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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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βPix controls the activity and localization of Cdc42 during 3D collagenmigration. (a) Active Rac1 and Cdc42 were isolated using GST-PBDfrom NS and βPix shRNA-expressing HFFs migrating on fibronectin (FN) orfibrillar collagen (FIB COL). (b) Quantification of western blotband intensity revealed collagen-specific losses in both Rac1 (~20%) andCdc42 (~30%) activity after depletion of βPix (n = 3 independentwestern blots, mean ± s.e.m, t-tests). (c)βPix also binds specifically to recombinant Cdc42G15A in lysates fromcells migrating on collagen, but not fibronectin. Result represents threeindependent experiments. (d) Single, independent siRNA treatments(10 nM) targeting Rac1 or Cdc42 were sufficient to deplete endogenous proteinlevels. (e) siRNA-treated HFFs were embedded in 3D collagen gelsand incubated overnight in complete media. Cells were then fixed and stainedwith rhodamine-phalloidin. Maximum projections of 150 μm sections of theactin-labeled gels revealed that knockdown of Cdc42 mimicked βPixknockdown morphology, with no defects observed with Rac1 knockdown. Scale bars,50 μm. (f) Higher-power images of actin-labeled (green),siRNA-treated fibroblasts in relation to the surrounding collagen fibers (red,reflection microscopy). Knockdown of Cdc42 mimics the morphology, protrusive,and highly contractile phenotype of βPix knockdown. Holes torn in thecollagen matrix are indicated by white asterisks; scale bars, 25 μm.(g) Maximum projections of confocal stacks of live-fibroblastmigration expressing a Cdc42 biosensor on fibronectin or fibrillar collagen.Active Cdc42 is polarized toward the leading edges during migration onfibronectin in fibroblasts expressing NS or βPix shRNA. After knockdownof βPix on collagen, polarization of Cdc42 activity is lost, and overallactivity is decreased. Pseudocolor intensity scales were maintained for eachmatrix condition; scale bars, 25 μm. White arrows designate direction ofleading edge protrusions. (h) Quantification of cell ellipticalfactor (maximal length/width) in 3D collagen after Rac1 or Cdc42 siRNAtreatments. n = 35, 30, 35, and 31 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. (i)Quantification of cell velocity in 3D collagen for Rac1 or Cdc42 siRNAtreatments. n = 25, 24, 22, and 24 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. For (h) and(i), data given as mean ± s.e.m., one-way ANOVA withBonferroni multiple comparisons correction. Statistical source data can be foundin Supplementary Table2, *** P < 0.001 * P< 0.05.
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Figure 2: βPix controls the activity and localization of Cdc42 during 3D collagenmigration. (a) Active Rac1 and Cdc42 were isolated using GST-PBDfrom NS and βPix shRNA-expressing HFFs migrating on fibronectin (FN) orfibrillar collagen (FIB COL). (b) Quantification of western blotband intensity revealed collagen-specific losses in both Rac1 (~20%) andCdc42 (~30%) activity after depletion of βPix (n = 3 independentwestern blots, mean ± s.e.m, t-tests). (c)βPix also binds specifically to recombinant Cdc42G15A in lysates fromcells migrating on collagen, but not fibronectin. Result represents threeindependent experiments. (d) Single, independent siRNA treatments(10 nM) targeting Rac1 or Cdc42 were sufficient to deplete endogenous proteinlevels. (e) siRNA-treated HFFs were embedded in 3D collagen gelsand incubated overnight in complete media. Cells were then fixed and stainedwith rhodamine-phalloidin. Maximum projections of 150 μm sections of theactin-labeled gels revealed that knockdown of Cdc42 mimicked βPixknockdown morphology, with no defects observed with Rac1 knockdown. Scale bars,50 μm. (f) Higher-power images of actin-labeled (green),siRNA-treated fibroblasts in relation to the surrounding collagen fibers (red,reflection microscopy). Knockdown of Cdc42 mimics the morphology, protrusive,and highly contractile phenotype of βPix knockdown. Holes torn in thecollagen matrix are indicated by white asterisks; scale bars, 25 μm.(g) Maximum projections of confocal stacks of live-fibroblastmigration expressing a Cdc42 biosensor on fibronectin or fibrillar collagen.Active Cdc42 is polarized toward the leading edges during migration onfibronectin in fibroblasts expressing NS or βPix shRNA. After knockdownof βPix on collagen, polarization of Cdc42 activity is lost, and overallactivity is decreased. Pseudocolor intensity scales were maintained for eachmatrix condition; scale bars, 25 μm. White arrows designate direction ofleading edge protrusions. (h) Quantification of cell ellipticalfactor (maximal length/width) in 3D collagen after Rac1 or Cdc42 siRNAtreatments. n = 35, 30, 35, and 31 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. (i)Quantification of cell velocity in 3D collagen for Rac1 or Cdc42 siRNAtreatments. n = 25, 24, 22, and 24 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. For (h) and(i), data given as mean ± s.e.m., one-way ANOVA withBonferroni multiple comparisons correction. Statistical source data can be foundin Supplementary Table2, *** P < 0.001 * P< 0.05.

Mentions: Because βPix is a dual specificity GEF21, we tested its effects on Rac1 and Cdc42 activity duringmigration in fibrillar collagen microenvironments. βPix bound specifically to thenucleotide-free mutant of Rac1, with no binding to recombinant wild-type or aconstitutively active mutant (Supplementary Fig. 2j). Consistent with its reported function as aRac1/Cdc42 GEF21, βPixknockdown resulted in collagen-specific decreases in both Rac1 (~20%) and Cdc42(~30%) activities (Fig. 2a,b). Similar toRacG15A, βPix differentially bound to recombinant Cdc42G15A (Fig. 2c). It displayed increased but partial co-localization withCdc42 in leading edge protrusions (Supplementary Fig. 3a) during migration on fibrillar collagen, but notfibronectin. Independent single-siRNA knockdowns for each protein were performed todetermine whether depletion of Rac1 or Cdc42 would recapitulate the βPixknockdown phenotype in 3D collagen (Fig. 2d, Supplementary Fig. 2d-i).Surprisingly, we found that knockdown of Cdc42, but not Rac1, fully mimicked βPixknockdown in 3D collagen. While Rac1 knockdown cells mirrored nonspecific siRNAcontrols, Cdc42 knockdowns displayed the rounded, hyper-contractile morphology observedwith loss of βPix (Fig. 2e,f,h). We usedmultiple Rac-isoform knockdowns to rule out compensatory roles of other Rac isoforms(Rac2, Rac3) after Rac1 knockdown (Supplementary Fig. 2d-i), indicating that the collagen-βPix knockdownphenotype was due to loss of Cdc42 activity, but not Rac1. This deregulated protrusivebehavior of Cdc42 or βPix-depleted cells was accompanied by defective migrationin both 3D and thin fibrillar collagen environments (Supplementary Movie 3, Fig. 2i), along with physical tearing of holes inthe surrounding matrix. These findings are consistent with a report that loss of Cdc42in 3D microenvironments leads to temporally and spatially deregulated protrusions andimpaired leading edge coordination22.We therefore investigated whether βPix regulates the localization and activity ofCdc42 under different ECM conditions. Imaging a single-chain Cdc42 biosensor based onintramolecular fluorescence resonance energy transfer (FRET)23 revealed that on fibronectin, Cdc42 activity remainspolarized toward the leading edge of migrating cells expressing either nonspecific orβPix shRNA (Fig. 2g). On collagen, Cdc42activity was also polarized to the leading edge in the same regions where βPixwas found to uniquely localize on the membrane. In contrast, βPix knockdown onfibrillar collagen led to a loss of this polarization and decreased overall Cdc42activity (Fig. 2g, Supplementary Fig. 2l,m).Additionally, we observed similar collagen-specific decreases in Cdc42 FRET and loss ofFRET polarization in 3D collagen, but not 3D cell-derived matrix (Supplementary Fig. 2k), furtherestablishing that βPix acts through Cdc42, but not Rac1, to coordinate migrationin 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)

βPix controls the activity and localization of Cdc42 during 3D collagenmigration. (a) Active Rac1 and Cdc42 were isolated using GST-PBDfrom NS and βPix shRNA-expressing HFFs migrating on fibronectin (FN) orfibrillar collagen (FIB COL). (b) Quantification of western blotband intensity revealed collagen-specific losses in both Rac1 (~20%) andCdc42 (~30%) activity after depletion of βPix (n = 3 independentwestern blots, mean ± s.e.m, t-tests). (c)βPix also binds specifically to recombinant Cdc42G15A in lysates fromcells migrating on collagen, but not fibronectin. Result represents threeindependent experiments. (d) Single, independent siRNA treatments(10 nM) targeting Rac1 or Cdc42 were sufficient to deplete endogenous proteinlevels. (e) siRNA-treated HFFs were embedded in 3D collagen gelsand incubated overnight in complete media. Cells were then fixed and stainedwith rhodamine-phalloidin. Maximum projections of 150 μm sections of theactin-labeled gels revealed that knockdown of Cdc42 mimicked βPixknockdown morphology, with no defects observed with Rac1 knockdown. Scale bars,50 μm. (f) Higher-power images of actin-labeled (green),siRNA-treated fibroblasts in relation to the surrounding collagen fibers (red,reflection microscopy). Knockdown of Cdc42 mimics the morphology, protrusive,and highly contractile phenotype of βPix knockdown. Holes torn in thecollagen matrix are indicated by white asterisks; scale bars, 25 μm.(g) Maximum projections of confocal stacks of live-fibroblastmigration expressing a Cdc42 biosensor on fibronectin or fibrillar collagen.Active Cdc42 is polarized toward the leading edges during migration onfibronectin in fibroblasts expressing NS or βPix shRNA. After knockdownof βPix on collagen, polarization of Cdc42 activity is lost, and overallactivity is decreased. Pseudocolor intensity scales were maintained for eachmatrix condition; scale bars, 25 μm. White arrows designate direction ofleading edge protrusions. (h) Quantification of cell ellipticalfactor (maximal length/width) in 3D collagen after Rac1 or Cdc42 siRNAtreatments. n = 35, 30, 35, and 31 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. (i)Quantification of cell velocity in 3D collagen for Rac1 or Cdc42 siRNAtreatments. n = 25, 24, 22, and 24 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. For (h) and(i), data given as mean ± s.e.m., one-way ANOVA withBonferroni multiple comparisons correction. Statistical source data can be foundin Supplementary Table2, *** P < 0.001 * P< 0.05.
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Figure 2: βPix controls the activity and localization of Cdc42 during 3D collagenmigration. (a) Active Rac1 and Cdc42 were isolated using GST-PBDfrom NS and βPix shRNA-expressing HFFs migrating on fibronectin (FN) orfibrillar collagen (FIB COL). (b) Quantification of western blotband intensity revealed collagen-specific losses in both Rac1 (~20%) andCdc42 (~30%) activity after depletion of βPix (n = 3 independentwestern blots, mean ± s.e.m, t-tests). (c)βPix also binds specifically to recombinant Cdc42G15A in lysates fromcells migrating on collagen, but not fibronectin. Result represents threeindependent experiments. (d) Single, independent siRNA treatments(10 nM) targeting Rac1 or Cdc42 were sufficient to deplete endogenous proteinlevels. (e) siRNA-treated HFFs were embedded in 3D collagen gelsand incubated overnight in complete media. Cells were then fixed and stainedwith rhodamine-phalloidin. Maximum projections of 150 μm sections of theactin-labeled gels revealed that knockdown of Cdc42 mimicked βPixknockdown morphology, with no defects observed with Rac1 knockdown. Scale bars,50 μm. (f) Higher-power images of actin-labeled (green),siRNA-treated fibroblasts in relation to the surrounding collagen fibers (red,reflection microscopy). Knockdown of Cdc42 mimics the morphology, protrusive,and highly contractile phenotype of βPix knockdown. Holes torn in thecollagen matrix are indicated by white asterisks; scale bars, 25 μm.(g) Maximum projections of confocal stacks of live-fibroblastmigration expressing a Cdc42 biosensor on fibronectin or fibrillar collagen.Active Cdc42 is polarized toward the leading edges during migration onfibronectin in fibroblasts expressing NS or βPix shRNA. After knockdownof βPix on collagen, polarization of Cdc42 activity is lost, and overallactivity is decreased. Pseudocolor intensity scales were maintained for eachmatrix condition; scale bars, 25 μm. White arrows designate direction ofleading edge protrusions. (h) Quantification of cell ellipticalfactor (maximal length/width) in 3D collagen after Rac1 or Cdc42 siRNAtreatments. n = 35, 30, 35, and 31 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. (i)Quantification of cell velocity in 3D collagen for Rac1 or Cdc42 siRNAtreatments. n = 25, 24, 22, and 24 cells for NS, βPix, Rac1, Cdc42 siRNAwere assessed across three independent experiments. For (h) and(i), data given as mean ± s.e.m., one-way ANOVA withBonferroni multiple comparisons correction. Statistical source data can be foundin Supplementary Table2, *** P < 0.001 * P< 0.05.
Mentions: Because βPix is a dual specificity GEF21, we tested its effects on Rac1 and Cdc42 activity duringmigration in fibrillar collagen microenvironments. βPix bound specifically to thenucleotide-free mutant of Rac1, with no binding to recombinant wild-type or aconstitutively active mutant (Supplementary Fig. 2j). Consistent with its reported function as aRac1/Cdc42 GEF21, βPixknockdown resulted in collagen-specific decreases in both Rac1 (~20%) and Cdc42(~30%) activities (Fig. 2a,b). Similar toRacG15A, βPix differentially bound to recombinant Cdc42G15A (Fig. 2c). It displayed increased but partial co-localization withCdc42 in leading edge protrusions (Supplementary Fig. 3a) during migration on fibrillar collagen, but notfibronectin. Independent single-siRNA knockdowns for each protein were performed todetermine whether depletion of Rac1 or Cdc42 would recapitulate the βPixknockdown phenotype in 3D collagen (Fig. 2d, Supplementary Fig. 2d-i).Surprisingly, we found that knockdown of Cdc42, but not Rac1, fully mimicked βPixknockdown in 3D collagen. While Rac1 knockdown cells mirrored nonspecific siRNAcontrols, Cdc42 knockdowns displayed the rounded, hyper-contractile morphology observedwith loss of βPix (Fig. 2e,f,h). We usedmultiple Rac-isoform knockdowns to rule out compensatory roles of other Rac isoforms(Rac2, Rac3) after Rac1 knockdown (Supplementary Fig. 2d-i), indicating that the collagen-βPix knockdownphenotype was due to loss of Cdc42 activity, but not Rac1. This deregulated protrusivebehavior of Cdc42 or βPix-depleted cells was accompanied by defective migrationin both 3D and thin fibrillar collagen environments (Supplementary Movie 3, Fig. 2i), along with physical tearing of holes inthe surrounding matrix. These findings are consistent with a report that loss of Cdc42in 3D microenvironments leads to temporally and spatially deregulated protrusions andimpaired leading edge coordination22.We therefore investigated whether βPix regulates the localization and activity ofCdc42 under different ECM conditions. Imaging a single-chain Cdc42 biosensor based onintramolecular fluorescence resonance energy transfer (FRET)23 revealed that on fibronectin, Cdc42 activity remainspolarized toward the leading edge of migrating cells expressing either nonspecific orβPix shRNA (Fig. 2g). On collagen, Cdc42activity was also polarized to the leading edge in the same regions where βPixwas found to uniquely localize on the membrane. In contrast, βPix knockdown onfibrillar collagen led to a loss of this polarization and decreased overall Cdc42activity (Fig. 2g, Supplementary Fig. 2l,m).Additionally, we observed similar collagen-specific decreases in Cdc42 FRET and loss ofFRET polarization in 3D collagen, but not 3D cell-derived matrix (Supplementary Fig. 2k), furtherestablishing that βPix acts through Cdc42, but not Rac1, to coordinate migrationin 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.

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Related in: MedlinePlus