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

Kutys ML, Yamada KM - Nat. Cell Biol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.

ABSTRACT
Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments 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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Fibrillar collagen activates βPix throughα2β1 integrin, leading to a criticaldephosphorylation at T526 through PP2A. (a) Loss of focal adhesionlocalization is a read-out of differential βPix function on fibrillarcollagen (Fig. 1c). Dishes were coated withmonoclonal integrin antibodies targeting β1 (9EG7),α5 (mAb 16), or α2 (P1E6) to mimicintegrin ligation. GFP-βPix knockdown/rescue cells were plated on thedishes and assayed for focal adhesion localization (red; yellow in overlay).Ligation of α2 results in a dramatic loss in GFP-βPix(grayscale) localization at paxillin (red)-containing adhesions with no changesin overall focal adhesion profile. Scale bars, 25 μm. (b)Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescuecells migrating on fibronectin or fibrillar collagen for phospho-threonineshowed a decrease in phosphorylation levels during migration on collagen.Immunoprecipitation of KDR-T526A βPix showed no change inphospho-threonine between FN and FIB COL, highlighting the functional importanceof this residue. (c) We generated phospho-mimetic (T526E) andphospho- (T526A) mutant βPix knockdown/rescue cells and assayed theirmorphology in 3D collagen. T526E βPix was insufficient to rescue themorphological and hypercontractile phenotype of βPix knockdown (collagenfibers, red, reflection microscopy). T526A mutants efficiently rescued theβPix morphological and contractile defects. Scale bars, 25 μm.(d) Quantification of cell velocity in βPixknockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cellsfor βPix sh#2, WT, T526E, and T526A were assessed across threeindependent experiments (mean ± s.e.m., one-way ANOVA with Bonferronimultiple comparisons correction). (e) GFP-βPix wasimmunoprecipitated from HFFs expressing knockdown/rescue phosphovariants atThr526 migrating on fibrillar collagen. We find that phosphorylationmimetic(T526E) inhibits binding to srGAP1, but not Cdc42. (f)Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cellsmigrating on fibronectin versus fibrillar collagen identified acollagen-specific interaction between βPix and PP2A regulatory subunit Aα isoform (PPP2R1A). (g) GFP-βPix knockdown/rescuefibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1AsiRNA #1. We observed that knockdown or inhibition (Supplementary Fig. 5g) ofPPP2R1A increased phosphothreonine levels on βPix during migration oncollagen. (h) Summary model of the collagen-specific role ofβPix during migration in fibrillar collagen environments. All westernblots are representative of at least three independent experiments. Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
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Figure 5: Fibrillar collagen activates βPix throughα2β1 integrin, leading to a criticaldephosphorylation at T526 through PP2A. (a) Loss of focal adhesionlocalization is a read-out of differential βPix function on fibrillarcollagen (Fig. 1c). Dishes were coated withmonoclonal integrin antibodies targeting β1 (9EG7),α5 (mAb 16), or α2 (P1E6) to mimicintegrin ligation. GFP-βPix knockdown/rescue cells were plated on thedishes and assayed for focal adhesion localization (red; yellow in overlay).Ligation of α2 results in a dramatic loss in GFP-βPix(grayscale) localization at paxillin (red)-containing adhesions with no changesin overall focal adhesion profile. Scale bars, 25 μm. (b)Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescuecells migrating on fibronectin or fibrillar collagen for phospho-threonineshowed a decrease in phosphorylation levels during migration on collagen.Immunoprecipitation of KDR-T526A βPix showed no change inphospho-threonine between FN and FIB COL, highlighting the functional importanceof this residue. (c) We generated phospho-mimetic (T526E) andphospho- (T526A) mutant βPix knockdown/rescue cells and assayed theirmorphology in 3D collagen. T526E βPix was insufficient to rescue themorphological and hypercontractile phenotype of βPix knockdown (collagenfibers, red, reflection microscopy). T526A mutants efficiently rescued theβPix morphological and contractile defects. Scale bars, 25 μm.(d) Quantification of cell velocity in βPixknockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cellsfor βPix sh#2, WT, T526E, and T526A were assessed across threeindependent experiments (mean ± s.e.m., one-way ANOVA with Bonferronimultiple comparisons correction). (e) GFP-βPix wasimmunoprecipitated from HFFs expressing knockdown/rescue phosphovariants atThr526 migrating on fibrillar collagen. We find that phosphorylationmimetic(T526E) inhibits binding to srGAP1, but not Cdc42. (f)Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cellsmigrating on fibronectin versus fibrillar collagen identified acollagen-specific interaction between βPix and PP2A regulatory subunit Aα isoform (PPP2R1A). (g) GFP-βPix knockdown/rescuefibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1AsiRNA #1. We observed that knockdown or inhibition (Supplementary Fig. 5g) ofPPP2R1A increased phosphothreonine levels on βPix during migration oncollagen. (h) Summary model of the collagen-specific role ofβPix during migration in fibrillar collagen environments. All westernblots are representative of at least three independent experiments. Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.

Mentions: Having identified a collagen-specific role for βPix, we searched formechanisms regulating βPix in different matrix conditions. We first tested forintegrin-specific regulation of βPix using certain anti-integrin monoclonalantibodies that can mimic full integrin ligation and adhesive function29. Using loss of focal adhesionlocalization as a read-out of signaling to βPix (as observed on fibrillarcollagen, Fig. 1c), we assayed the localization ofGFP-βPix in knockdown/rescue cells migrating on substrates coated with antibodiestoward β1, α5, and α2 integrin. GFP-βPix stronglyco-localized to focal adhesions stained for paxillin on glass or substrates targetingβ1 and α5 integrin (Fig. 5a). However, on substrates targeting α2integrin, GFP-βPix localization to focal adhesions was greatly diminished, eventhough paxillin-containing focal adhesions were formed normally. Conversely, treatmentof cells migrating in 3D collagen with inhibitory antibodies against specific integrinsconfirmed specificity for the α2β1 integrin byblocking migration (Supplemental Fig.4b). Thus, the α2 subunit ofα2β1 integrin is important for mediatingβPix function during migration in fibrillar collagen environments.


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

Kutys ML, Yamada KM - Nat. Cell Biol. (2014)

Fibrillar collagen activates βPix throughα2β1 integrin, leading to a criticaldephosphorylation at T526 through PP2A. (a) Loss of focal adhesionlocalization is a read-out of differential βPix function on fibrillarcollagen (Fig. 1c). Dishes were coated withmonoclonal integrin antibodies targeting β1 (9EG7),α5 (mAb 16), or α2 (P1E6) to mimicintegrin ligation. GFP-βPix knockdown/rescue cells were plated on thedishes and assayed for focal adhesion localization (red; yellow in overlay).Ligation of α2 results in a dramatic loss in GFP-βPix(grayscale) localization at paxillin (red)-containing adhesions with no changesin overall focal adhesion profile. Scale bars, 25 μm. (b)Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescuecells migrating on fibronectin or fibrillar collagen for phospho-threonineshowed a decrease in phosphorylation levels during migration on collagen.Immunoprecipitation of KDR-T526A βPix showed no change inphospho-threonine between FN and FIB COL, highlighting the functional importanceof this residue. (c) We generated phospho-mimetic (T526E) andphospho- (T526A) mutant βPix knockdown/rescue cells and assayed theirmorphology in 3D collagen. T526E βPix was insufficient to rescue themorphological and hypercontractile phenotype of βPix knockdown (collagenfibers, red, reflection microscopy). T526A mutants efficiently rescued theβPix morphological and contractile defects. Scale bars, 25 μm.(d) Quantification of cell velocity in βPixknockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cellsfor βPix sh#2, WT, T526E, and T526A were assessed across threeindependent experiments (mean ± s.e.m., one-way ANOVA with Bonferronimultiple comparisons correction). (e) GFP-βPix wasimmunoprecipitated from HFFs expressing knockdown/rescue phosphovariants atThr526 migrating on fibrillar collagen. We find that phosphorylationmimetic(T526E) inhibits binding to srGAP1, but not Cdc42. (f)Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cellsmigrating on fibronectin versus fibrillar collagen identified acollagen-specific interaction between βPix and PP2A regulatory subunit Aα isoform (PPP2R1A). (g) GFP-βPix knockdown/rescuefibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1AsiRNA #1. We observed that knockdown or inhibition (Supplementary Fig. 5g) ofPPP2R1A increased phosphothreonine levels on βPix during migration oncollagen. (h) Summary model of the collagen-specific role ofβPix during migration in fibrillar collagen environments. All westernblots are representative of at least three independent experiments. Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
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Figure 5: Fibrillar collagen activates βPix throughα2β1 integrin, leading to a criticaldephosphorylation at T526 through PP2A. (a) Loss of focal adhesionlocalization is a read-out of differential βPix function on fibrillarcollagen (Fig. 1c). Dishes were coated withmonoclonal integrin antibodies targeting β1 (9EG7),α5 (mAb 16), or α2 (P1E6) to mimicintegrin ligation. GFP-βPix knockdown/rescue cells were plated on thedishes and assayed for focal adhesion localization (red; yellow in overlay).Ligation of α2 results in a dramatic loss in GFP-βPix(grayscale) localization at paxillin (red)-containing adhesions with no changesin overall focal adhesion profile. Scale bars, 25 μm. (b)Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescuecells migrating on fibronectin or fibrillar collagen for phospho-threonineshowed a decrease in phosphorylation levels during migration on collagen.Immunoprecipitation of KDR-T526A βPix showed no change inphospho-threonine between FN and FIB COL, highlighting the functional importanceof this residue. (c) We generated phospho-mimetic (T526E) andphospho- (T526A) mutant βPix knockdown/rescue cells and assayed theirmorphology in 3D collagen. T526E βPix was insufficient to rescue themorphological and hypercontractile phenotype of βPix knockdown (collagenfibers, red, reflection microscopy). T526A mutants efficiently rescued theβPix morphological and contractile defects. Scale bars, 25 μm.(d) Quantification of cell velocity in βPixknockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cellsfor βPix sh#2, WT, T526E, and T526A were assessed across threeindependent experiments (mean ± s.e.m., one-way ANOVA with Bonferronimultiple comparisons correction). (e) GFP-βPix wasimmunoprecipitated from HFFs expressing knockdown/rescue phosphovariants atThr526 migrating on fibrillar collagen. We find that phosphorylationmimetic(T526E) inhibits binding to srGAP1, but not Cdc42. (f)Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cellsmigrating on fibronectin versus fibrillar collagen identified acollagen-specific interaction between βPix and PP2A regulatory subunit Aα isoform (PPP2R1A). (g) GFP-βPix knockdown/rescuefibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1AsiRNA #1. We observed that knockdown or inhibition (Supplementary Fig. 5g) ofPPP2R1A increased phosphothreonine levels on βPix during migration oncollagen. (h) Summary model of the collagen-specific role ofβPix during migration in fibrillar collagen environments. All westernblots are representative of at least three independent experiments. Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
Mentions: Having identified a collagen-specific role for βPix, we searched formechanisms regulating βPix in different matrix conditions. We first tested forintegrin-specific regulation of βPix using certain anti-integrin monoclonalantibodies that can mimic full integrin ligation and adhesive function29. Using loss of focal adhesionlocalization as a read-out of signaling to βPix (as observed on fibrillarcollagen, Fig. 1c), we assayed the localization ofGFP-βPix in knockdown/rescue cells migrating on substrates coated with antibodiestoward β1, α5, and α2 integrin. GFP-βPix stronglyco-localized to focal adhesions stained for paxillin on glass or substrates targetingβ1 and α5 integrin (Fig. 5a). However, on substrates targeting α2integrin, GFP-βPix localization to focal adhesions was greatly diminished, eventhough paxillin-containing focal adhesions were formed normally. Conversely, treatmentof cells migrating in 3D collagen with inhibitory antibodies against specific integrinsconfirmed specificity for the α2β1 integrin byblocking migration (Supplemental Fig.4b). Thus, the α2 subunit ofα2β1 integrin is important for mediatingβPix function during migration in fibrillar collagen environments.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.

ABSTRACT
Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments 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.

Show MeSH
Related in: MedlinePlus