Figure 1. Effects of mEGFP-Lifeact on endocytosis in fission yeast cells.
The 3nmt1 promoter controlled the expression of mEGFP-Lifeact. (a) Fluorescence micrograph of a field of cells (pseudo-colored) grown in 5 μg/ml thiamine to induce expression of mEGFP-Lifeact. Lifeact labels actin patches (circle), contractile rings (arrow) and actin cables (arrowhead). This wide range of expression of mEGFP-Lifeact was observed in >10 independent experiments. (b) Distribution of the cytoplasmic mEGFP-Lifeact concentrations expressed by 325 cells across 4 independent experiments. (c) Growth of dilution series of wild type cells and the cells expressing mEGFP-Lifeact at 25°C under either repressing (+ thiamine) or inducing (− thiamine) conditions, repeated in two independent experiments. (d) Fluorescence micrographs of fixed cells stained with Alexa568-phalloidin. (Left) pseudo-colored mEGFP fluorescence. Asterisks: cells expressing low levels of Lifeact. (Middle and Right) Alexa568-phalloidin fluorescence. Staining with rhodamine-phalloidin or Bodipy-phallacidin produced similar results. (e) Fluorescence micrographs of a cell expressing mEGFP-Lifeact. Upper: circles mark seven actin patches used for measuring fluorescence over time. Lower: time course in seconds of the appearance and disappearance of the first patch. Observations were similar in >100 cells across 3 independent experiments. (f) Time courses of average numbers of mEGFP-Lifeact molecules in actin patches of four cells expressing mEGFP-Lifeact at the indicated cytoplasmic concentrations including the cell shown in e (arrowhead in f). Data show the mean of 4 independent experiments. (g) Dependence of the time for mEGFP-Lifeact fluorescence to peak and to disappear (insert) in patches on the cytoplasmic concentration of mEGFP-Lifeact. The gray dots are average times of 5 or more patches in a cell. The blue dots are the average times in bins of 50 μM mEGFP-Lifeact. The red lines are the best linear fits of the binned data points. Data were obtained from n=20 independent experiments.(h) Dependence of the average peak number (blue circles) of mEGFP-Lifeact molecules per actin patch (> 3 patches/cell) on the concentrations of free mEGFP-Lifeact (n = 16 cells). Data were obtained and pooled across 15 independent experiments. The red curve is the best fit of Equation 1 (see Methods) to the data giving a Kd of 20.0 μM. Inset: best fit with constraining the Kd at 9.3 μM (insert). All the error bars = mean ± 1 standard deviation. All scale bars are 5 μm.