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Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration

Nature Cell Biology volume 23, pages 1148–1162 (2021)Cite this article

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Abstract

Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.

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Fig. 1: WRC diffusion trapping at the lamellipodium tip correlates with its activation state.
Fig. 2: WRC displays lateral movements along the lamellipodium tip.
Fig. 3: WRC lateral movements are driven by actin filaments elongation and correlate with fast turnover.
Fig. 4: Mechanical forces exerted by physical contact with elongating actin filaments drive FMNL2 lateral movements.
Fig. 5: Characterization of a functional transmembrane WRC enabling optical tweezers in situ manipulation.
Fig. 6: WRC activation correlates with its mechanical connections to the lamellipodium tip.
Fig. 7: Positive correlation between WRC increased dwell time at the lamellipodium tip and rates of membrane protrusion.

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All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank B. Tessier, R. Sterling and J. Carrere for technical assistance, M. Garcia, M. Lagardère and T. Orré for helpful discussions, C. Poujol and S. Marais (Bordeaux Imaging Center, BIC) for technical help and F. Cordelières (BIC) for support with kymograph analysis (Kymo Tool Box). We thank E. Verdier and N. Retailleau for technical help (IINS Cell Biology Facility, grant no. ANR-10-LABX-43). We would also like to thank J. B. Sibarita (IINS) for his support with sptPALM analysis and B. Wehrle-Haller (University of Geneva) and T. Stradal (Helmholtz Centre for Infection Research) for providing GFP-K80-β3-integrin expression plasmid and anti-Nap1 antibodies, respectively. J. Lakins (Center for Bioengineering and Tissue Regeneration, UCSF) is gratefully acknowledged for his help in using the PiggyBac system as well as for providing the Piggybac inducible and Hyperactive transposase plasmids. We acknowledge financial support from the French Ministry of Research and CNRS, ANR grant Integractome (G.G.), ANR grant FastNano (G.G.), Ligue Contre le Cancer (A.M.), Conseil Régional Aquitaine (A.M.), Fondation pour la Recherche Médicale (G.G. and A.M.) and the Deutsche Forschungsgemeinschaft (K.R.). We also acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04).

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Authors and Affiliations

  1. University Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Amine Mehidi, Zeynep Karatas, Maureen Cercy, Matthieu Sainlos, Olivier Rossier & Grégory Giannone

  2. Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Braunschweig, Germany

    Frieda Kage, Matthias Schaks & Klemens Rottner

  3. Department of Cell Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany

    Frieda Kage, Matthias Schaks & Klemens Rottner

  4. CNRS, Ecole Polytechnique, Institut Polytechnique de Paris, Palaiseau, France

    Anna Polesskaya & Alexis M. Gautreau

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Contributions

G.G. conceived and coordinated the project. G.G, A.M., K.R. and O.R. conceptualized the experiments. A.M. performed and analysed the sptPALM and laser trap experiments. O.R. performed pilot sptPALM experiments. F.K. designed and generated Nap1-KO cells and performed rescue experiments. M. Schaks carried out rescue experiments in Sra-1/PIR121 double-KO cells. M. Sainlos generated biotinylated nanobody. Z.K. and A.G. designed and generated protein constructs. Z.K. designed and generated TM-Nap1 and TM-Sra1 constructs and stable TM-Nap1 cell lines for biochemical analysis. M.C. performed sptPALM experiments using TM-Sra1, LatA and CytoD at lower concentrations. A.P. and A.G. performed immunoprecipitation and western blotting experiments. G.G., K.R. and A.M. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Grégory Giannone.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Prototypic trajectories corresponding to the different diffusion modes: immobile, confined and free-diffusive.

a, Trajectories obtained from a sptPALM acquisition on mEos2-Nap1 in the lamellipodium of a spreading MEF (same cell as in Fig. 1a, 50 Hz, duration: 150 s). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow) and immobile (red) (top). b, Prototypic trajectories corresponding to distinct diffusion modes (immobile, confined and free-diffusive) highlighted in a. c, Illustration of the Mean Squared Displacement (MSD) computation from Δt to 4 x Δt using the equation in the inset, where xi and yi are the coordinates of the label position at time \(i \times {\Delta}t\). We defined the measured diffusion coefficient D as the slope of the affine regression line fitted to the n=1 to 4 values of the\(MSD\left( {n \times {\Delta}t} \right)\) (dotted line in the graph). d, Variation of the MSD over time for selected and highlighted trajectories in a and b. e, Variation of the MSD over time for all the trajectories of the sptPALM sequence with the same color-code as in a, mean ± s.e.m. for trajectories. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 2 Diffusive behavior of WRC subunits at the lamellipodium tip and the membrane outside of the lamellipodium.

a, Super-resolution intensity image of mEos2-Abi1 in a MEF lamellipodium obtained by sptPALM (50 Hz, duration: 150 s) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). b, Same as a for mEos2-Sra1. c, Same as a for mEos2-Wave2. d, Same as a for mEos2-Actin. e, Same as a for mEos2-CAAX. f, Same as a for cytosolic mEos2. g, Distribution of Log(D) at the Tip versus Outside (mean for cells). h, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. i, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). Images in a-f are representative of 3, 3, 6, 8, 6 and 2 independent experiments that yielded similar results. g-h (for inside and outside), data pooled from independent experiments: mEos2-Nap1, n = 22 cells (pooled over 6 experiments); mEos2-Abi1, n = 11 cells (over 3 experiments); mEos2-Wave2, n = 15 cells (over 6 experiments); mEos2-Sra1, n = 11 cells (over 3 experiments) and cytosolic mEos2, n = 5 cells (over 2 experiments). i, Data pooled from cells in g-h and Fig. 1d,e, n is the number of trajectories: mEos2-ArpC5A, nTip = 1035, nOutside = 691; mEos2-Actin, nTip = 1948, nOutside = 6764; cytosolic mEos2, nTip = 26, nOutside = 111; mEos2-Nap1, nTip = 2976, nOutside = 12493; mEos2-Abi1, nTip = 4183, nOutside = 7233; mEos2-Wave2, nTip = 1936, nOutside = 6885; mEos2-Sra1, nTip = 1715, nOutside = 12564 and mEos2-CAAX, nTip = 455, nOutside = 4605. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (h) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (i). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (h-i). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (h-i). Note that a significant fraction of the Wave2 protein displays a faster membrane free-diffusion which differs from all the other subunits of WRC that we tested in this study. This could correspond to a fraction of the Wave2 protein not incorporated in WRC12, which might diffuse as single proteins at the plasma membrane. Also see Supplementary Table 1. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 3 WRC and FMNL2 lateral movements are driven by actin filaments elongation.

a, Fluorescence image of a MEF lamellipodium labeled with α-actinin-GFP (left). Right, mEos2-Abi1 kymographs generated from a sptPALM time-lapse sequence (2 Hz) perpendicular to the edge (left panel, dashed lines). mEos2-Abi1 motions highlighted (white lines) and corresponding velocities (nm.s-1). b, Distributions of anterograde and retrograde perpendicular movements (duration > 3 s) for mEos2-ArpC5A (n = 535 movements, pooled over 8 cells from 5 independent experiments); mEos2-Nap1 (n = 161 movements, over 6 cells from 3 experiments) and mEos2-Abi1 (n = 143 movements, over 3 cells from 2 experiments). Fractions of anterograde and retrograde movements superior to displacements measured in fixed cells are shown in the plot. c, Fluorescence image of a MEF lamellipodium labeled with α-actinin-GFP (left). Scale bar, 3 µm. Right, mEos2-Abi1 kymographs generated from a sptPALM time-lapse sequence (2 Hz) tangential to the edge (left panel, dashed lines). mEos2-Abi1 motions highlighted (white lines) and corresponding velocities (nm.s-1). d, Same as c for mEos2-Wave2. e, Same as c for mEos2-Abi1 in a fixed MEF. f, Speed of lateral movements (duration > 3 s) for mEos2-Wave2, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). g, Dwell times of lateral movements (duration > 1.5 s) for mEos2-Wave2, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). f, g, Data pooled from independent experiments: mEos2-Wave2, nSpeed = 165 movements, nDwell-Time = 303 movements (pooled over 5 cells from 3 experiments). h, α-actinin-GFP images of a spreading MEF before (left) and after (middle left) acute treatment with LatA (200 nM). Same for LatA (1 µM) (before: middle right, after: right). i, Same as h for CytoD (100 nM, 1 µM). j, Same as c for mEos2-Abi1 in a live MEF treated with LatA (1 μM). k, Same as c for mEos3.2-Sra1 treated with LatA (200 nM). l, Same as c for mEos3.2-Sra1 treated with CytoD (100 nM). m, Same as c for mEos3.2-Sra1-AD treated with LatA (1 µM). n, Distribution of left and right lateral movements (duration > 3 s) for mEos2-Nap1 (n = 263 movements, pooled over 5 cells from 3 independent experiments);mEos2-Nap1 LatA 1 µM (n = 639 movements, over 6 cells from 3 experiments); mEos2-Abi1 (n = 342 movements, over 3 cells from 2 experiments); mEos2-Abi1 LatA 1 µM (n = 210 movements, over 4 cells from 2 experiments); mEos3.2-Sra1 (n = 164 movements, over 5 cells from 2 experiments); mEos3.2-Sra1 LatA 200 nM (n = 275 movements, over 5 cells from 2 experiments); mEos3.2-Sra1 LatA 1 µM (n = 562 movements, over 5 cells from 3 experiments); mEos3.2-Sra1 CytoD 100 nM (n = 327 movements, over 6 cells from 3 experiments); mEos3.2-Sra1 CytoD 1 µM (n = 361 movements, over 6 cells from 3 experiments); mEos3.2-Sra1-WCA* (n = 471 movements, over 6 cells from 3 experiments); mEos3.2-Sra1-AD (n = 62 movements, over 6 cells from 4 experiments); mEos3.2-Sra1-AD LatA 1 µM (n = 145 movements, over 6 cells from 2 experiments); mEos2-Wave2 (n = 165 movements, over 5 cells from 3 experiments); mEos2-Wave2-ΔWCA (n = 270 movements, over 3 cells from 2 experiments); mEos2-Wave2-ΔWCA LatA 1 µM (n = 155 movements, over 3 cells from 2 experiments); FMNL2-mEos2 (n = 479 movements, over 5 cells from 2 experiments); FMNL2-mEos2 LatA 1 µM (n = 206 movements, over 3 cells from 1 experiments); FMNL2-A272E-mEos2 (n = 488 movements, over 4 cells from 2 experiments); FMNL2-I704A-mEos2 (n = 200 movements, over 6 cells from 3 experiments) and fixed mEos2-Abi1 (n = 103 movements, over 2 cells from 1 experiment). Images in a, c-e and h-m are representative of 2, 2, 3, 1, 3, 3, 2, 3, 2, 3 and 3 independent experiments that yielded similar results. Fractions of left and right movements superior to displacements measured in fixed cells are shown in the plot. All results for each condition correspond to pooled data from several independent experiments. Also see Supplementary Table 2. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 4 FRAP experiments show a decreasing order of turnover for Sra1-AD, Sra1 and WCA*.

a, GFP fluorescence images of GFP-Sra1 transfected in Sra1/PIR121-KO B16 cells (upper). GFP-Sra1 time-lapse images during a FRAP experiment (bottom). The outlined area in the upper panel is shown at a higher magnification in the bottom panel. Scale bar, 5 µm. b, Analysis of GFP-Sra1 fluorescence recovery in FRAP experiments performed as in a (mean ± s.e.m. for cells) (n = 24 cells). The recovery is quantified from the outlined area in a bottom. c, d, Same as a,b for GFP-Sra1-WCA* (n = 25 cells). e, f, Same as a,b for GFP-Sra1-AD (n = 30 cells). Images in a, c, d, are representative of 2 independent experiments that yielded similar results. b, d, f, Data pooled from 2 independent experiments. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 5 Diffusive behavior of WRC subunits after LatA treatment.

a, Super-resolution intensity image of mEos2-Nap1 in the lamellipodium of a MEF treated with LatA (1 μM) (50 Hz, duration: 150 s) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). b, Same as a for mEos2-Abi1 in a MEF treated with LatA (1 μM). c, Same as a for mEos2-CAAX in a MEF treated with LatA (1 μM). d, Distribution of Log(D) at the Tip versus Outside (mean for cells). e, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. f, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). Images in a-c are representative of 3, 5 and 2 independent experiments that yielded similar results. d-e, (for inside and outside), data pooled from independent experiments: mEos2-Nap1, n = 22 cells (pooled over 6 experiments); mEos2-Nap1 treated with 1 µM of LatA, n = 6 cells (over 3 experiments); mEos2-Abi1, n = 11 cells (over 3 experiments); mEos2-Abi1 treated with 1 µM of LatA, n = 9 cells (over 5 experiments); mEos2-CAAX, n = 7 cells (over 6 experiments) and mEos2-CAAX treated with 1 µM of LatA, n = 4 cells (over 2 experiments). k, Data pooled from cells in d-e, n is the number of trajectories: mEos2-Nap1, nTip = 2976, nOutside = 12493; mEos2-Nap1 treated with 1 µM of LatA, nTip = 399, nOutside = 667; mEos2-Abi1, nTip = 4183, nOutside = 7233; mEos2-Abi1 treated with 1 µM of LatA, nTip = 383, nOutside = 1191; mEos2-CAAX, nTip = 455, nOutside = 4605 and mEos2-CAAX treated with 1 µM of LatA, nTip = 314, nOutside = 1671. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (e) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (f). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (e,f). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (e,f). Also see Supplementary Table 1. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 6 Diffusive behavior of Wave2-ΔWCA, FMNL2, Sra1-AD after LatA treatment.

a, Super-resolution intensity image of mEos2-Wave2-ΔWCA in a MEF lamellipodium obtained by sptPALM (50 Hz, duration: 150 s) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). b, Same as a for mEos2-Wave2-ΔWCA in a MEF treated with LatA (1 μM). c, Same as a for mEos2-Sra1-AD in a MEF treated with LatA (1 μM). d, Same as a for FMNL2-mEos2 in a MEF treated with LatA (1 μM). e, Distribution of Log(D) at the Tip versus Outside (mean for cells). f, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. g, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). Images in a-d are representative of 3, 2, 4 and 3 independent experiments that yielded similar results. e-f, (for inside and outside), data pooled from independent experiments: mEos3.2-Sra1-AD, n = 7 cells (pooled over 4 experiments); mEos3.2-Sra1-AD treated with 1 µM of LatA, n = 7 cells (over 2 experiments); mEos2-Wave2, n = 15 cells (over 6 experiments); mEos2-Wave2-ΔWCA, n = 8 cells (over 3 experiments); mEos2-Wave2-ΔWCA treated with 1 µM of LatA, n = 4 cells (over 2 experiments); FMNL2-mEos2, n = 19 cells (over 5 experiments) and FMNL2-mEos2 treated with 1 µM of LatA, n = 6 cells (over 3 experiments). k, Data pooled from cells in i-j, n is the number of trajectories: mEos3.2-Sra1-AD, nTip = 398, nOutside = 2971; mEos3.2-Sra1-AD treated with 1 µM of LatA, nTip = 202, nOutside = 738; mEos2-Wave2, nTip = 1936, nOutside = 6885; mEos2-Wave2-ΔWCA, nTip = 1033, nOutside = 8281; mEos2-Wave2-ΔWCA treated with 1 µM of LatA, nTip = 69, nOutside = 255; FMNL2-mEos2, nTip = 3388, nOutside = 19567 and FMNL2-mEos2 treated with 1 µM of LatA, nTip = 743, nOutside = 3245. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (f) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (g). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (f,g). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (f,g). Also see Supplementary Table 1. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 7 Fast directed motions of FMNL2 inside and outside the lamellipodium.

a, Fluorescence image of α-actinin-GFP in a MEF co-transfected with FMNL2-mEos2 (left). Kymographs generated from a raw sptPALM acquisition (50 Hz) to measure FMNL2-mEos2 fast directed motions occurring in the lamellipodium (1) or outside the lamellipodium (2,3) (left panel, dashed lines). Fast motions of FMNL2-mEos2 highlighted (white lines) and corresponding velocities (nm.s-1). b, Distributions of speed for fast directed motions occurring in the lamellipodium (n = 29 events), or outside the lamellipodium (n = 38 events) for FMNL2-mEos2 (pooled over 3 cells from 2 experiments), box plots displaying median (notch) and mean (square) ± percentiles (25-75%). c, Super-resolution intensity image of FMNL2-mEos2 in the a MEF lamellipodium obtained by sptPALM (50 Hz, duration: 150 s) (left). Right, FMNL2-mEos2 kymographs generated from a sptPALM time-lapse sequence (2 Hz) along LOI corresponding to directed motions occurring within or outside the lamellipodium. d, same as c for FMNL2-I704A-mEos2. e, same as c for FMNL2-A272E-mEos2. f, density of directed motions, box plots displaying median (horizontal line) and mean (square) ± percentiles (25-75%) for each cell. Data pooled from independent experiments: mEos3.2-Sra1, n = 5 cells (pooled over 2 experiments); mEos3.2-Sra1-WCA*, n = 4 cells (over 2 experiments) and mEos3.2-Sra1-AD, n = 6 cells (over 3 experiments). Images in a, c-e are representative of 2, 2, 2 and 3 independent experiments that yielded similar results. Where indicated, statistical significance were obtained using two-tailed Mann-Whitney rank sum test for speed (b) and density (f) of directed motions. Also see Supplementary Table 3. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 8 Sra1 lateral movements in B16 FMNL2/3 KO.

a, sptPALM image of mEos3.2-Sra1 in the lamellipodium of a FMNL2/3 KO B16 cell (50 Hz) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). b, Same as a for mEos3.2-Sra1 in a FMNL2/3 KO B16 cell expressing FMNL2-GFP (insets: α-actinin-GFP and FMNL2-GFP fluorescence images). c, Distribution of Log(D) at the Tip versus Outside (mean for cells). d, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. e, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). c,d, (for inside and outside), Data pooled from 2 independent experiments: mEos3.2-Sra1 (n = 11 cells) and mEos3.2-Sra1 in cells expressing FMNL2-GFP (n = 6 cells). e, Data pooled from cells in c,d, n is the number of trajectories: mEos3.2-Sra1, nTip = 3714, nOutside = 9469 and mEos3.2-Sra1 in cells expressing FMNL2-GFP, nTip = 1682, nOutside = 4311. f, Fluorescence image of the lamellipodium labeled with α-actinin-GFP (left) in a FMNL2/3 KO B16 cell. Scale bar, 3 µm. Right, mEos3.2-Sra1 kymographs generated from a sptPALM time-lapse sequence (2 Hz) tangential to the edge (left panel, dashed lines). mEos3.2-Sra1 motions highlighted (white lines) and corresponding velocities (nm.s-1). g, Fluorescence image of the lamellipodium labeled with FMNL2-GFP (left) in a FMNL2/3 KO B16 cell. Right, mEos3.2-Sra1 kymographs. h, Speed of lateral movements (duration > 3 s), box plots displaying median (notch) and mean (square) ± percentiles (25-75%). i, Dwell times of lateral movements (duration > 1.5 s), box plots displaying median (notch) and mean (square) ± percentiles (25-75%). j, Distribution of left and right of lateral movements (duration > 3 s). h-j, Data pooled from 2 independent experiments: mEos3.2-Sra1, nSpeed = 186 movements, nDwell-Time = 372 movements, nDisplacement = 186 movements (over 5 cells) and mEos3.2-Sra1, nSpeed = 124 movements, nDwell-Time = 309 movements, nDisplacement = 124 movements (over 5 cells). Images in a-b and f-g are representative of 2 independent experiments that yielded similar results. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (d) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (e). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (d,e). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (d,e). Statistical significance were obtained using two-tailed Mann-Whitney rank sum test for speed (h) and dwell time (i) of lateral movements. Also see Supplementary Table 1 and Supplementary Table 2. Numerical source data are provided in Source data.

Source data

Extended Data Fig. 9 Diffusive behavior of transmembrane-Sra1 versus controls.

a, Schematic representation of TM-Sra and the transmembrane control protein, TM-ctr. b, Super-resolution intensity image of TM-ctr in a MEF lamellipodium obtained by sptPALM (50 Hz, duration: 150 s) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). c, Same as b for TM-Sra1. d, Distribution of Log(D) at the Tip versus Outside (mean for cells). e, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. f, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). d, e (for inside and outside), data pooled from independent experiments: mEos3.2-Sra1, n = 9 cells (pooled over 4 experiments); TM-Sra1, n = 9 cells (over 2 experiments) and mEos2-TM-ctr, n = 10 cells (over 2 experiments). f, Data pooled from cells in d-e and Fig. 5c,d, n is the number of trajectories: mEos2-Nap1, nTip = 2976, nOutside = 12493; TM-Nap1, nTip = 1820, nOutside = 6729; mEos3.2-Sra1, nTip = 921, nOutside = 3713; TM-Sra1, nTip = 629, nOutside = 3174 and mEos2-TM-ctr, nTip = 1893, nOutside = 16442. g, Western blotting of Nap1-deficient (Nap1-KO) and control B16-F1 cells expressing TM-Nap1 and using anti-Nap1 or anti-GFP antibodies, as indicated; tubulin expression was used as loading control. Blot in g is representative of 3 independent experiments that yielded similar results. h, Filamentous actin labeled with ATTO-594-labeled phalloidin in Sra1/PIR121-KO B16 cells (upper left and right). Arrowheads point to lamellipodia. GFP fluorescence images of TM-Sra1 (lower left) transfected into Sra1-KO B16-F1 melanoma cells or untransfected cells (lower right). i, Quantification of cells displaying lamellipodia in Sra1/PIR121-KO B16 cells transfected with TM-Sra1 (n = 80 cells pooled over 3 independent experiments) or untransfected (n = 55 cells pooled over 3 independent experiments) (mean ± s.e.m. for experiments). Images in b, c and h are representative of 2, 2 and 3 independent experiments that yielded similar results. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (e) or t-test for lamellipodia formation (i), or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (f). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (e,f). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (e,f). Also see Supplementary Table 1. Numerical source data are provided in Source data. Unprocessed blot from g is available in the Source Data.

Source data

Extended Data Fig. 10 Optical tweezer calibrations for pulling and connection-breaking assays.

a, Schematic representation of the optical tweezers binding assay. Nanobody-beads were held for 3 s on the cell surface ∼ 1 µm behind the tip of the lamellipodium using the laser trap. Beads were scored as bound if they remained focused in the plane of the membrane for more than 10 s after turning the laser tweezers off. b, Binding fractions as a function of the Nanobody-Biotin/BSA-Biotin ratio on beads for GFP-β3-integrin (n0/1 = 24 cells, n1/2 = 11 cells and n1/0 = 43 cells, pooled over 8, 3 and 15 independent experiments), TM-Nap1 (n0/1 = 6 cells, n1/2 = 46 cells and n1/2 = 29 cells, pooled over 2, 9 and 5 experiments) and TM-ctr (n0/1 = 3 cells, n1/2 = 12 cells and n1/0 = 5 cells, pooled over 1, 7 and 2 experiments), (mean ± s.e.m. for cells). c, Fractions of pulled beads below 1 pN (light histograms), between 1 pN-2 pN (mid shaded histograms) and above 2 pN (dark histograms), for TM-Nap1 (n = 5 experiments), TM-Sra1 (n = 3 experiments), TM-Sra1-WCA* (n = 3 experiments), TM-Sra1-AD (n = 3 experiments) and TM-ctr (n = 4 experiments), (mean ± s.e.m. for experiments). d, Pulled fraction as function of the maximal escapement force determined for defined trap stiffnesses. TM-Nap1 (n = 329 pulling assay), TM-Sra1 (n = 399 pulling assay), TM-Sra1-WCA* (n = 372 pulling assay), TM-Sra1-AD (n = 211 pulling assay) and TM-ctr (n = 157 pulling assay). c, d, Data pooled from independent experiments: TM-Nap1 (pooled over 28 cells from 5 experiments), TM-Sra1(pooled over 11 cells from 3 experiments), TM-Sra1-WCA* (pooled over 12 cells from 3 experiments), TM-Sra1-AD (pooled over 18 cells from 3 experiments) and TM-ctr (pooled over 9 cells from 4 experiments). e, Fractions of breaking forces bellow 1 pN (dark histograms), between 1 pN-2 pN (mid shaded histograms) and above 2 pN (light shaded histograms). TM-Nap1 (11 cells), TM-Sra1(9 cells), TM-Sra1-WCA* (22 cells), TM-Sra1-AD (4 cells) and TM-ctr (5 cells), (mean ± s.e.m. for cells). f, Distributions of breaking forces for nanobody-beads bound to TM-Nap1 (n = 54 breaking events), TM-Sra1 (n = 65 breaking events), TM-Sra1-WCA* (n = 218 breaking events), TM-Sra1-AD (n = 8 breaking events) and TM-ctr (n = 16 breaking events). e, f, Data pooled from independent experiments: TM-Nap1 (pooled over 11 cells from 5 experiments), TM-Sra1 (pooled over 9 cells from 5 experiments), TM-Sra1-WCA* (pooled over 22 cells from 8 experiments), TM-Sra1-AD (pooled over 4 cells from 3 experiments) and TM-ctr (pooled over 5 cells from 3 experiments). g, Using a piezoelectric motor, the sample was moved at increasing velocities. The increasing viscous drag force exerted on the bead triggered larger displacements until escapement from laser trap (left). Displacement versus time plots from the trap center as function of the viscous drag force at different laser powers (2.4, 3.9 and 7.9 mW) (right). h, Displacement amplitudes as function of the viscous drag force. Trap stiffnesses were calculated for different laser powers, and corresponded to the slope of displacement versus force curves. i, Trap stiffness as a function of laser power. j, Maximal trap force as function of laser power. Also see Supplementary Table 4. Numerical source data are provided in Source data.

Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Video 1

From a raw sptPALM acquisition to a super-resolution intensity image and corresponding trajectories.

Supplementary Video 2

Super-resolution time-lapse sequence of ArpC5A.

Supplementary Video 3

Super-resolution time-lapse sequence of Nap1.

Supplementary Video 4

Super-resolution time-lapse sequence of Abi1.

Supplementary Video 5

Super-resolution time-lapse sequence of Nap1 after LatA treatment.

Supplementary Video 6

Super-resolution time-lapse sequence of Abi1 after LatA treatment.

Supplementary Video 7

Super-resolution time-lapse sequence of Wave2-ΔWCA.

Supplementary Video 8

Super-resolution time-lapse sequence of Wave2-ΔWCA after LatA treatment.

Supplementary Video 9

Fast directed motions of FMNL2 at the lamellipodium tip and outside the lamellipodium.

Supplementary Video 10

Super-resolution time-lapse sequence of FMNL2-mEos2.

Supplementary Video 11

Super-resolution time-lapse sequence of FMNL2-mEos2 after LatA treatment.

Supplementary Video 12

Pulling assay for a ‘pulled’ nanobody-bead.

Supplementary Video 13

Pulling assay for a ‘not pulled’ nanobody-bead.

Supplementary Video 14

Breaking assay for TM-Nap1.

Supplementary Video 15

Breaking assay for TM-ctr.

Supplementary Video 16

TM-Sra1 crosslinking increases membrane protrusion rates.

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Mehidi, A., Kage, F., Karatas, Z. et al. Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration. Nat Cell Biol 23, 1148–1162 (2021). https://doi.org/10.1038/s41556-021-00786-8

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