Abstract
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia1,2. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks3. This machine is activated at the leading edge of migrating cells by Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia4,5,6. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac–Arpin–Arp2/3 inhibitory circuit with the Rac–WAVE–Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
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Acknowledgements
A.G. acknowledges his PhD supervisor M. Arpin, the name of the here identified protein is a tribute to her mentoring. We thank G. Romet-Lemonne, E. Portnoy and F. Marletaz for suggestions. We acknowledge support from Agence Nationale pour la Recherche (ANR-08-BLAN-0012-CSD 8 to A.G. and L.B., ANR-08-PCVI-0010-03 to A.G., ANR-11-BSV8-0010-02 to A.G., J.C. and S.Z.-J.), Association pour la Recherche sur le Cancer (SFI20101201512 to A.G., PDF20111204331 to R.G., SFI20111203770 to N.B.D.), the Bio-Emergences IBISA facility and Fundacao para a Ciencia e a Tecnologia (SFRH/BPD/46451/2008 to C.S.-B.), the Austrian Science Fund (FWF P21292-B09 to J.V.S.), the Deutsche Forschungsgemeinschaft (FA 330/5-1 to J.F.) and grant number 8066, code 2012-1.1-12-000-1002-064 from the Russian Ministry of Education and Science to A.Y.A.
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Contributions
I.D., R.G. and C.S.-B. performed videomicroscopy, analysed cell migration, analysed biochemical interactions of Arpin and its localization. E.D. wrote the bioinformatics programme that first identified Arpin. C.G. and L.B. performed in vitro actin polymerization and fluorescence anisotropy assays. J.L. and J.F. isolated knockout amoeba and analysed their migration. M.N. and J.V.S. micro-injected fish keratocytes. J.G.D., F.A.G. and N.B.D characterized the Arpin phenotype in zebrafish. A.B. and N.P. determined the Arpin expression profile in zebrafish. I.H. and S.Z.-J. contributed the NMR spectrum. T.A.C., V.D.E., A.Y.A., S.V., I.B., V.C., V.D., G.L., K.O., F.P., A.-G.P., S.F. and V.H. generated DNA constructs, isolated stable cell clones, purified and characterized recombinant proteins, and performed crucial experiments for our understanding of Arpin function. A.S. and K.R. isolated the Rac1 knockout MEFs. All authors designed experiments. N.P., K.R., S.Z.-J., J.C., N.B.D., I.B., A.Y.A., J.V.S., J.F., L.B. and A.G. supervised the work in their respective research group. A.G. coordinated the study and wrote the paper.
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Extended data figures and tables
Extended Data Figure 1 Prediction of secondary structure elements and disordered regions of Arpin.
A multiple alignment of the Arpin orthologues was performed with MUSCLE41 and displayed with Jalview42. Two methods relying on multiple alignments of Arpin orthologues were used to predict secondary structures and disordered regions, Psipred43 and Disopred44, respectively. The predicted secondary structure (SS) elements are indicated by green arrows for β-strands, red cylinders for α-helices, and a black line for coils; the associated confidence (conf) score is displayed below, ranging from 0 to 9 for poor and high confidence, respectively. Amino acid conservation is indicated by a 0 to 10 score, and highlighted by brown to yellow histogram bars. The confidence in predicting disorder is also scored from 0 to 10, by multiplying tenfold the Disopred probability. Arpin is predicted to be a structured protein with the notable exception of the 20 C-terminal residues, which are predicted to be disordered with high confidence.
Extended Data Figure 2 NMR analysis of 15N-labelled human Arpin.
Both views represent 1H–15N heteronuclear single quantum coherence (HSQC) spectra. Each peak corresponds to the 1H–15N backbone amide bond of a specific residue. The position of a 1H–15N peak in the spectrum depends on the chemical environment of the corresponding residue. a, Such a scattered distribution of peaks is characteristic of a folded protein. The last 20 residues were assigned to individual peaks and are displayed on the spectrum. These residues are clustered in the centre of the spectrum. b, Same HSQC spectrum displayed with a higher threshold to display only high peaks. The height of a peak depends on the mobility of the residue on a picosecond to millisecond timescale. This spectrum experimentally demonstrates that the 20 C-terminal residues are highly mobile. This result confirms that, as predicted, the Arp2/3-binding site of Arpin is exposed as a poorly structured tail of the protein.
Extended Data Figure 3 Characterization of recombinant Arpin.
a, Full-length Arpin or ArpinΔA from human or zebrafish cDNA was expressed in E. coli and purified. Purity was assessed by SDS–PAGE and coomassie staining. These proteins were used for in vitro actin polymerization assays and for fish keratocyte injection, respectively. b, Analysis of the molar mass of full-length Arpins by size-exclusion chromatography coupled to multiangle light scattering (SEC–MALS). The ultraviolet measurement (left axis, dashed line) and the molar mass (right axis, horizontal solid line) were plotted as a function of column elution volume. c, SEC–MALS measures of masses indicate that both proteins are monomeric in solution. d, GST pull-down using a lysate of 293 cells overexpressing PC-tagged Arpin and purified GST–Rac1 wild type, GST–Rac1(Gln61Leu) or GST alone as a negative control. Arpin did not associate with either type of Rac. By contrast, the endogenous WAVE complex bound to Rac(Gln61Leu), but not Rac wild type, as expected from a Rac effector. e, Untagged Rac1 was purified from E. coli and then loaded with either GDP or GTPγS. Human Arpin (60 μM), Rac1 (120 μM) and mixture of these two proteins were analysed by SEC–MALS as above. SEC was run in 20 mM HEPES, 100 mM NaCl, pH 7.4. The height of ultraviolet peaks was normalized to 1 to be displayed on the same figure. A single peak was detected in all cases. The measured masses indicate that no complex is formed between Arpin and Rac and that the single peak observed in the mixture corresponds to cofractionation of the two proteins of similar mass by SEC.
Extended Data Figure 4 Arpin directly binds to the Arp2/3 complex.
a, Fluorescence anisotropy measurements of labelled ArpinA peptide binding at equilibrium to the purified Arp2/3 complex at the indicated concentrations. b, Labelled ArpinA peptide bound to the Arp2/3 complex was then titrated with purified Arpin full-length, ArpinΔA or unlabelled ArpinA peptide as indicated. Full-length Arpin displaces the labelled ArpinA peptide more efficiently than the A peptide. ArpinΔA is unable to displace the ArpinA peptide. Curves that best fit the values yield the indicated equilibrium constants. c, Arpin inhibits Arp2/3 activation in the pyrene-actin assay. Part of this experiment is displayed in Fig. 1c, more curves are plotted here. d, From curves in c, the number of actin barbed ends is calculated from the slope at half-polymerization using the relationship described previously45. Best fit of the values indicate an apparent Kd value of 760 ± 156 nM for the Arp2/3 complex in a mixture including actin and the VCA. e, ArpinA inhibits Arp2/3 activation in a dose dependent manner in the pyrene-actin assay. Conditions: 2 μM actin (10% pyrene-labelled), 500 nM VCA, 20 nM Arp2/3 and ArpinA at the indicated concentrations. f, ArpinA competes with the NPF for Arp2/3 binding. Arp2/3 is displaced from its interaction with 5 μM GST N-WASP VCA immobilized on glutathione beads by the Arpin acidic peptide (304 μM and serial twofold dilutions).
Extended Data Figure 5 Specific localization of Arpin at the lamellipodium tip.
a, Arpin overlaps with the Arp2/3 complex at the lamellipodium tip. b, Arpin overlaps with cortactin at the lamellipodium tip. Arpin staining is lost after short interfering RNA (siRNA)-mediated depletion. Intensity profiles along multiple line scans encompassing the cell periphery were registered to the outer edge of the staining of a lamellipodial marker. This marker was Arpin in a and cortactin in b. The multiple line scans were then averaged and displayed as an intensity plot, where the y axis represents fluorescent intensity, arbitrary units (mean ± s.e.m., n = 17, 16 and 17, respectively). Scale bar, 20 μm. Arp2/3 localization extends rearwards relative to Arpin localization. This result is because the Arp2/3 complex becomes a branched junction when activated by the WAVE complex at the lamellipodium tip. The branched junction undergoes retrograde flow like actin itself due to actin filament elongation9,12. Cortactin recognizes Arp2/3 at the branch junction and is thought to stabilize branched actin networks13. As a marker of the branched junction, cortactin stains the width of lamellipodia, like the Arp2/3 complex. c, Rac1 knockout MEFs that lack lamellipodia14 are completely devoid of Arpin staining at the cell periphery, in line with the complete absence of lamellipodia indicated here by the absence of cortactin staining. Arpin is normally expressed in the Rac1 knockout MEFs (see Fig. 2c). Intensity profiles along multiple line scans encompassing the cell periphery were averaged after manual drawing of the cell edge (mean ± s.e.m., n = 16). Scale bar, 20 μm.
Extended Data Figure 6 Arpin regulates cell spreading through its interaction with the Arp2/3 complex.
Arpin was depleted from human RPE1 cells after transient transfection of shRNA plasmids and blasticidin-mediated selection of transfected cells. After 5 days, cells were either analysed by western blot or used for the spreading assay. Cells were serum-starved for 90 min in suspension in polyHEMA-coated dishes and then allowed to spread on collagen-I-coated coverslips for 2 h. Phalloidin staining was used to calculate cell surface area of individual cells using ImageJ. Mean ± s.e.m.; **P < 0.01, ***P < 0.001; t-test or ANOVA when more than two conditions. a, Arpin depletion increases cell spreading (n = 57 and 52, respectively). The same effect is obtained with three shRNAs targeting Arpin (n = 51, 48 and 59, respectively). b, This effect is rescued by GFP–Arpin expression in knockdown cells, but not by GFP–ArpinΔA expression(n = 63, 56, 63 and 52, respectively). c, Combined depletion of Arpin and the Arp2/3 complex reverses the phenotype of Arpin depletion. The effect is seen with two shRNAs targeting ArpC2 (n = 56, 60, 69, 68, 63 and 66, respectively). The last two experiments indicate that Arpin exerts its effect on cell spreading through its ability to regulate the Arp2/3 complex.
Extended Data Figure 7 Arpin regulates protrusion frequency of prechordal plate cells and their collective migration in zebrafish embryos.
a, In situ hybridization of arpin probe in zebrafish embryos at different stages. arpin mRNAs are maternally deposited. During gastrulation, arpin is expressed in hypoblast, which includes the prechordal plate. b, Three-dimensional trajectories of prechordal plate cells in embryos injected with control or arpin morpholino. During fish gastrulation, prechordal plate cells migrate collectively in a straight direction from the margin of the embryo towards the animal pole38,46. Loss of arpin function induces dispersion as evidenced by increased lateral cell displacement (n = 1,516 and 1,546) and a higher distance between cells (n = 194 and 235). Lateral displacement is the cell movement perpendicular to main direction of the migration. Distance between cells refers to the average distance of the nucleus of a given cell to the nuclei of its five closest neighbours. Mean ± s.e.m.; ***P < 0.001, t-test. c, At the onset of gastrulation prechordal plate cells derived from morpholino injected embryos were transplanted into the prechordal plate of an untreated host embryo at the same stage in order to allow imaging of cell autonomous effects on protrusion formation. Donor embryos are injected with control or arpin morpholinos and mRNAs encoding Lifeact-mCherry as well as GFP–Arpin for the rescue. Time-lapse imaging of injected cells is performed by epifluorescence to reveal Lifeact, a marker of filamentous actin, which stains actin-based protrusions. For each cell, presence of a protrusion was assessed at each frame to deduce probability of protrusion presence and protrusion lifetimes. arpin loss of function increases the probability of presence of protrusions (n = 8, 8 and 10, respectively; *P < 0.05, ANOVA) and their duration (in this case, n corresponds to the number of protrusions (n = 42, 41 and 40, respectively; *P < 0.05, Kruskal–Wallis). Protrusions are indicated by arrowheads. Scale bar, 50 μm.
Extended Data Figure 8 Arpin depletion increases cell migration in three dimensions.
Stable MDA-MB-231 clones depleted of Arpin or not were embedded in a collagen gel. a, Single-cell trajectories illustrate that control cells hardly move in this dense environment (see Supplementary Video 4), unlike Arpin-depleted cells, which explore a significant territory, albeit at lower pace than in two dimensions, as evidenced by mean square displacement (Extended Data Fig. 9). b, Cell speed is significantly increased in the Arpin-depleted clones. Mean ± s.e.m.; n = 27, 25, 26 and 17, respectively, *P < 0.05, Kruskal–Wallis, two experiments. Directional persistence, calculated by d/D, is not significantly different in the clones depleted of Arpin or not. Direction autocorrelation (Extended Data Fig. 10), however, shows an increased directionality in the Arpin-depleted cells at the earliest time points.
Extended Data Figure 9 Analysis of mean square displacement of the different migration experiments.
The mean square displacement gives a measure of the area explored by cells for any given time interval. By setting a positional vector on the cellular trajectory at time t, the MSD is defined as: 
, in which brackets 
indicate averages over all starting times t0 and all cells N. For each time interval Δtime, mean and s.e.m. are plotted. Error bars corresponding to s.e.m. are plotted, even if too small to be visible. The grey area excludes the noisy part of curves corresponding to large time intervals where less data points are available. a, MDA-MB-231 depleted or not of Arpin in a two- or three-dimensional environment. Arpin-depleted MDA-MB-231 cells explore a larger territory than the controls in time intervals examined (for two dimensions, n as indicated in Fig. 3a; P < 0.001, two-way ANOVA with time and conditions; for three dimensions, n as indicated in Extended Data Fig. 8; P < 0.001, two-way ANOVA with time and conditions). b, Dictyostelium discoideum knockout amoebae explore a larger territory than controls and rescued amoebae (n as indicated in Fig. 3b, P < 0.001, two-way ANOVA with time and conditions). c, Arpin-injected fish keratocytes explore a smaller territory than the controls (n as indicated in Fig. 4b; P < 0.001, two-way ANOVA with time and conditions).
Extended Data Figure 10 Analysis of direction autocorrelation of the different migration experiments.
a, Principle of the analysis. A hypothetical cell trajectory is depicted. Each step is represented by a vector of normalized length. θ is the angle between compared vectors. The plot illustrates the cosθ values for the putative trajectory of four steps (colour-coded). Averaging these cosθ values yields the direction autocorrelation (DA) function of time that measures the extent to which these vectors are aligned over different time intevals. The DA function is defined as: 
, in which υ(t0) is the vector at the starting time t0, and υ(t0 + t) the vector at t0 + t. Brackets indicate that all calculated cosines are averaged for all possible starting times (t0) over all cells (N). For each time interval t, vectors from all cell trajectories were used to compute average and sem. Error bars corresponding to s.e.m. are plotted, even if too small to be visible. b, Arpin-depleted MDA-MB-231 clones turn less than control cells (for two dimensions, n as indicated in Fig. 3a; P < 0.05 between 10 and 40 min, Kruskal–Wallis; for three dimensions, n as indicated in Extended Data Fig. 8; P < 0.05 at time 10 min, Kruskal–Wallis). c, Arpin knockout amoebae turn less than wild-type amoebae, and GFP–Arpin overexpressing knockout amoebae (rescue) turn more than wild type (n as indicated in Fig. 3b; P < 0.05 between 5 and 85 s, Kruskal–Wallis). e, Arpin-injected fish keratocytes turn more than buffer-injected cells, and ArpinΔA-injected keratocytes turn more than buffer-injected but less than full-length-Arpin-injected keratocytes (n as indicated in Fig. 4b; P < 0.05 between 16 and 272 s, Kruskal–Wallis).
Supplementary information
Arpin inhibits the formation of branched actin networks by the Arp2/3 complex
1 µM actin, 150 nM VCA, 80 nM Arp2/3 and Arpin at 5 µM when indicated. TIRF microscopy. Scale bar : 30 µm and 5 µm in the inset. (AVI 3294 kb)
Arpin depletion increases the speed of lamellipodial protrusions
RPE1 cells were recorded with one phase contrast image every 3 s for 10 min (using a Plan Apochromat 63x/1.40 oil immersion objective) while spreading on collagen I coated dish (Ibidi) in serum free medium. Scale bar: 3 µm. The insets showing lamellipodial protrusions are magnified 4-fold. (AVI 3559 kb)
arpin loss of function increases protrusion lifetime of zebrafish prechordal plate cells.
At the onset of gastrulation, prechordal plate cells derived from morpholino injected embryos were transplanted into the prechordal plate of an untreated host embryo at the same stage. Donor embryos are injected with control or arpin morpholinos and mRNAs encoding Lifeact-mCherry as well as GFP-Arpin for the rescue. Z-stacks were recorded every 30 seconds. Protrusion lifetime was measured from the appearance of the protrusion (green arrowhead) to its retraction (red arrowhead). Scale bar: 20 µm. (AVI 486 kb)
Arpin depletion increases migration of MDA-MB-231 cells
Stable Arpin knock-down cells were compared to control cells. Cells were allowed to spread on fibronectin-coated 8 well µ-slide (Ibidi) for 2 h, before imaging for 24 h with an image every 10 min using phase contrast and a Plan-Apochromat 20x/0.80 air objective. Scale bar : 50 µm. (AVI 3164 kb)
Arpin depletion increases migration of MDA-MB-231 cells in 3D
Stable Arpin knock-down cells were compared to control cells. Cells were sandwiched between two 3D collagen gels so as to have most cells in a single plane of view at the beginning of movie acquistion. Cells were imaged for 48 h with an image every 20 min using phase contrast and a Plan-Apochromat 20x/0.80 air objective. Scale bar: 50 µm. (AVI 2668 kb)
Arpin knock-out increases migration of Dictyostelium discoideum
KO amoeba were compared to wild type and KO re-expressing GFP-DdArpin. Amoeba were imaged onto 3 cm glass-bottom dishes (Matek) in Soerensen-phosphate buffer (17 mM Na/K-phosphatebuffer, pH 6.1) for 15 min with an image every 5 s using phase contrast or green fluorescence and a UPlanFL 10x NA 0.3 objective. Scale bar : 40 µm. (AVI 861 kb)
Arpin microinjection into fish keratocytes induces lamellipodium instability and cell turning
Keratocytes were imaged with phase contrast optics at x63 magnification with one frame every 8 s for 10 minutes. They were microinjected at the time 24 s. Scale bar: 10 µm. (AVI 1964 kb)
Arpin microinjection into fish keratocytes induces cycles of protrusion and retraction of the lamellipodium
This video shows the same kymograph as fig. 4c but corrected from cell movement. To register the video to the cell rather than to the substratum, fiduciary marks within the cell nucleus were manually tracked and the reverse movement was applied to the entire image using a custom imageJ macro. A kymograph on the corrected video was then generated along the white line using imageJ. This video highlights the oscillatory behavior of the leading edge upon Arpin microinjection. Scale bar: 10µm. (AVI 2512 kb)
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Dang, I., Gorelik, R., Sousa-Blin, C. et al. Inhibitory signalling to the Arp2/3 complex steers cell migration. Nature 503, 281–284 (2013). https://doi.org/10.1038/nature12611
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DOI: https://doi.org/10.1038/nature12611
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