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A Trio–Rac1–Pak1 signalling axis drives invadopodia disassembly

Nature Cell Biology volume 16, pages 571–583 (2014)Cite this article

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A Corrigendum to this article was published on 27 February 2015

This article has been updated

Abstract

Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, we developed a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. We describe here an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.

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Figure 1: Rac1 depletion increases invadopodia degradation activity.
Figure 2: Rac1 biosensor.
Figure 3: Rac1 activity at invadopodia.
Figure 4: Localized photoactivation of Rac1 induces invadopodia disassembly.
Figure 5: Pak1 links Rac1 to cortactin for invadopodia disassembly.
Figure 6: TrioGEF is involved in invadopodia activity.
Figure 7: TrioGEF affects Rac1 biosensor activation levels.
Figure 8: Trio–Rac1–Pak1 signalling axis is necessary for efficient invasion and 3D motility.

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Change history

  • 23 January 2015

    In the version of this Article originally published, the Author Contributions section was incorrect and should have read: 'Y.M. and L.H. designed and built the biosensor. Y.M., J-J.B.C., J.C. and L.H. designed the biological experiments. Y.M., J-J.B.C. and V.M. performed the experiments. J.C. and L.H. advised and gave critical feedback. Y.M. wrote the original manuscript. J-J.B.C. and L.H. revised the manuscript. J-J.B.C., V.M., J.C. and L.H. finalized the manuscript.' This has now been corrected in the online versions of the Article.

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Acknowledgements

This work was supported by GM093121 (Y.M., J.J.B-C., L.H.), T32GM007491 (Y.M., V.M.), and CA150344 (J.J.B-C., J.C.). We acknowledge G. Rosenberger (Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf Campus, Germany) for providing the α-Pix–GFP construct64, J. van Buul (Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Academic Medical Center, University of Amsterdam, The Netherlands) for providing the Trio constructs49, and M. Way (Cell Motility Laboratory, Cancer Research UK, UK) for providing the c-Src constructs65. This work is in partial fulfilment of the PhD requirements for Y.M. We thank the J. Condeelis, J. Segall and D. Cox laboratory members for their helpful discussions.

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  1. Yasmin Moshfegh and Jose Javier Bravo-Cordero: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA

    Yasmin Moshfegh, Jose Javier Bravo-Cordero, Veronika Miskolci, John Condeelis & Louis Hodgson

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Contributions

Y.M. and L.H. designed and built the biosensor. Y.M., J-J.B.C., J.C. and L.H. designed the biological experiments. Y.M., J-J.B.C. and V.M. performed the experiments. J.C. and L.H. advised and gave critical feedback. Y.M. wrote the original manuscript. J-J.B.C. and L.H. revised the manuscript. J-J.B.C., V.M., J.C. and L.H. finalized the manuscript.

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Correspondence to Louis Hodgson.

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Integrated supplementary information

Supplementary Figure 1 Trio-Rac1-Pak1-axis promote invasion in human invasive tumor cells but not in normal breast epithelial cells, and RNAi specificity.

(A) Representative images from human MDA-MB-231 showing cortactin (invadopodia marker) and changes in 405-gelatin matrix-degradation with siRNA against: control, Rac1, Pak1, and Trio. Scale Bar, 10 μm. Representative image set is from 40 image sets for Control, from 40 image sets for Rac1 siRNA, from 39 image sets for Pak1 siRNA, and from 41 image sets for Trio siRNA. (B) Left: Quantification of the degradation area per field in MDA-MD-231 from (A). n = 39 fields per condition, pooled from 3 independent experiments. Students t-test: ***P = 3.18 × 10−6 (Rac1), ***P = 1.67 × 10−5 (Pak1), ***P = 2.81 × 10−6 (Trio). Right: MDA-MD-231 with control, Rac1, Pak1 and Trio-siRNA show reduced invasion through matrigel. n = 24 fields per condition, pooled from 6 independent experiments. Students t-test: ***P = 6 × 10−5 (Rac1), ***P = 6.9 × 10−5 (Pak1), **P = 0.005 (Trio). (C) Representative images from human BT-549 showing cortactin (invadopodia marker) and changes in 405-gelatin matrix-degradation with siRNA against: control, Rac1, Pak1, and Trio. Scale Bar, 10 μm. Representative image set is from 29 image sets for Control, from 28 image sets for Rac1 siRNA, from 35 image sets for Pak1 siRNA, and from 31 image sets for Trio siRNA. (D) Left: Quantification of the degradation area per field in BT-549 from (C). n = 29 fields per condition, pooled from 3 independent experiments. Students t-test: *P = 0.042 (Rac1), *P = 0.048 (Pak1), **P = 0.008 (Trio). Right: BT-549 with control, Rac1, Pak1 and Trio-siRNA show reduced invasion through matrigel. n = 16 fields pooled from 4 independent experiments per condition. Students t-test: *P = 0.01(Rac1), **P = 0.009(Pak1), *P = 0.02(Trio). (E) Representative images from human MCF10A showing cortactin and the 405-gelatin matrix with siRNA against: control, Rac1, Pak1, and Trio. Scale Bar, 10 μm. Representative image set is from 40 image sets for Control, from 40 image sets for Rac1 siRNA, from 43 image sets for Pak1 siRNA, and from 40 image sets for Trio siRNA. (F) Left: Quantification of the degradation area per field in MCF10A from (E). n = 40 fields per condition, pooled from 3 independent experiments. Students t-test: P = 0.3(Rac1), P = 0.7(Pak1), P = 0.7(Trio). Right: Invasion through matrigel in MCF10A with control, Rac1, Pak1 and Trio-siRNA. n = 24 fields per condition, pooled from 6 independent experiments. Students t-test: P = 0.4(Rac1), P = 0.059(Pak1), P = 0.09(Trio). (G) Top: Western blot of Rac1, Pak1, Trio, β-actin, mDia1, GAPDH levels in control, Rac1, Pak1, and Trio-siRNA-treated MDA-MB-231. Middle: Western blot of Rac1, Pak1, Trio, β-actin, mDia1 levels in control, Rac1, Pak1, and Trio-siRNA-treated BT-549. Bottom: Western blot of Rac1, Pak1, Trio, mDia1, GAPDH levels in control, Rac1, Pak1, and Trio-siRNA-treated MCF10A. All blots shown are from single blots. (H) Western blots from MTLn3 with control and single oligonucleotides of Rac1 (top), Pak1 (middle) or TrioGEF (bottom)-siRNA and detecting Rac1, β-actin (top), Pak1, β-actin (middle) or Trio, mDia1 (bottom). Right: Quantifications of matrix-degradation for single oligonucleotide-knockdown condition. n = 29 fields for each condition, pooled from 3 independent experiments, with s.e.m. Students t-test: ***P = 1.8 × 10−17(#2); P = 5.7 × 10−12(#4:Rac1-siRNA), ***P = 0.0001(#1); ***P = 8.7 × 10−8(#3:Pak1-siRNA), ***P = 5.6 × 10−7(#1); ***P = 9.4 × 10−6(#3:TrioGEF-siRNA). Representative blots are shown from 1 blot for Rac1 panel, 1 blot for Pak1 panel and 2 blots for the TrioGEF panel.

Supplementary Figure 2 Rac1 is inactive in, but does not accumulate at mature invadopodia, the structures are non-vesicular, Rac1 activity in disappearing invadopodia, and control RhoA biosensor at steady-state invadopodia.

(a) Still-frames taken from a live-cell movie of MTLn3 expressing TagRFP-cortactin and CFP-Rac1. Insets show zoom on a single invadopodium (white box) during formation up to steady-state (top) and before and during disassembly (bottom). Throughout the invadopodium’s lifetime Rac1 does not accumulate at the cortactin spot, and maintains its homogeneous distribution. Scalebar, 10 μm. Representative image set from 11 image sets. (b) Representative image of MTLn3 plated on 405-gelatin matrix and stained for Transferrin, cortactin and Tks5. Insets show magnified invadopodium precursor with no matrix degradation activity (left white box), and a mature invadopodium actively degrading the gelatin matrix (right white box). In both cases there is no enrichment of the vesicle marker transferrin. Scalebars, 10 μm. Representative image set from 10 image sets. (c) Representative image of an MTLn3 cell plated on 405-gelatin matrix and stained with clathrin and cortactin antibodies to show endogenous localization. Scalebar, 10 μm. Insets show magnified invadopodium precursor with no matrix degradation activity (left white box), and a mature invadopodium actively degrading the gelatin matrix (right white box). In both cases there is no enrichment of the vesicle marker clathrin. Representative image set from 12 image sets. (d) Additional examples of Rac1 activity changes at invadopodia before the disappearance. White and black circles show the invadopodium location and corresponding Rac1 activity levels. Red arrows indicate the moment of elevated Rac1 activity before the disappearance of invadopodium. Pseudocolor scale: 1.0 (Black) to 1.62 (Red). Scalebar, 2 μm. Representative image set from 4 image sets showing burst of Rac1 activity. (e) Left: RhoA activity dynamics in RhoA biosensor-expressing MTLn3 cells, transfected with TagRFP-cortactin and imaged under steady state conditions on gelatin matrix. Images were acquired every minute. Tag-RFP-cortactin (top), RhoA activity (middle) and biosensor localization (bottom) images are shown at different time points. White boxes are magnified on the right. Black and white circles indicate invadopodia. Scalebar, 10 μm and 5 μm (insets). Pseudocolor scale: 1.0 (Black) to 3.0 (White). Right: Quantification of RhoA activity (normalized to time = 0) during steady-state invadopodia lifetimes. No significant change in RhoA activity was observed between any of the time points (P > 0.05). n = 16 invadopodia, pooled from 3 independent experiments, with s.e.m. Student’s t-test was used. Representative image set from 6 cell movies.

Supplementary Figure 3 Pak1 is recruited to invadopodia in Rac1-depleted cells, photo-uncaging of Rac1 at invadopodia containing phosphorylation deficient cortactin, and Pak1 modulates serine phosphorylation of cortactin.

(A) Representative image of MTLn3 cell transfected with Rac1 siRNA, then plated on Alexa-405 conjugated gelatin and fixed and stained with cortactin and Pak1 antibodies to show endogenous protein localization. Arrows point to sites of mature, degrading invadopodia. Scale bar, 10 μm. Representative image set is from 30 image sets. (B) Representative images of MTLn3 cell transfected with PA-Rac1, S113A TagRFP-cortactin, and Tks5-GFP before and after photoactivation. Blue circles show photoactivated region. Blue arrows point to invadopodia after photoactivation. Scale bars, 10μm. Representative image set is shown from 51 image sets. (C) Expression of cortactin S113A mutant alone increases matrix degradation area. Quantification of mean degradation area of control and cortactin mutant-overexpressing cells plated on fluorescent matrix overnight. n = 30 fields, pooled from 3 independent experiments, with s.e.m. Students t-test was used. **P = 3.7571 × 10−5. (D) Quantification of mean invadopodia lifetime in control and cortactin S113A overexpressing cells, from time-lapse movies over a period of 6 h. Invadopodia lifetime was quantified as the time in minutes from the appearance to the disappearance of RFP-cortactin and Tks5-GFP co-localized spots. n = 11 invadopodia for each condition, pooled from 3 independent experiments, error bars are s.e.m. Students t-test was used. **P = 9.193 × 10−13. (E) Quantification of percentage of invadopodia that disappear as a result of photoactivation with PA-Rac1, in control, Pak1 knockdown, and cortactin S113A mutant conditions. n = 46 invadopodia (PA-Rac) pooled from 3 independent experiments, n = 27 invadopodia (Pak1 siRNA) pooled from 3 independent experiments, n = 61 invadopodia (Cortactin S113A) pooled from 3 independent experiments, error bars are s.e.m. Students t-test was used. **P = 3.278 × 10−5 (Pak1 siRNA), **P = 1.91167 × 10−5 (Cortactin S113A). (F) Representative images of MTLn3 cell fixed and stained with antibodies to cortactin, pSerine, and 488-phalloidin (F-actin) showing: Proximity ligation assay spots (cortactin-phosphoserine interaction), cortactin and F-actin. Insets show sites of colocalization of cortactin with F-actin, and cortactin with the phosphoserine-cortactin interaction detected by PLA. Scale bar, 10μm. Representative image set is from 46 image sets. (G) Quantification of the percentage of invadopodia containing positive PLA staining for control, Rac1, Pak1, and TrioGEF-knockdowns. Invadopodia were identified as F-actin and cortactin positive spots. n = 300 invadopodia per condition, pooled from 3 independent experiments, error bars are s.e.m. Students t-test was used. *P = 0.0397 (Pak1 KD), *P = 0.019 (Trio KD).

Supplementary Figure 4 Tiam1 does not accumulate at invadopodia and is not required for matrix degradation activity.

(A) MTLn3 cell plated on Alexa-405 conjugated gelatin matrix and fixed and stained with antibodies for cortactin and Tiam1. Arrows point to sites of mature invadopodia. Endogenous Tiam1 is uniformly distributed and does not focally accumulate at invadopodia. Scale Bar, 10 μm. Representative image set is from 30 image sets. (B) Still images from a time lapse movie of MTLn3 cell transfected with Tiam1-FL-GFP (FL: Full-Length) and TagRFP-cortactin, and starved and stimulated with EGF. Insets show an invadopodium precursor formed after stimulation (bottom panel), while there is no corresponding enrichment of Tiam1 (top panel). Scale bar, 10 μm. Representative image set is from 16 cell movies. (C) Quantification of average matrix degradation area per field from MTLn3 cells transfected with GFP or a dominant negative Tiam1 mutant (GFP-Tiam1-PH-CC-EX) and plated overnight on fluorescent matrix. n = 31 fields for each condition, pooled from 3 independent experiments, error bars are s.e.m. Students t-test was used. P = 0.347.

Supplementary Figure 5 TrioGEF recruitment and regulation at invadopodia.

(a) Representative images of MTLn3 cells transfected with Trio-FL-GFP (FL: Full-Length) or Trio-D1ΔSH3-DN-GFP (D1SH3 fragment lacking the SH3 domain and with a point mutation at N1406AD1407A making it dominant negative (DN)). Cells were fixed and stained with cortactin and Tks5 antibodies. White boxes are magnifications of the indicated areas. Arrows point to single invadopodia. Scalebar, 1 μm. Representative image set from 55 image sets for Cortactin-Tks5-Trio-FL–GFP, and from 70 image sets for Cortactin-Tks5-Trio-D1ΔSH3–GFP. (b) Quantification of the percentage of invadopodia that show Trio at invadopodia. The bars represent the percentage of Trio positive invadopodia. n = 100 invadopodia per condition pooled from 5 independent experiments, error bars are s.e.m. Student’s t-test was used. *P = 0.031 (TrioFL-GFP versus TrioD1ΔSH3-DN-GFP), **P = 0.0075 (TrioFL-DN-GFP versus TrioD1ΔSH3-DN-GFP), *P = 0.011 (TrioD1SH3-DN-GFP versus TrioD1ΔSH3-DN-GFP). (c) Quantification of the percentage of invadopodia that show Trio at invadopodia before and after EGF stimulation. n = 30 invadopodia per condition pooled from 3 independent experiments, error bars are s.e.m. Student’s t-test was used. P = 0.6. (d) Examples of Trio recruitment to invadopodium before invadopodium disappearance. MTLn3 cells were transfected with cortactin-mRFP and Trio-GFP and image every 2 min for 3 h. Scalebar, 1 μm. Representative image set from 10 cell movies. (e) Trio recruitment to invadopodia in MTLn3 cells treated with control or Rac1 siRNA. n = 150 invadopodia pooled from 3 independent experiments, with s.e.m. Student’s t-test was used. ***P = 2.25 × 10−34.

Supplementary Figure 6 Depletion of α-Pix or β-Pix inhibits matrix degradation by invadopodia and paxillin does not accumulate at invadopodia.

(A) Representative images of MTLn3 cell transfected with α-Pix-GFP and stained for cortactin and Tks5 as invadopodia markers. White squares boxes are magnified. Arrows point to invadopodia. Scale bar, 10 μm. Representative image set is from 9 image sets. (B) Representative images of MTLn3 cell transfected with β-Pix-GFP and stained for cortactin and Tks5 as invadopodia markers. White square boxes are magnified. Arrows point to invadopodia. Scale bar, 10 μm. Representative image set is from 13 image sets. (C) Western blot of MTLn3 cells transfected with α-Pix,β-Pix or control siRNA and blotted for α-Pix,β-Pix, and β-actin. Representative blot from 1 blot each for the α- and β-Pix. (D) Quantification of the degradation area per field in MTLn3 cells transfected with control, α-Pix, or β-Pix siRNA. n = 40 fields per condition, pooled from 4 independent experiments, with s.e.m. Students t-test was used. ***P = 0.0004 (α-Pix), ***P = 3.37 × 10−5 (β-Pix). (E) Quantification of percentage of invadopodia with α-Pix (left) or β-Pix (right) before and after EGF stimulation for 3 minutes. n = 40 invadopodia for each condition, pooled from 3 independent experiments, with s.e.m. Students t-test was used. P = 0.59 (α-Pix), P = 0.39 (β-Pix). (F) Representative image of an MTLn3 cell plated on Alexa-405 conjugated gelatin matrix and fixed and stained with paxillin, cortactin and Tks5 antibodies. White squares show magnifications of an invadopodium precursor with no matrix degradation activity (left), and a mature invadopodia actively degrading the gelatin matrix. No enrichment of paxillin is seen at the core of the invadopodia in both cases. Scale bar, 10 μm. Representative image set is from 10 image sets.

Supplementary Figure 7 Pak1 and Trio are not required for EGF-stimulated lamellipodia protrusion but Rac1 is required, and Src regulation of cortactin-Tks5-containing structures is independent of Trio-Rac1-Pak1-axis.

(A) MTLn3 cells transfected with control, Rac1, Pak1 or Trio siRNA. Mean cell area over time is plotted for serum-starved MTLn3 cells stimulated with 5nM EGF, n = 10 cells each from one experiment were analyzed, with s.e.m. Students t-test was used. P = 5.06493 × 10−08 (Rac1 KD), P = 1.29769 × 10−05 (Pak1 KD), P = 0.058908959 (Trio KD). (B) Representative images of MTLn3 cell transfected with GFP, wildtype c-Src-GFP (Src-WT-GFP), constitutively activated (Y528F; Src-CA-GFP) or dominant negative (K296R-Y528F; Src-DN-GFP). Cortactin staining is shown. Scale bar, 10 μm. Representative image set is from 44 image sets for Cortactin-GFP-control, from 53 image sets for Cortactin-Src-WT-GFP, from 43 image sets for Cortactin-Src-CA-GFP, and 47 image sets for Cortactin-Src-DN-GFP. (C) Quantification of the average number of cortactin and Tks5 positive structures per cell in MTLn3 cells treated with control, Rac1, Pak1 or Trio siRNA and transfected with GFP, Src-WT-GFP, Src-CA-GFP or Src-DN-GFP. n = 20 cells per condition, pooled from 7 independent experiments, error bars are s.e.m. Students t-test was used. Control siRNA p-values: ***P = 3.3 × 10−15 (Src-WT), ***P = 1.28 × 10−14 (Src-CA), *P = 0.011 (Src-DN). Rac1 siRNA p-values: ***P = 7.38 × 10−18 (Src-WT), ***P = 6.84 × 10−25 (Src-CA), *P = 0.035 (Src-DN). Pak1 siRNA p-values: ***P = 2.35 × 10−10 (Src-WT), ***P = 3.16 × 10−12 (Src-CA), *P = 0.001 (Src-DN). Trio siRNA p-values: ***P = 6.87 × 10−12 (Src-WT), ***P = 4.08 × 10−16 (Src-CA), *P = 0.003 (Src-DN).

Supplementary Figure 8 RhoG is not required for invadopodia matrix degradation activity.

(a) Western blot of RhoG and β-actin protein levels in control and RhoG siRNA treated cells. Representative blot from 1 blot. (b) Quantification of average degradation area per field in control and RhoG siRNA treated cells. n = 40 fields per condition, pooled from 4 independent experiments, error bars are s.e.m. Student’s t-test was used. P = 0.77. Representative image set from 9 cell movies. (c) Representatives images from a time lapse movie of MTLn3 cell transfected with cortactin-mRFP and RhoG-CFP, serum starved then stimulated with EGF for 8 min. White boxes are magnifications showing no enrichment of RhoG at invadopodia. Scalebar, 10 μm.

Supplementary Figure 9 Uncropped images of western blots.

Black box indicated the cropped part included in the corresponding figure.

Supplementary information

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Invadopodia lifetimes are dramatically increased when Rac1 is silenced.

Rac1-depleted MTLn3 cell transfected with two invadopodia markers, TagRFP-cortactin and Tks5-GFP, and time-lapse imaged every 2 min over 6 h. Cortactin and Tks5 fluorescence co-localization show invadopodia. (MOV 8745 kb)

Rac1 activity at invadopodia is abrogated during invadopodium formation.

Serum-starved MTLn3 cell expressing Rac1 biosensor and TagRFP-cortactin stimulated with EGF (zoomed view of single invadopodium). Imaged every 10 s for 10 min. (MOV 705 kb)

Rac1 activity inside the invadopodium is significantly lower compared to the region outside, until when the invadopodium disappears.

MTLn3 cell expressing Rac1 biosensor and TagRFP-cortactin at steady state (zoomed view of single invadopodium). Imaged every 1 min. (MOV 359 kb)

Focal photo-uncaging of PA-Rac1 induces disappearance of invadopodium.

Uncaging Rac1 at a single invadopodium (arrow) in MTLn3 cell expressing PA-Rac1 and TagRFP-cortactin. Imaged every 5 s for 10 min. Photoactivation pulses start at 25 s time point. (MOV 2649 kb)

Photo-uncaging the dark-mutant (C450A) of PA-Rac1 has no effect on invadopodium.

Photoactivation of single invadopodium (arrow) using the C450A light-insensitive PA-Rac1 mutant in an MTLn3 cell expressing TagRFP-cortactin. Imaged every 5 s for 10 min. Photoactivation pulses start at 25 s time point. (MOV 2731 kb)

Depletion of Pak1 prevents disappearance of invadopodium on focal photo-uncaging of PA-Rac1.

Uncaging Rac1 at a single invadopodium (arrow) in an MTLn3 cell treated with Pak1 siRNA and expressing TagRFP-cortactin. Imaged every 5 s for 10 min. Photoactivation pulses start at 25 s time point. (MOV 2476 kb)

Phosphorylation deficient mutant (S113A) of cortactin prevents disappearance of invadopodium on focal photo-uncaging of PA-Rac1.

Uncaging Rac1 at a single invadopodium (arrow) in a cell overexpressing S113A-mutated TagRFP-cortactin. Imaged every 5 s for 10 min. Photoactivation pulses start at 25 s time point. (MOV 2415 kb)

Invadopodia turnover dynamics under control conditions in MTln3 cells.

MTLn3 control cell transfected with two invadopodia markers, TagRFP-cortactin and Tks5-GFP, and time lapse-imaged every 2 min over 6 h. Cortactin and Tks5 fluorescence co-localization show invadopodia. (MOV 3990 kb)

Invadopodia turnover dynamics in MTln3 cells treated with Rac1 inhibitor NSC23766.

MTLn3 cell transfected with two invadopodia markers, TagRFP-cortactin and Tks5-GFP, and time-lapse imaged every 2 min over 6 h, with 100 μM NSC23766 (Rac1 Inhibitor) treatment. (MOV 3802 kb)

Invadopodia turnover dynamics in MTln3 cells treated with Rac1 inhibitor Z62954982.

MTLn3 cell transfected with two invadopodia markers, TagRFP-cortactin and Tks5-GFP, and time-lapse imaged every 2 min over 6 h, with 25 μM Z62954982 (Rac Inhibitor II) treatment. (MOV 4618 kb)

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Moshfegh, Y., Bravo-Cordero, J., Miskolci, V. et al. A Trio–Rac1–Pak1 signalling axis drives invadopodia disassembly. Nat Cell Biol 16, 571–583 (2014). https://doi.org/10.1038/ncb2972

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