Right here we varied the transformation and parameter. the next cycles.(TIF) pbio.1001788.s001.tif (3.4M) GUID:?DCFF8BCA-C3DF-47BE-9451-A707DE2A136F Body S2: pT161CCdk1 levels correlate very well with cyclin B1 levels, linked to Body 2 . (A) Organic pictures for quantitative Traditional western blots for pT161CCdk1 and cyclin B1. Examples extracted from embryos in the next and initial cycles are shown. The red container highlights examples of unfertilized eggs utilized being a titration series to check on for linearity from the Traditional western blot. For the embryos used within the initial cell routine, the proper time labeled corresponds to time after fertilization. For the embryos used within the next cell routine, the proper time labeled corresponds to enough time following the first division. (B) Relationship of cyclin B1 amounts with pT161 amounts. The same development line matches data from interphase (cyclin B1 levels increasing, blue points) and M-phase (cyclin B1 levels decreasing, red points). (C) The same trend line also fits data from the first cycle (blue points) and the second and third cycles (red points). The correlation coefficient egg and embryo extracts were prepared as described in Materials and Methods. One aliquot of the egg extract was treated with the MEK inhibitor U0126 (130 M). Samples were collected at various times after taking the extracts off ice. The extracts reached interphase after 30 min at room temperature, at which point most of the endogenous cyclin B1 had been degraded (not shown). 65Ccyclin B1 (50 nM) was BAY 293 then added to drive the extracts into mitosis, and samples were taken at various times for immunoblotting with a phospho-MAPK antibody.(TIF) pbio.1001788.s003.tif (458K) GUID:?492E30D6-0E58-47F9-87E3-DB749998972B Physique S4: Estimating the reduction of the Wee1/Cdc25 ratio during the transition between the first cycle and the subsequent cycles, related to Physique 5 . (A, B) Inferring the change in from the amplitude of pY15CCdk1 oscillations. (A) Time courses of pY15CCdk1 oscillations in the first four cell cycles, from two impartial experiments. (A, inset) Modeled relationship between and the amplitude of pY15CCdk1 oscillations. (B) Approximate amplitude of pY15CCdk1 oscillations as a function of cell cycle number. falls by 8-fold. (C, D) The relationship between cyclin B1 levels and Cdk1 Y15 phosphorylation during the first four interphases. Assuming that Y15 phosphorylation equilibrates quickly relative to the changes in cyclin B1 levels, the relationship between pY15CCdk1 and cyclin B1 is usually given by: Thus, when is small, it is approximately given by the slope of the pY15CCdk1 versus cyclin B1 plot (pY15CCdk1/cyclin B1). One plot showing cyclin B1 versus pY15CCdk1 for BZS two cell cycles is usually shown in (C). (D) Slope (pY15CCdk1/cyclin B1) versus cycle number. The slopes were normalized to the slope of the first cycle. The open circles represent the best fit values of the slopes, and the error bars correspond to one standard error. The dashed line corresponds to an 8-fold decrease of the slope, which is what BAY 293 we inferred from the pY15CCdk1 amplitudes in (B), and assumed in our computational BAY 293 model of the transition between the first cycle and the subsequent cycles.(TIF) pbio.1001788.s004.tif (790K) GUID:?51000DCF-1EC5-4E06-AB04-A5CA4D1D8B75 Figure S5: Modeled robustness and tunability in oscillators with strong and weak positive feedback, related to Figure 6 . (A, B) An alternative way of varying positive feedback strength. Here we varied the parameter and change. The red and blue curves show the calculated periods and amplitudes resulting from changes of each of the models other parameters. (E) Lack of tunability in the negative-feedback-only oscillator. As an example, the parameter tuned here is the APC inactivation rate constant still occurs on schedule.(TIF) pbio.1001788.s006.tif (527K) GUID:?1B1A484C-B424-4DEE-AB06-2C86B41E0CCB Movie S1: Time-lapse movies of embryos, the first mitotic cell cycle is long (85 min) and the subsequent 11 cycles are short (30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1.