An extracellular-matrix-specific GEF–GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration

Abstract

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices1,2,3. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments4,5 and discovered a form of Cdc42–RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF–GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

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Figure 1: Loss of βPix leads to collagen-specific morphological and migratory defects.
Figure 2: βPix controls the activity and localization of Cdc42 during 3D collagen migration.
Figure 3: βPix acts through Cdc42 to suppress and localize RhoA activity during migration in 3D collagen.
Figure 4: A collagen-specific GEF–GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity.
Figure 5: Fibrillar collagen activates βPix through α2β1 integrin, leading to a critical dephosphorylation at Thr 526 through PP2A.

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Acknowledgements

The authors would like to thank W. Daley, R. Petrie and D. Tran for critical reading of the manuscript. This study was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, NIH.

Author information

M.L.K. designed and carried out experiments. M.L.K. and K.M.Y. wrote the manuscript. K.M.Y. directed the project.

Correspondence to Matthew L. Kutys or Kenneth M. Yamada.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1

(a) Schematic diagram of the screen for ECM-specific GEFs. Briefly, HFFs were plated on ECM-coated dishes, allowed to reach steady-state migration overnight in the absence of serum, lysed, and incubated with GST-RacG15A conjugated to beads to extract active GEFs. Beads were analyzed by SDS-PAGE, Coomassie staining, and mass spectrometry of excised protein bands for identification. (b) Western blot confirms up-regulation of SmgGDS binding to RacG15A in the presence of collagen or fibronectin (result was confirmed with three independent experiments). (c) Knockdown of βPix was achieved by generating HFF lines stably expressing either NS shRNA or two βPix shRNA hairpins (shRNA#2 or shRNA#4). Migration experiments were performed using each hairpin and a single siRNA toward βPix, resulting in identical phenotypes. (d) In maximum intensity confocal projections showing all βPix in a cell, the strong nuclear/intracellular membrane localization on both fibronectin and collagen substrates obscures the unique membrane localization in each ECM condition (e.g., Supplementary Fig. 1e). Consequently, we focused on the single confocal section showing plasma membrane-localized βPix (green) and paxillin (red) using composite images at the plasma membrane plane of HFFs migrating on fibronectin (5 image segments in these relatively flat cells) or fibrillar collagen (21 image segments) of 0.2 μm confocal slices to visualize βPix localization at the cell-ECM interface. On fibronectin, βPix was concentrated in focal adhesions (yellow co-localization) with the nuclear staining visible because the cells were flatter. On fibrillar collagen, βPix was uniformly distributed along the plasma membrane with distinct, non-paxillin containing aggregates (green) observed toward the leading edge; nuclear staining was not in the plane of the plasma membrane in these less-flat cells. (e) Maximum confocal projections of βPix (green) and paxillin (red) in fibroblasts migrating on FN and FIB COL. Scale bars, 25 μm. (f) HFFs in 3D collagen and 3D CDM immunostained for endogenous paxillin (red) and βPix (green) display the same loss of adhesion localization as observed on fibronectin and fibrillar collagen (Fig. 1c); yellow indicates co-localization. Scale bars, 25 μm. (g) Live GFP-βPix knockdown/rescue cells expressing mApple-paxillin (red) migrating on fibronectin or fibrillar collagen display loss of adhesion localization; yellow indicates co-localization. Scale bars, 25 μm. (h) Single siRNA knockdown of βPix in human breast adenocarcinoma cells, primary human osteoblasts, human aortic smooth muscle cells, and human umbilical vein endothelial cells revealed collagen-specific morphological and migratory defects between 3D collagen and 3D cell-derived matrix (data not shown for CDM) and (i) 2D fibronectin and fibrillar collagen (green, actin; red, collagen). (j) Western blot confirmation of βPix knockdown using a single βPix siRNA. (k) Quantification of morphology of MDA-MB-231 cells with βPix knockdown in 3D collagen versus 3D cell-derived matrix. Elongated cells defined as having an elliptical factor >1.5. n = 30, 30, 26, and 27 cells for CDM NS, CDM KD, COL NS, and COL KD were assessed across three independent experiments (mean ± s.e.m., t-tests). (l) Quantification of MDA-MB-231 cell velocity with βPix knockdown in 3D cell-derived matrix or 3D collagen. n = 19, 19, 19, and 21 cells for NS CDM, βPix si#1 CDM, NS COL, and βPix si#1 COL were assessed across three independent experiments (mean ± s.e.m., t-tests). Statistical source data can be found in Supplementary Table 2. P < 0.001.

Supplementary Figure 2

(a) Immunostaining of endogenous paxillin, actin, and β-tubulin in HFFs on fibrillar collagen expressing NS or βPix shRNA. The multiple protrusions in βPix knockdown cells have paxillin-containing adhesions, enriched actin fibres, and efficient microtubule targeting. Scale bars, 20 μm. (b) Western blot of fibroblasts expressing NS shRNA, βPix shRNA#2, or βPix knockdown with a GFP-βPix rescue (βPix KDR-WT). GFP marker indicates the successful expression of the rescue construct at near endogenous levels. (c) Quantification of cell velocity of fibroblasts expressing NS shRNA, βPix shRNA, or βPix knockdown/rescue constructs in 3D collagen. n = 25, 24, and 25 cells for NS, βPix sh#2, and KDR-WT were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction). Single siRNA knockdown controls toward (d) Rac1, (e) Cdc42, and (f) Rac3. HFFs do not express Rac2. (g) Knockdown of Rac1 led to no compensatory increase in Rac3 (left) or Rac2 (right) protein levels. (h) Max projections of phalloidin stained HFFs in 3D collagen treated with single siRNAs toward βPix, Rac1, Rac3, or Rac1, Rac2 and Rac3. No Rac siRNA treatment was capable of recapitulating the βPix knockdown morphological phenotype. Scale bars, 25 μm (i) Quantification of migration velocities of GTPase siRNA-treated HFFs in 3D collagen. Two independent siRNAs toward Cdc42 mimic βPix knockdown. Additionally, Rac1 and Rac3 knockdown had no significant effect on HFF migration in 3D collagen. n = 18-25 cells for NS, Rac1 #1, Rac1 #2, Cdc42 #1, Cdc42 #2, Rac3 #1, Rac3 #2, and Rac1-3 #1 siRNAs were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction. (j) βPix specifically binds dominant negative RacG15a and not wild-type Rac1 or constitutively active Rac1 (Q61L) in lysates extracted from cells migrating on collagen. (k) Maximum projections of confocal stacks of live-fibroblast migration expressing a Cdc42 biosensor in 3D cell-derived matrix or 3D collagen. Knockdown of βPix in 3D leads to collagen-specific decreases in Cdc42 activity and loss of leading edge polarization. Scale bars, 25 μm. (l) Average integrated whole cell Cdc42 FRET intensity on FN versus FIB COL. n = 10 cells for NS FN, βPix sh#2 FN, NS FIB COL, and βPix sh#2 FIB COL were assessed across three independent experiments (mean ± s.e.m., t-test). (m) Quantification Cdc42 FRET polarization index on FN versus FIB COL. n = 10 cells for NS FN, βPix sh#2 FN, NS FIB COL, and βPix sh#2 FIB COL were assessed across three independent experiments (mean ± s.e.m., t-test). Statistical source data can be found in Supplementary Table 2. P < 0.001.

Supplementary Figure 3

(a) Immunostaining of βPix (green) and Cdc42 (red) at the leading edge of HFFs on fibronectin or fibrillar collagen. Migration on fibrillar collagen revealed increased but partial co-localization between βPix and Cdc42 (yellow, white arrows) in comparison to cells on fibronectin Scale bars, 10 μm. (b) Maximum projections of confocal stacks of live-fibroblast migration expressing a RhoA biosensor in 3D cell-derived matrix or 3D collagen. Knockdown of βPix in 3D leads to similar collagen-specific increases in RhoA activity and loss of front-back polarization of RhoA activity. Scale bars, 25 μm. (c) Low-level overexpression of GFP-RhoAQ63L (greyscale, green) during 3D collagen migration (left). We find that similar to βPix and Cdc42 knockdown, RhoAQ63L leads to a rounded, notably hyper-protrusive cell with significant remodelling of collagen fibres (red; holes in the collagen matrix, white asterisks). Expressing RhoAQ63L at comparable levels in HFFs migrating in cell-derived matrix (right) does not perturb morphology or lead to hyper-protrusive behaviors. Scale bar, 25 μm. (d) Max projections of phalloidin-stain (green) βPix knockdown cells in 3D collagen (red) treated with inhibitors of RhoA (cell-permeable C3 transferase, 2 μg ml−1) or myosin II (blebbistatin, 20 μM) (top). Representative migratory tracks of each condition. We find that direct inhibition of RhoA with C3 transferase significantly rescues the motility of βPix knockdown, while blebbistatin rescues to a lesser degree (bottom). (e) RhoA activity determined using GST-RBD binding from NS and βPix shRNA-expressing fibroblasts migrating in 3D or fibrillar collagen environments. Representative of three independent experiments. (f) Single siRNA knockdown controls toward srGAP1. (g) Quantification of migration velocities of srGAP1 siRNA-treated HFFs in 3D collagen. 21, n = 24, 22, and 19 cells for NS, βPix si#1, srGAP1 si#1, and srGAP1 si#2 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction). (h) srGAP1 has been reported to have GAP activity toward Rac1. Active Rac1 was isolated using GST-PBD from NS and srGAP1 siRNA-treated fibroblasts migrating on fibronectin (FN) or fibrillar collagen (FIB COL). Confirming previous reports, we observed an increase in Rac1 activity with srGAP1 knockdown during migration on fibronectin, but not during collagen migration. Blot representative of two independent experiments. Statistical source data can be found in Supplementary Table 2, P < 0.001.

Supplementary Figure 4

(a) GFP-βPix knockdown/rescue cells were allowed to reach steady-state migration on fibronectin (FN) or fibrillar collagen (FIB COL). GFP-βPix was immunoprecipitated from cell lysates under each condition to search for matrix-specific associated proteins. Coomassie blue staining of protein bound to βPix revealed a unique 130 kDa band (∼65 kDa band (b) HFFs were cultured overnight in 3D collagen gels, incubated with inhibitory integrin antibodies, (β1-mAb 13, α5-mAb 16, or α2-P1E6) and allowed to migrate for a further 12–16 h. Inhibition of β1 orα2, but not α5, inhibited cell migration and spreading in 3D collagen. Experiment was performed independently at least twice with identical observations. (c) GFP-βPix isolated from knockdown/rescue fibroblasts migrating on fibronectin or fibrillar collagen was analyzed for candidate phosphopeptides that were unique to each ECM. The resulting phosphopeptides are displayed in tabular form, showing the peptide sequence with modified residue in lowercase letters, MASCOT ion score, specific modifications, and delta PPM of each peptide spectra. The two unique phospho-threonine peptides isolated while on fibronectin are outlined in red. (d) Quantification of cell elliptical factor 3D collagen of βPix knockdown/rescue cells expressing wild-type βPix and the phosphorylation variants. n = 24, 20, 23, and 19 cells for βPix sh#2, KDR-WT, T526E, and T526A were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). (e) Max projections of phalloidin-stained MDA-MB-231 knockdown/rescue cells expressing wild-type βPix or the βPix phosphorylation variants migrating in 3D collagen. Scale bars, 50 μm. (f) Quantification of morphology of MDA-MB-231 knockdown/rescue cells expressing wild-type βPix and the phosphorylation variants migrating in 3D collagen. Elongated cells defined as having an elliptical factor > 1.5. n = 34, 36, and 38 cells for WT, T526E, and T526A were assessed across three independent experiments. (g) Quantification of migration velocities of MDA-MB-231 knockdown/rescue cells expressing wild-type βPix or the phosphorylation variants migrating in 3D collagen. n = 23, 21, and 25 cells for WT, T526E, and T526A were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction). (h) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue MDA-MB-231 migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) additionally shows collagen-specific associations between βPix and PPP2R1A/srGAP1. Blot representative of three independent experiments. Statistical source data can be found in Supplementary Table 2, P < 0.001.

Supplementary Figure 5

(a) Single siRNA knockdown of PPP2R1A with two independent sequences. (b) Maximum projection of phalloidin-stained (green) HFFs in 3D collagen (red, reflection) or 3D cell-derived matrix (red, reflection) treated with DMSO or with the PP2A inhibitor okadaic acid (1 nM) overnight prior to fixation. Inhibition of PP2A resulted in collagen-specific morphological defects. Scale bars 25 μm. (c) Maximum projection of phalloidin-stained (green) HFFs in 3D collagen (red, reflection) or 3D cell-derived matrix (red, fibronectin immunostaining) treated with NS or with PPP2R1A siRNA #1. Scale bars 25 μm. (d) Quantification of morphology of PPP2R1A siRNA-treated HFFs in 3D collagen. n = 19-32 cells for NS, PPP2R1A si#1, PPP2R1A si#2 (both CDM and COL) were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction). (e) Quantification of migration velocities of PPP2R1A siRNA-treated HFFs in 3D collagen. n = 16-22 cells for NS, PPP2R1A si#1, PPP2R1A si#2 (both CDM and COL) were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni correction). (f) Quantification of migration velocities of okadaic acid-treated (1 nM) HFFs in 3D collagen and 3D cell-derived matrix. n = 19, 20, 20, and 20 cells for DMSO CDM, OKA CDM, DMSO COL, and OKA COL were assessed across three independent experiments (mean ± s.e.m., t-tests). (g) GFP-βPix knockdown/rescue fibroblasts migrating on fibrillar collagen were treated with DMSO or the PP2A inhibitor okadaic acid (OKA, 1 nM). Inhibition of PP2A with okadaic acid increased phospho-threonine levels on βPix during migration on collagen. Representative of at least three independent experiments. (h) Maximum projection of phalloidin-stained KDR-WT or KDR-T526A HFFs treated with NS or PPP2R1A siRNA #1 migrating in 3D collagen. Scale bars, 50 μm. (i) Morphological quantification of KDR-WT or KDR-T526A HFFs treated with NS or PPP2R1A siRNA #1 in 3D collagen. n = 40, 31, 35, and 38 cells for WT, WT + PPP2R1A si#1, T526A, and T526A + PPP2R1A si#1 were assessed across three independent experiments (mean ± s.e.m., t-tests). (j) Quantification of cell velocities in KDR-WT or KDR-T526A HFFs treated with NS or PPP2R1A siRNA #1 in 3D collagen. n = 26, 24, 24, and 24 cells for WT, WT + PPP2R1A si#1, T526A, and T526A + PPP2R1A si#1 were assessed across three independent experiments (mean ± s.e.m., t-tests). Statistical source data can be found in Supplementary Table 2, P < 0.001,P < 0.05.

Supplementary Figure 6

Full scans of all blots that were presented in cropped form in figures in the primary and supplemental manuscript texts.

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Loss of βPix leads to severe, matrix-specific migratory defects in 3D collagen matrices.

NS and βPix shRNA-expressing fibroblasts were allowed to spread in 3D collagen gels overnight and assayed for motility the following day. We find that βPix knockdown cells lack any form of persistent cell motility, and they are characterized by hyperactive spatially and temporally deregulated protrusions, rounded cell morphology, lack of defined leading and trailing edges, and elevated matrix contractility. Cells were allowed to migrate overnight in collagen and 24 hour timelapse was started the following morning, 5 min frame rate, accelerated 5000x for display. Scale bar indicates 50 μm. (MOV 660 kb)

βPix is critical for migration in fibrillar collagen environments.

Thin, fibrillar collagen substrates improved for optical imaging, but they retain the fibrillar structure of 3D collagen gels. We find that fibrillar collagen substrates are sufficient to recapitulate the βPix knockdown phenotype observed in 3D collagen, including the spatially and temporally deregulated protrusions, rounded morphology, lack of defined leading and trailing edges, and especially increased collagen contraction. Notably, the βPix knockdowns can be observed tearing holes in the fibrillar collagen matrix. Cells were allowed to migrate overnight in collagen and 24 hour timelapse was started the following morning, 5 minute frame rate, accelerated 5000x for display. Scale bars indicate 50 μm. (MOV 705 kb)

Cdc42, but not Rac1, phenocopies βPix knockdown in fibrillar collagen environments.

Fibroblasts treated with NS, βPix, Cdc42, or Rac1 siRNA were plated on fibrillar collagen substrates and assayed for migration. Cdc42 knockdown effectively mimics βPix knockdown in fibrillar collagen environments, while Rac1 knockdown does not. Both βPix and Cdc42 knockdowns adhere to the surrounding collagen fibres and visibly tear physical holes in their adjacent regions. Cells were allowed to migrate overnight in collagen and 24 hour timelapse was started the following morning, 10 minute frame rate, accelerated 5000x for display. Scale bar indicates 50 μm. (MOV 388 kb)

Low-level overexpression of constitutively-active RhoA is sufficient to mimic βPix knockdown in fibrillar collagen environments.

GFP-RhoAQ63L was transfected into HFFs, which were then plated on fibrillar collagen (FIB COL) substrate (center cell) or 3D collagen gels. Cells expressing low levels of GFP-RhoAQ63L as indicated by fluorescence intensity exhibited hyper-protrusive activity, increased collagen gel contraction (Supplementary Fig. 3c), and inability to migrate efficiently. Cells were allowed to migrate overnight in collagen and 24 hour timelapse was started the following morning, 10 minute frame rate, accelerated 5000x for display. Scale bars indicate 50 μm. (MOV 206 kb)

Knockdown of srGAP1 phenocopies the migratory defects of Cdc42 or βPix knockdown in 3D collagen.

HFFs were treated with NS or srGAP1 siRNA and allowed to spread overnight in 3D collagen gels. Migration assays revealed hyper-protrusive activity, increased collagen gel contraction (Fig. 4d and evident in movie), and an inability to migrate efficiently. Cells were allowed to migrate overnight in collagen and 24 hour timelapse was started the following morning; 5 min frame rate, accelerated 5000x for display. Scale bar indicates 50 μm. (MOV 1132 kb)

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Kutys, M., Yamada, K. An extracellular-matrix-specific GEF–GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration. Nat Cell Biol 16, 909–917 (2014). https://doi.org/10.1038/ncb3026

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