Cytosolic concentrations of actin binding proteins and the implications for in vivo F-actin turnover

Figures

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  Figure S1.. Strategy for quantifying cellular concentrations of S. cerevisiae ABPs. (A) Example standard curve for quantifying the cellular abundance of actin. Signals (AU) on immunoblots for known quantities of purified actin were plotted (filled triangles) to generate a standard curve. Signals from different quantities of total cell lysate on the same immunoblot were plotted (open circles) to determine the amount of actin in the lysates (ng per µg total protein). (B) Calculation of the cytosolic volume of a haploid S. cerevisiae cell using known values for the weight and density of a haploid yeast cell (Baldwin and Kubitschek, 1984; Sherman, 2002). (C) Standard curve used to estimate the cellular concentrations of Pfy1-GFP and Abp140-GFP, since we did not have reliable antibodies for Pfy1 or Abp140. For eight ABPs, confocal microscopy was used to determine total ABP-GFP concentrations in cells. These values were plotted against cellular concentrations determined by quantitative Western blotting (as in B; also see Materials and methods). Linear regression was used to generate the line shown, which has an equation (y = 102× +35.22) from which we estimated the cellular concentrations of Pfy1…

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  Figure 1.. Cellular concentrations of S. cerevisiae actin and ABPs. (A) Equation used to calculate cellular concentrations of actin and ABPs (example shown is for actin). The starting value, actin abundance in cells (ng/µg) comes from quantitative immunoblotting (Fig. 1, A and B; and Table 1). The µg of total protein in a haploid S. cerevisiae cell is known (Johnston et al., 1977; Baroni et al., 1989). The cytosolic volume was calculated in Fig. S1 B. (B) Graph comparing the total cellular concentrations of actin plus 14 ABPs (mean ± SD). Abundances of actin and 11 of the ABPs were determined by quantitative immunoblotting as described in A. Asterisks denote the other three ABPs (Pfy1, Tpm2, and Abp140), for which antibodies were not available for immunoblotting. Therefore, the total cellular concentrations of these three proteins were calculated by GFP-tagging and comparison to GFP-tagged ABPs of known quantity (see Materials and methods). The sample size for each Western blot in descending order (excluding proteins marked with an asterisk): 11, 6, 3, 2, 5, 2, 7, 6, 2, 8, 4, 3 (also listed in Table 1). (C) Landscape view of S. cerevisiae actin and ABP quantities and their interactions. The…

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  Figure S2.. Comparison of total ABP cellular concentrations quantified by Western blotting versus GFP fluorescence. (A) Graph comparing the total cellular concentrations of 12 different ABPs. Western blot data (mean ± SD) are from multiple experiments (as listed in Table 1 and Fig. 1 B). GFP data shown (mean ± SD) are from three independent experiments (n > 20 cells per experiment), with each data point representing one cell. GFP intensity values were converted to cellular concentrations (µM) as in Fig. S1 D. (B) Table comparing ABP cellular concentrations determined by Western blotting (this study), GFP fluorescence (this study), and mass spectrometry (Ho et al., 2018).

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  Figure S3.. Correlation between cellular levels of GFP-tagged ABPs in yeast strains with or without additional Arc15-mScarlet marker. (A) Graph comparing the mean GFP fluorescence intensity values for yeast strains co-expressing Arc15-mScarlet and the GFP-tagged ABP (y-axis) and yeast strains expressing only the GFP-tagged ABP (x-axis). For each cell, an average intensity projection was generated, giving the mean GFP fluorescence intensity for that cell. Data from all cells were averaged to give the values graphed (circles). For strains co-expressing Arc15-mScarlet (n > 80 cells per condition) and for strains not expressing Arc15-mScarlet (n > 35 cells per condition). (B) Table showing the average GFP fluorescence values for the cells and the number of cells analyzed.

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  Figure 2.. Strategy for quantifying distributions of ABPs between cytosol and cellular F-actin structures. (A) Scheme for ABPs that localize to F-actin patches. Yeast strains co-expressing an actin patch marker (Arc15-mScarlet) along a GFP-tag on the ABP of interest were imaged by confocal microscopy, taking optical sections through the entire cell. The mean fluorescence intensity of each ABP-GFP fusion was quantified for each cell. Then the patch marker was used to generate a “mask,” which was superimposed on the ABP-GFP image. The “unmasked” region was defined as the cytosol and its mean intensity was measured and used to calculate the fraction of the total cellular ABP-GFP signal in the cytosol. (B) Scheme for ABPs that localize to actin cables. Same as in A, except strains used coexpress a cable marker (Lifeact-mScarlet) along with an ABP of interest tagged with mNeon. (C) Large changes in the threshold value used to make F-actin masks lead to only small changes in the percentage of GFP signal in the cytosol. Shown are patch masks obtained using three different threshold values overlaid on the ABP-GFP image. The “set point” (center) is the threshold value used for generating masks of…

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  Figure 3.. Distributions of ABPs between the cytosol and F-actin structures. (A) Representative average intensity projections from confocal imaging of cells expressing fluorescently tagged patch or cable ABPs. The top panel shows cells expressing fluorescently tagged ABPs with a range of different intensities, and due to their different cellular abundances; images were acquired and processed identically. Bottom panel shows the same cell images but with the brightness adjusted to improve visualization of the fluorescence distribution between cytosol and F-actin structures. Note these strains also express either a patch marker (Arc15-mScarlet) or a cable marker (Lifeact-mScarlet) for generating an F-actin mask (see strategy in Fig. 2). (B) Percentage of GFP signal in the cytosol (not bound to F-actin) for each ABP (mean ± SD). Data are from three independent experiments (n > 20 cells per experiment). Each data point represents one cell. Total number of cells analyzed for each ABP (left to right): 96, 98, 95, 96, 102, 116, 112, 122, 113, 99, 137, 92, 112. (C) Cytosolic concentrations (mean ± SD) of each ABP in descending order, calculated by multiplying the cellular concentration of each ABP…

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  Figure S4.. Cell-to-cell variance in ABP-GFP fluorescence levels. (A) Total ABP-GFP signal for actin patch and cable proteins (mean ± SD). Data shown are from three independent experiments (n > 20 cells per experiment), with each data point representing one cell. Total number of cells analyzed for each ABP (left to right): 117, 116, 112, 96, 92, 99, 136, 102, 112, 113, 122, 98, 96, 102. Note that the actin cable proteins (Tpm1, Tpm2, and Abp140) were imaged on a different microscope from the patch proteins, which was better suited for imaging cables. Therefore, the intensities of the cable ABPs cannot be directly compared to the intensities of the patch ABPs. (B) Data from A normalized to the mean of each strain in order to more accurately assess variance. (C) Table showing the total fluorescence signal (mean ± SD) and the coefficient of variation (CV) for each ABP.

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  Figure 4.. Cytosolic concentrations of ABPs show no major fluctuations over the cell cycle. For each ABP, the data from Fig. 3 B, showing the percentage of ABP in the cytosol for each individual cell, was replotted against the area (µm2) of that cell. Linear regression analysis was used to correlate cytosolic concentration of ABP with cell area (µm2) within the population.

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  Figure S5.. Total cellular levels of ABPs do not change significantly over the cell cycle. For each ABP, the total GFP fluorescence value in a cell was plotted against the area (µm2) of the same cell. Then, linear regression analysis was used to correlate the total cellular concentration of ABP with increasing cell area (µm2) within the population.

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  Figure 5.. Cartoon showing cytosolic concentrations of ABPs available to regulate the formation and turnover of F-actin patches and cables. Cartoon depicting cytosolic ABPs (circles) and their interactions with cellular F-actin structures: cortical patches (above) and cables (below). ABPs have been spatially organized to highlight their known effects on F-actin assembly, organization, remodeling, and disassembly. The area of each ABP circle is proportional to its cytosolic concentration listed in Table 1. Cytosolic ABPs are color-coded to match ABPs decorating F-actin networks (with shapes that reflect their known structures). Actin cables are shown decorated by bundlers (light gray) of unknown identity. Bidirectional arrows indicate the rapid exchange of ABPs between F-actin structures and cytosol (Lacy et al., 2019). Dashed lines between circles represent known physical interactions. Concentrations of select ABPs are shown as points of reference. For Cof1, we could not directly measure its cytosolic fraction, and instead it was estimated operating under the assumption that it falls within the range of the other ABPs (61–91% in the cytosol). Since profilin (Pfy1) binds specifically to…