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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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A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulatessuppression of RhoA activity. (a) Immunoprecipitation ofGFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin(FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAPinteraction between βPix and srGAP1. (b) Concurrentdecreased association of βPix with known effector Pak1 when migrating onfibrillar collagen. Blots are representative of three independent experiments.(c) RhoA activity determined by GST-RBD binding from NS andsrGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagenenvironments. (d) Quantification of bands again revealed a 40-60%collagen-specific increase in RhoA activity after loss of srGAP1 (mean ±s.e.m., n = 3 independent western blots, t-tests).(e) srGAP1 knockdown HFFs were cultured overnight in 3Dcollagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded,protrusive (white arrowheads) morphology akin to βPix knockdown.Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiberarrangement (red, reflection microscopy) adjacent to the cell. Hole in matrixmarked by white asterisk; scale bar, 25 μm. (f)Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3Dcollagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). (g)Quantification of cell velocity in cells treated with srGAP1 siRNA in 3Dcollagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
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Figure 4: A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulatessuppression of RhoA activity. (a) Immunoprecipitation ofGFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin(FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAPinteraction between βPix and srGAP1. (b) Concurrentdecreased association of βPix with known effector Pak1 when migrating onfibrillar collagen. Blots are representative of three independent experiments.(c) RhoA activity determined by GST-RBD binding from NS andsrGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagenenvironments. (d) Quantification of bands again revealed a 40-60%collagen-specific increase in RhoA activity after loss of srGAP1 (mean ±s.e.m., n = 3 independent western blots, t-tests).(e) srGAP1 knockdown HFFs were cultured overnight in 3Dcollagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded,protrusive (white arrowheads) morphology akin to βPix knockdown.Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiberarrangement (red, reflection microscopy) adjacent to the cell. Hole in matrixmarked by white asterisk; scale bar, 25 μm. (f)Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3Dcollagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). (g)Quantification of cell velocity in cells treated with srGAP1 siRNA in 3Dcollagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.

Mentions: To address mechanistically how βPix acts through Cdc42 to suppress RhoA,we utilized GFP-βPix knockdown/rescue fibroblasts to isolate the proteins thatbound differentially to βPix from cells undergoing migration on fibronectinversus fibrillar collagen (Supplementary Fig. 2b, 4a). Immunoblotting for known binding partners such as Pak1 revealeddecreased association on collagen compared to fibronectin, confirming differentialbinding to βPix (Fig. 4b). Unexpectedly,mass spectrometry analysis of a strong ~130 kDa band revealed a collagen-specificassociation between βPix and the RhoGAP srGAP1 in both HFFs (Fig. 4a) and MDA-MB-231cells (Supplementary Fig. 4h). GAPsinactivate Rho proteins by stimulating their intrinsic GTPase activity and are criticalelements in inhibitory Rho family crosstalk4,25, 26. Depending on the context, srGAP1 can promote the GTPhydrolysis of RhoA, Cdc42, or Rac127,and overexpression of srGAP1 can suppress protrusive plasma membrane dynamics28.


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

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

A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulatessuppression of RhoA activity. (a) Immunoprecipitation ofGFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin(FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAPinteraction between βPix and srGAP1. (b) Concurrentdecreased association of βPix with known effector Pak1 when migrating onfibrillar collagen. Blots are representative of three independent experiments.(c) RhoA activity determined by GST-RBD binding from NS andsrGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagenenvironments. (d) Quantification of bands again revealed a 40-60%collagen-specific increase in RhoA activity after loss of srGAP1 (mean ±s.e.m., n = 3 independent western blots, t-tests).(e) srGAP1 knockdown HFFs were cultured overnight in 3Dcollagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded,protrusive (white arrowheads) morphology akin to βPix knockdown.Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiberarrangement (red, reflection microscopy) adjacent to the cell. Hole in matrixmarked by white asterisk; scale bar, 25 μm. (f)Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3Dcollagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). (g)Quantification of cell velocity in cells treated with srGAP1 siRNA in 3Dcollagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
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Figure 4: A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulatessuppression of RhoA activity. (a) Immunoprecipitation ofGFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin(FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAPinteraction between βPix and srGAP1. (b) Concurrentdecreased association of βPix with known effector Pak1 when migrating onfibrillar collagen. Blots are representative of three independent experiments.(c) RhoA activity determined by GST-RBD binding from NS andsrGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagenenvironments. (d) Quantification of bands again revealed a 40-60%collagen-specific increase in RhoA activity after loss of srGAP1 (mean ±s.e.m., n = 3 independent western blots, t-tests).(e) srGAP1 knockdown HFFs were cultured overnight in 3Dcollagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded,protrusive (white arrowheads) morphology akin to βPix knockdown.Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiberarrangement (red, reflection microscopy) adjacent to the cell. Hole in matrixmarked by white asterisk; scale bar, 25 μm. (f)Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3Dcollagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). (g)Quantification of cell velocity in cells treated with srGAP1 siRNA in 3Dcollagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1were assessed across three independent experiments (mean ± s.e.m.,one-way ANOVA with Bonferroni multiple comparisons correction). Statisticalsource data can be found in Supplementary Table 2, *** P < 0.001.
Mentions: To address mechanistically how βPix acts through Cdc42 to suppress RhoA,we utilized GFP-βPix knockdown/rescue fibroblasts to isolate the proteins thatbound differentially to βPix from cells undergoing migration on fibronectinversus fibrillar collagen (Supplementary Fig. 2b, 4a). Immunoblotting for known binding partners such as Pak1 revealeddecreased association on collagen compared to fibronectin, confirming differentialbinding to βPix (Fig. 4b). Unexpectedly,mass spectrometry analysis of a strong ~130 kDa band revealed a collagen-specificassociation between βPix and the RhoGAP srGAP1 in both HFFs (Fig. 4a) and MDA-MB-231cells (Supplementary Fig. 4h). GAPsinactivate Rho proteins by stimulating their intrinsic GTPase activity and are criticalelements in inhibitory Rho family crosstalk4,25, 26. Depending on the context, srGAP1 can promote the GTPhydrolysis of RhoA, Cdc42, or Rac127,and overexpression of srGAP1 can suppress protrusive plasma membrane dynamics28.

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