mTOR controls transferrin uptake by regulation of the number of transferrin receptors per vesicle and not endocytosis or recycling rates. (a) Effect of rapamycin on the time-course of fluorescent transferrin endocytosis (left) and recycling (right). Means ± standard error of the mean (SEM; n = 4 replicates). (b) Lack of effect of rapamycin on the rate constants for endocytosis and recycling. The rate constants, tau-1, were calculated from a single exponential fit of the time-course data. (c) Effect of the PtdIns(3,4,5)P3-mTOR-targeting d-siRNAs on the number of transferrin receptors (TFRC) per cell (measured by immunofluorescence on permeabilized cells). Means ± SEM (n = 4). (d) Correlation between transferrin uptake (F, vertical axis) and transferrin receptor numbers (immunofluorescence measurements on permeabilized cells, horizontal axis) in HeLa cells in which PtdIns(3,4,5)P3-mTOR regulators were silenced. Means ± SEM (n = 4). (e) Selective effect of rapamycin on transferrin receptor staining (TFRC) compared to tubulin and actin staining. Means ± SEM (n = 6 replicates). (f) Rapamycin preferentially reduces the number of transferrin molecules per vesicles. Histogram of the relative transferrin fluorescence intensity per vesicle for control (CTR) and rapamycin (RAPA)-treated cells. Means ± SEM (n = 6 replicates). Inset shows the average intensity measured for the 30 brightest vesicles per cell. (g) Rapamycin has only a minor effect on the number of endocytic vesicles. Bar diagram shows means ± SEM (n = 6 replicates).