate of biogenesis is increasing over time. (H) Estimation of the experimental birth rates using maximum likelihood estimation fitting. An exponential distribution with rate λ > 0 was fitted by maximum likelihood estimation to the CDF of each observed waiting time. The estimated rate of de novo centriole assembly is represented in the graph as a function of the number of centrioles previously/already present in the volume.

Spatio-temporal kinetics of de novo centriole biogenesis. (A) Schematic representation of the experimental data analysis for distances. The first four centrosomes formed de novo in the explants were tracked in 3D using the intensity signal from the Jupiter (MT reporter) channel (first tracking round) and Spd2 (centrosomal reporter) channel (second tracking round) combined. For each of the de novo birth events, an XYZT coordinate matrix was retrieved, from which the inter-event distances were calculated. Experimental n = 68 explants/eggs. (B) Scatter plot of observed inter-event distances for all pairwise combinations of the first four de novo biogenesis events. Horizontal lines and error bars represent median and interquartile distance, respectively. (C) Cumulative distribution functions (CDF) of inter-event distance. Distributions were not significantly (ns) different (Kruskal–Wallis mean rank test, P = 0.467). (D) In silico simulations were performed to test if the observed experimental data deviates from a theoretical scenario in which all four birth events occurred at independent and identically distributed random positions with a uniform probability density distribution, within explants with similar geometry as in the experiments. Four random events were obtained in 100 simulations of 68 explants. The graph depicts the median CDF of all experimentally observed (obs, solid line) and all simulated (sim, dashed line) inter-events distances, while the gray envelope indicates the 95% confidence interval (from quantile 0.025 to 0.975) for the simulated data. The experimental observations do not deviate from random simulations. (E) Schematic representation of the experimental data analysis for time. For each of the four de novo birth events, an XYZT coordinate matrix was retrieved, from which the inter-event time were calculated. Experimental n = 68 explants/eggs. (F) Scatter plot of observed inter-event time between the first four de novo biogenesis events. Horizontal lines and error bars represent median and interquartile range, respectively. The first event time is significantly different (**) from subsequent inter-event times (Kruskal–Wallis mean rank test, P = 0.0047). Note that this first event time exhibits high (systematic) variability due to an ill-defined time reference (see Materials and methods). (G) Cumulative distribution functions of observed (continuous) and in silico obtained inter-event time (dashed). Simulations were performed to test if the observed experimental data deviates from a theoretical scenario where all four birth events occurred independently at a constant rate within an explant with similar geometry as in the experiments. Four random events were obtained in 100 simulations of 68 explants. In the simulation, the first event rate of birth was approximated to the inter-event time between the first and second events. The graph depicts the median CDF of the experimentally observed (obs, continuous line) and simulated (sim, dashed line) waiting times between the first and second, second and third, and third and fourth events, while the gray envelope indicates the 95% confidence interval (from quantile 0.025 to 0.975) for the simulations. The observed and simulated waiting time distributions do not overlap, and differ more as centriole number increases, suggesting that the rate of biogenesis is increasing over time. (H) Estimation of the experimental birth rates using maximum likelihood estimation fitting. An exponential distribution with rate λ > 0 was fitted by maximum likelihood estimation to the CDF of each observed waiting time. The estimated rate of de novo centriole assembly is represented in the graph as a function of the number of centrioles previously/already present in the volume.

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