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Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

Nature Cell Biology volume 23, pages 147–159 (2021)Cite this article

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A Publisher Correction to this article was published on 02 March 2021

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Abstract

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

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Fig. 1: KO of twinfilins leads to F-actin accumulation in the lamellipodia and perinuclear region.
Fig. 2: Twinfilin regulates actin dynamics in lamellipodia.
Fig. 3: Twinfilin does not accelerate barbed-end depolymerization of ADP-actin filaments.
Fig. 4: Twinfilin and CP display different localizations and dynamics in lamellipodia.
Fig. 5: Twinfilin regulates CP dynamics.
Fig. 6: Twinfilin accelerates filament barbed end uncapping in vitro.
Fig. 7: The actin-binding function of twinfilin is required for filament uncapping.
Fig. 8: Localization of twinfilin in lamellipodia and its role in regulation of CP dynamics.

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Data availability

Examples of all FRAP experiments over 5–9 time points as well as Excel spreadsheets summarizing the measured fluorescence intensity values in each cell are available at FigShare (https://doi.org/10.6084/m9.figshare.13221638.v1). Other original data of the study are available on reasonable request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank V. Paavilainen and J. Saarikangas for reading the manuscript. We acknowledge staff at the Institute of Biotechnology Light Microscopy Unit, Biomedicum Imaging Unit and Institute of Molecular Medicine Finland High Content Imaging and Analysis Unit for providing support in imaging and image analysis. We thank M. Tirkkonen for technical assistance. This study was supported by grants from the Academy of Finland (no. 302161) and Cancer Society Finland (no. 4705949; to P.L.); the Doctoral School in Health Sciences at the University of Helsinki (to M.H. and T.K.) and from the Agence Nationale de la Recherche (Grant Muscactin; to G.R.-L.) and from the European Research Council (no. StG-679116; to A.J.).

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Authors and Affiliations

  1. Institute of Biotechnology and Helsinki Institute of Life Sciences, University of Helsinki, Helsinki, Finland

    Markku Hakala, Mari Tolonen, Tommi Kotila & Pekka Lappalainen

  2. Université de Paris, CNRS, Institut Jacques Monod, Paris, France

    Hugo Wioland, Antoine Jegou & Guillaume Romet-Lemonne

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Contributions

P.L. and M.H. crafted the original idea, and M.H., H.W., T.K., A.J., G.R.-L. and P.L. designed the experiments. M.H., M.T., T.K., A.J. and H.W. performed the experiments, and M.H., A.J. and H.W. analysed the data. M.H. and P.L. drafted the manuscript with contributions from all of the authors. P.L., A.J. and G.R.-L. acquired the funding.

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Correspondence to Pekka Lappalainen.

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Extended data

Extended Data Fig. 1 Generation of twinfilin-1, twinfilin-2, and twinfilin-1/twinfilin-2 knockout B16-F1 cells.

a-b, Western blot analysis of wild-type and twinfilin knockout cells performed with anti-TWF-1 (panel a) and anti-TWF-2 (panel b) antibodies. Anti-α-tubulin antibody was used as a loading control. c, Sanger sequencing of the third exon of twinfilin-1 gene from genomic DNA of wild-type and twf1/twf2-KO g3 knockout cell lines. Sequences obtained with forward and reverse primers for the third exon of twinfilin-1 are shown. d, Sanger sequencing of the third exon of twinfilin-2 from genomic DNA of wild-type and twf1/twf2-KO g3 knockout cell lines. Sequences with forward and reverse primers for the third exon of twinfilin-2 are shown. e-f, MiSeq NGS sequencing results of twinfilin-1 exon 3 (panel e), and twinfilin-2 exon 3 (panel f) of wild-type and twf1/twf2-KO-g3 cells. Due to chromosome amplification, twinfilin-1 knockout was a result of two allele variants and twinfilin-2 knockout a result of four allele variants. All mutant alleles lead to premature stop-codons in the region encoding twinfilins’ N- terminal ADF-H domain. Numbering of nucleotide and amino acid sequences represent the first nucleotide and amino acid residues shown, respectively. All Western blot experiments were repeated three times. Unprocessed Western blots are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Knockout of twinfilins does not affect cellular levels of actin and actin-binding proteins.

Identical amounts of lysates of wild-type and twf1/twf2 KO B16-F1 cells were loaded on SDS-PAGE gels and protein levels were determined with Western blot. a, Levels of β-actin. Anti-α-tubulin antibody was used as a loading control. We note that anti-β-actin antibody remained in the blot even after vigorous stripping of the membrane and was thus visible also in anti-α-tubulin detection. Protein levels of b, Capping Protein, c, cofilin-1, d, profilin-1, e, cyclase-associated protein-1, and f, CARMIL-1. Anti-α-tubulin or anti-histone H3 antibodies were used as a loading controls. All Western blot experiments were repeated three times, except in panel c where experiment was repeated two times. Unprocessed Western blots are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region.

a, Representative examples of phalloidin staining of wild-type B16-F1, and twf1/twf2-KO cells after 7.5 min uptake of 20 µg/ml AlexaFluor-647 transferrin. Scale bar = 20 µm. The dotted square indicates the perinuclear region magnified in the insert. b, Pearson’s correlation analysis of AlexaFluor-568 phalloidin and AlexaFluor-647 transferrin. Each data point represents individual cell (n = 19 for wild type and n = 21 for knockout cells) from one experiment with mean and standard deviations shown. c, Normalized F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 µg/ml AlexaFluor-647 phalloidin. The median, 25th and 75th percentiles and the minimum and maximum values of the data are shown. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158 from two experiments. Statistical source data are provided in Source Data Extended Data Fig. 3.

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Extended Data Fig. 4 Twinfilin slows down the barbed end depolymerization of ADP-actin filaments anchored to the glass bottom of open chambers.

The depolymerization of individual ADP-actin filaments was recorded during 8 minutes in the presence or absence of 2 µM TWF1 in F-buffer (see Methods). Filament surface anchoring induces pauses during depolymerization that need to be excluded from the analysis in order to quantify filament depolymerization rates. We note that the presence of twinfilin reduces the frequency of observable pauses. a, Depolymerization rate measurements excluding pauses are in agreement with measurements performed on single filaments in microfluidics experiments shown Fig. 3b (n = 30 filaments for both conditions, N = 2 biological repeats). The median, 25th and 75th percentiles and the minimum and maximum values of the data are shown. b, The global depolymerization rates, including pauses, are lower and very similar to the values reported from previous experiments in open chambers assays43,48,49 (n = 30 filaments for both conditions, N = 2 biological repeats). The median, 25th and 75th percentiles and the minimum and maximum values of the data are shown. c, Two representative kymographs showing the depolymerization of actin filaments in buffer (left), exhibiting a 3-minute-long pause, or in the presence of 2 µM TWF1 (right). d, Depolymerization of 3.4 µM actin filaments (5% pyrene-label) capped at their pointed ends with 0.5 µM tropomodulin-1 was followed by decrease in pyrene-actin fluorescence. In absence of TWF1 (black squares), no depolymerization was observed. Addition of 7 µM C-terminal ADF-H domain of TWF1, which binds ADP-actin monomers with nanomolar affinity, induces filament barbed end depolymerization (magenta circles) due to ADP-actin monomer sequestration. In the presence of 7 µM full-length TWF1, a slower actin filament depolymerization was detected (cyan triangles), indicating that full-length TWF1 slows down actin depolymerization by associating with filament barbed ends. A representative example from two independent experiments is shown. Statistical source data are provided in Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 CARMIL-1 increases CP dynamics in vitro.

a, Survival fraction of capped filament barbed ends, exposed to 1 µM G-actin with either 0 or 0.5 µM twinfilin, and stabilized by phalloidin or not. Survival fractions measured in depolymerization conditions are shown (from Fig. 6b) as comparison. Following label order, n = 480, 170, 32, 46, 31, 50 filaments from N = 8, 3, 1, 1, 1, 1 experiments. Filaments were exposed to rhodamine-phalloidin throughout the experiment, and were estimated to be >70% phalloidin-saturated, based on rhodamine fluorescence intensity. Fraction of uncapped filament barbed ends are shown as thick lines with 95% confidence intervals as shaded surfaces and with single exponential fits as thin lines. b, CARMIL and TWF1 accelerate uncapping in vitro, CARMIL being more efficient. Intermediate uncapping efficiency was detected, when both proteins were included in the assay. In the same order as figure labels, n = 480, 170, 60, 120, 120 filaments from N = 8, 3, 1, 2, 2 experiments. Fraction of uncapped filament barbed ends are shown as thick lines with 95% confidence intervals as shaded surfaces and with single exponential fits as thin lines. Statistical source data are provided in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 CARMIL-1 cannot rescue CP localization and dynamics in twf1/twf2-knockout cells.

a, Representative examples of localization of EGFP-CARMIL1 in B16-F1 wild-type and twf1/twf2-KO cells. Scale bar = 10 µm. b, Line profile analysis of EGFP-CARMIL1 localization compared to AlexaFluor-568-phalloidin staining. Data represent mean of n = 13 (wild type) and n = 14 (knockout) cells from two experiments with error bars representing standard deviation. c, CARMIL-1 was depleted by siRNA in twf-1/twf-2 knockout cells, and the protein levels were detected by Western blot with anti-CARMIL-1 antibody. Histone H3 antibody was used as a loading control (left). Representative Western blot and immunofluorescence images from two experiments of twf1/twf2 KO and twf/twf2 KO + CARML-1 siRNA cells (right). Scale bar = 10 µm. d, Representative example of FRAP experiments of EGFP-CP dynamics in twf-1/twf-2 knockout cells over-expressing mCherry-CARMIL-1 (left). Scale bars = 5 µm. Recovery of EGFP-CP in knockout cells over-expressing mCherry-CARMIL-1 was compared to EGFP-CP recovery in wild-type and twf-1/twf-2 KO B16-F1 cells (data from Fig. 5) (right). Data represent the mean of n = 13 measurements from two experiments with shaded surfaces indicating standard deviations. Halftime of EGFP-CP fluorescence recovery in twf-1/twf-2 knockout cells overexpressing mCherry-CARMIL-1 was 14.58 s. Statistical source data are provided in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 Overexpression of cofilin-1 does not rescue CP dynamics in twinfilin-deficient cells.

a, Representative example of EGFP-CP dynamics in lamellipodia of twf-1/twf-2 knockout cells overexpressing mCherry-cofilin-1 examined by a FRAP assay. Scale bars = 5 µm. b, Analysis of FRAP data from the mean of n = 9 individual measurements from two experiments with shaded surfaces indicating standard. Data of B16-F1 wild-type cells and twf-1/twf-2-knockout cells is the same as in Fig. 5. Half-time of EGFP-CP fluorescence recovery in twf-1/twf-2 knockout cells overexpressing mCherry-cofilin-1 was 20.95 s for bound fraction and 0.24 s for diffuse fraction. We note that longer observation time (60 s, see Fig. 5) is required for EGFP-CP recovery to reach the plateau. c, Western blot analysis of Cherry-cofilin-1 overexpression in twf1/twf2-KO B16-F1 cells. Anti-cofilin-1 antibody detection is shown. Based on band intensities in two experiments, we estimate 3-fold overexpression of cofilin-1 to normal levels. Unprocessed Western blot and statistical source data are provided in Source Data Extended Data Fig. 7.

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Supplementary information

Reporting Summary

Supplementary Video 1

Lamellipodia protrusion of WT (left) and Twf1/Twf2-KO (right) B16-F1 cells on laminin-coated glass-bottom dishes imaged using DIC microscopy.

Supplementary Video 2

Representative examples of random migration of WT and Twf1/Twf2-KO-g3 B16-F1 cells imaged using phase-contrast microscopy. The tracks of cells used in the analysis are indicated by lines. Please note that tracking was stopped when cells divided or collided with each other.

Supplementary Video 3

Fluorescence recovery of eGFP–actin after photobleaching in WT (left) and Twf1/Twf2-KO (right) B16-F1 cells.

Supplementary Video 4

Fluorescence decay of PA–GFP–actin (left) after photoactivation in WT (top) and Twf1/Twf2-KO (middle) B16-F1 cells, and in Twf1/Twf2-KO cells expressing mCherry–TWF1 (rescue; bottom). Lamellipodia were marked with either mCherry–LifeAct or mCherry–TWF1 expression (right).

Supplementary Video 5

Fluorescence recovery of eGFP–TWF1 in a WT B16-F1 cell.

Supplementary Video 6

Fluorescence recovery of eGFP–CP in a WT B16-F1 cell.

Supplementary Video 7

Fluorescence recovery of eGFP–CP in a Twf1/Twf2-KO B16-F1 cell.

Supplementary Video 8

Fluorescence recovery of eGFP–CP (left) after photobleaching in WT (top) and Twf1/Twf2-KO (bottom) B16-F1 cells. Endosomal F-actin structures were visualized with coexpression of mCherry–LifeAct (right).

Supplementary Video 9

Fluorescence recovery of eGFP–CP (left) after photobleaching in Twf1/Twf2-KO B16-F1 cells overexpressing mCherry–CARMIL-1 (right).

Supplementary Video 10

Fluorescence recovery of eGFP–CP (left) after photobleaching in Twf1/Twf2-KO B16-F1 cells overexpressing mCherry–cofilin-1 (right).

Supplementary Video 11

Fluorescence recovery of eGFP–CP (left) after photobleaching in Twf1/Twf2-KO B16-F1 cells coexpressing mCherry–TWF1 WT (right).

Supplementary Video 12

Fluorescence recovery of eGFP–CP (left) after photobleaching in Twf1/Twf2-KO B16-F1 cells coexpressing mCherry–TWF1 tail mutant (F323A/K325A/K327A) (right).

Supplementary Video 13

Fluorescence recovery of eGFP–CP (left) after photobleaching in Twf1/Twf2-KO B16-F1 cells coexpressing mCherry–TWF1 ADF-H-domain mutant (R96A/K98A/R267A/R269A) (right).

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Unprocessed western blots.

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Hakala, M., Wioland, H., Tolonen, M. et al. Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks. Nat Cell Biol 23, 147–159 (2021). https://doi.org/10.1038/s41556-020-00629-y

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