Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway
Figure 3
Schematic Illustration of Commitment Size Threshold Assay
The light/dark phases of synchronized cultures are indicated by the white and black bar on top. Aliquots of G1 cells are removed at different times (t1, t2, t3, t4), for size measurements (schematized in top row), and for dark-incubation in liquid and on plates. The dashed vertical line labeled CST represents a potential Commitment Size Threshold. After dark-incubation for 18 to 24 h in liquid, the daughter cell size distribution is measured (schematized in lower set of size distribution curves). The light-colored dashed curve represents the size distribution prior to the dark shift, and the dark solid line represents the cell size distribution after dark shifting and cell division. The aliquot of cells that was incubated on plates in the dark was scored microscopically to determine the fraction of committed cells in the population. The arrowhead in the picture inset indicates a cell that was not committed, and the stars indicate cells that were committed and divided either once or twice to produce two and four daughters, respectively. The CST is empirically determined by finding a size threshold value that best explains the behavior of the cells with respect to the fraction that were committed in each sample. The reliability of this value is reflected in the standard error (see Table 1).