FIGURE 2.

Experimental testing of centriole copy number homeostasis. (A) The vfl2^ts mutant allows reversible generation of centriole number errors. Graph shows distribution of centriole copy numbers in vfl2ts grown at 21°C (blue) and 34°C (red). Copy-number distribution in constitutive vfl2 mutant is included for comparison (gold). (Inset) Images to show recovery of centrin fibers (34°C) on left lacks recognizable fibers when cells are stained with anticentrin antibodies, but after one day of growth following downshift to permissive temperature (21°C) assembly of centrin fibers has been recovered. (B) Dynamic recovery of copy number in vfl2^ts following downshift. Copy number determined by counting flagella and computing rms error relative to the nominal wild-type copy number of two per cell. Blue diamonds indicate measured data points. Red dotted line indicates rms error in cells grown continuously at permissive temperature. Graph indicates that copy number error returns to the resting level within six generations following restoration of VFL2 gene function after temperature downshift. Error bars represent 95% confidence intervals. An average of 472 cells were scored per time point. (C) Copy-number restoration confirmed by immunofluorescence. Cells were fixed and stained with rabbit anti-FLA10 kinesin and monoclonal antiacetylated tubulin antibodies, both of which recognize centrioles. Centrioles were detected as foci that stained with both antibodies. Plots indicate fraction of cells containing either too few centrioles (less than two per cell, indicated by blue diamonds) or too many centrioles (more than two per cell, indicated by red circles). Gray shaded box represents the time interval during which cells lack the centrin-based connecting fibers responsible for proper centriole segregation as judged by centrin immunofluorescence. Plot shows that centriole number begins returning to the wild-type distribution as soon as centriole segregation is restored. An average of 212 cells were scored per time-point. (D) Copy-number restoration is not due to selective cell death. We examined individual cell divisions and determined the frequency with which vfl2 cells produce inviable daughter cells. Results from a total of 401 live cell divisions are reported.