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Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins

Nature Cell Biology volume 18, pages 676–683 (2016)Cite this article

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Abstract

When tagged with a fluorescent protein, actin is not fully functional1, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells2. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP–LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP–LifeAct and LifeAct–mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct–mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct–mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.

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Figure 1: Effects of mEGFP–LifeAct on endocytosis in fission yeast cells.
Figure 2: Effects of mEGFP–LifeAct on cytokinesis.
Figure 3: LifeAct–fluorescent-fusion-protein binding to actin filaments and effects on barbed end elongation.
Figure 4: Nanomolar concentrations of LifeAct–mCherry inhibit filament severing by cofilin.
Figure 5: Quantitative analysis of actin filaments during maturation and constriction of contractile rings.

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Acknowledgements

The research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM026338. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank C. Lamoureux, J. Johnson and C. McGuinness for carrying out preliminary experiments for this project.

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Author notes

  1. Naomi Courtemanche & Qian Chen

    Present address: Present addresses: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA (N.C.); Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606, USA (Q.C.).,

  2. Naomi Courtemanche and Qian Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA

    Naomi Courtemanche, Thomas D. Pollard & Qian Chen

  2. Departments of Molecular Biophysics and Biochemistry, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA

    Thomas D. Pollard

  3. Department of Cell Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA

    Thomas D. Pollard

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Contributions

N.C., Q.C. and T.D.P. designed the experiments. N.C. performed the biochemistry experiments. Q.C. performed the experiments in yeast. N.C., Q.C. and T.D.P. analysed the data. N.C., Q.C. and T.D.P. wrote the paper.

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Correspondence to Thomas D. Pollard.

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Integrated supplementary information

Supplementary Figure 2 Lifeact-mCherry does not affect the time course of spontaneous actin assembly, but decreases the critical concentration.

(a, b) Normalized time courses of spontaneous assembly of (a) 1.5 μM and (b) 4 μM actin monomers (20% pyrene-labelled) in the absence and presence of a range of concentrations of Lifeact-mCherry. Bulk assembly assays were repeated in 3 independent experiments. (c) Pyrene-actin fluorescence following overnight incubation in polymerization conditions in the absence (black) or presence (red) of 1.5 μM Lifeact-mCherry. Data sets were not normalized and were repeated in 3 independent experiments. Lines are linear fits to the data. (d) Unpolymerized actin as a function of the concentration of Lifeact-mCherry. Samples of 3 μM actin were polymerized with a range concentrations of Lifeact-mCherry. After pelleting filaments supernatant fractions were run on SDS-PAGE, stained with Coomassie blue and band intensities analysed using ImageJ. Data represent 1 out of 3 independent experiments. (e) Dependence of the concentration of additional nucleated filaments required to increase the polymerization rate three-fold on the time it takes each filament to anneal. Concentrations of nuclei were calculated using the length-dependent annealing rate measured by Andrianantoandro et al. 20 and assuming that nucleated filaments elongate with normal rate constants (that is, k+ = 12.9 μM−1 s−1, k− = 1.3 s−1) before annealing together. This experiment demonstrates that subnanomolar to low nanomolar concentrations of nucleated filaments are sufficient to increase the elongation rate of growing filament ends. Data represent the results of a single simulation, which was performed once.

Supplementary Figure 3 Fits to Lifeact-mCherry binding data in the presence of 30 μM Hs cofilin and to barbed end elongation rates.

(a) Binding of a range of Lifeact-mCherry concentrations to 3 μM actin filaments in the presence of 30 μM Hs cofilin (same data as Fig. 4c, which was produced from 3 independent experiments). Pellets were run on SDS-PAGE and band intensities were corrected for molecular weight to determine molar ratios of Lifeact-mCherry to actin. The band intensities were fit with a standard bimolecular binding curve (black dashed line, Kd = 114 μM) and a cooperative binding curve (red line). The solid line is a fit of the binding equation to the Lifeact-mCherry data (Kd = 67 μM and cooperativity factor ω = 8.8). (b) Barbed end elongation rates of actin filaments polymerized in the presence of 0.5 μM actin monomers and micromolar concentrations of Lifeact-mCherry (same data as Fig. 5b, which was produced from 4 independent experiments). Data were normalized with respect to the elongation measured in 100 μM Lifeact-mCherry and fit with a bimolecular binding curve (red line).

Supplementary Figure 4 Comparison of two methods to count the maximum number of mEGFP-Lifeact in contractile rings.

The graph compares counts of peak numbers of mEGFP-Lifeact in 51 cells with the segmentation method on the X-axis and the patch subtraction method on the Y -axis. The red line is the best linear fit of the data (R2 = 0.71). The mEGFP-Lifeact counts are about 50% higher with the patch subtraction method owing to actin filaments in whiskers and cables adjacent to the rings. Data were pooled across 6 independent experiments.

Supplementary Table 1 Comparison of two actin structures in fission yeast.
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Courtemanche, N., Pollard, T. & Chen, Q. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat Cell Biol 18, 676–683 (2016). https://doi.org/10.1038/ncb3351

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