| The homogeneity in cell size observed in many normal proliferating tissues, and the contrasting size disparities characteristic of several cancers, suggest that control mechanisms coordinate growth and cell cycle progression, but the existence of such mechanisms has not been firmly established. To address this problem, we used quantitative fluorescence microscopy to measure cell cycle position, total protein content, and the level of cell growth regulators such as phosphorylated ribosomal protein S6, in tandem in single cells. Measurements were made on large numbers of cells drawn from proliferating populations of both non-transformed and cancerous cells. Analysis of the joint distribution of cell size and cell cycle position revealed a control mechanism that restricts cells to a specified size range at several points in the cell cycle. Combining our measurements with live-cell imaging showed that this restriction is the result of a negative correlation between cell size and subsequent growth rate, indicating that cells can sense their own size and modulate their growth accordingly. We also observed cell-size-dependent adjustments of cell cycle length, which further reduced size variability. We then identified drugs that change the mean cell size without disrupting the cell-autonomous control mechanism, as well as drugs that weaken the size-dependence of either growth rate or cell cycle progression and increase cell-to-cell size variability. In particular, long- and short-term drug treatments revealed that mTORC1 inhibition decouples the rate of cell growth from cell size, impairing the efficiency of cell size specification and increasing size variability. Our measurements of mTORC1 activity as a function of size and cell cycle position suggest that mTORC1 assumes its role in growth control following S-phase entry. Although mTORC1 activity, assayed by levels of mTORC1-target phosphorylation, increases abruptly during G1, it becomes correlated with cell size only upon G1 exit. The mTORC1 inhibitor rapamycin preferentially inhibits growth in S-phase cells. Taken together, these results indicate that the mTOR pathway maintains size homogeneity by stimulating growth in a cell-size-dependent manner after G1 exit. The screening and analysis methods developed here will be used to further elucidate the cellular size-control mechanism. |
