Extended Data Fig. 5: Kinetic analysis of Pi release from actin filaments and dynamics of actin variants in yeast.
From: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments
a, Time courses of the normalized fluorescence intensity of 30 µM MDCC-PBP (Pi sensor) from 10 µM N111S actin containing either 1% (black) or 10% (red) wild-type, pyrene α-actin, seeded with 100 nM spectrin-actin seeds after initiation of polymerization (t = 0s). b, Characteristic half-times of polymerization for indicated actin variants as determined from mono-exponential fits to the observed polymerization time-courses (Fig. 3b). Dots are values from individual experiments, whereas lines represent the mean from three experiments. Error bars are SD. c, Semi-logarithmic plot of simulated Pi release reaction kinetics (gray to black) depending on the enhancement of Pi release rate constant (x-fold over wild-type actin as indicated) compared to the observed time courses of polymerization (cyan) and of Pi release (red) for N111S actin. d, Apparent rates of Pi release, obtained from mono-exponential fits of the simulated data shown in c as a function of the enhancement of Pi release rate constant (x-fold over wild-type actin as indicated). The cyan and red areas indicate the observed apparent rates of polymerization (cyan) and Pi release (red) with the center of the error bands (dark colors) being the average from three experiments and the error bands (light colors) representing SD. Note that an enhancement of the Pi release rate constant by at least 15-fold is required for the apparent rate of Pi release to fall within the error margin of the observed polymerization rate. e, Effect of varying window sizes on the average prediction error of the three fit parameters from the kinetic model. The average prediction error is large (>10%) for small and large window sizes with a shallow optimum in between (grey shaded region, size 17 to 29). At smaller window sizes the prediction error is large as a result of a higher variance in calculated velocities and for larger window sizes the prediction error increases with decrease in the number of data points available for fitting. f, Exemplary fits (grey dashed lines) calculated from velocities (black points) obtained with different window sizes from within (shaded grey, size 20) and flanking (size 14 and 30) the optimum. The exponential decay function used for these fits and their fit parameters are provided in the bottom-left and top-right parts of each sub-plot respectively. g, Cumulative distribution probability of pauses as a function of time from the onset of depolymerization. Blue line indicates the fraction of tracked filaments that have paused at any given time and the black line is obtained by fitting an analytic model of pause probability (described previously in ref. 70) as a function of time. The model is described by the equations in the bottom-right corner of the plot. From this fit we obtained for the fit parameter \(\omega\) (protomer transition rate) a value of 5.8 × 10−7 s−1. h, Estimation of lower bounds for the phosphate release rate of N111S mutant. Rates were calculated by fitting exponential decay functions to the observed depolymerization velocity data, so that the observed velocities converged to the mutant’s vdepol,ADP and the vdepol,ADP-Pi rate was either assumed to be equal (high estimate, blue) or twice that of the wild type (low estimate, turquoise). The latter assumption was motivated by the observation that the vdepol,ADP rate of N111S mutant was about 2.2 times that of wild-type actin (Fig. 5d). i, Time-lapse series of confocal microscopy images showing Lifeact-mCherry fluorescence every 5 seconds for actin path detection in yeast cells expressing either wild-type or N111S-actin. The white arrow highlights an exemplary dynamic actin path. The scale bar is 4 µm. The shown images of yeast cells are example images from a total of eight cells per strain. Four independent colonies per strain were imaged. The full experiment was performed in duplicate, obtaining similar results. j, Plot of endocytic actin patch lifespans per strain. Each color dot represents one patch. The average lifespans are 12 s ± 7 s (wild-type actin) and 14 s ± 7 s (N111S-actin). Error bars correspond to mean ± standard deviation. Sample size corresponds to n = 86 patches for WT and n = 89 patches for N111S, from a total of eight cells per strain from four independent colonies. There are no large differences in actin-patch lifespans between the two yeast strains.
