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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 acts through Cdc42 to suppress and localize RhoA activity duringmigration in 3D collagen. (a, b) RhoA activity determined usingGST-RBD binding from NS and βPix shRNA-expressing HFFs migrating infibronectin or fibrillar collagen environments; collagen-specific increases(40-60%) in RhoA activity with loss of βPix (mean ± s.e.m, n = 3independent western blots, t-tests). (c, d)Similarly, knockdown of Cdc42, but not Rac1, during migration on fibrillarcollagen leads to increased intracellular RhoA activity (mean ± s.e.m, n= 3 independent western blots, one-way ANOVA with Bonferroni correction).(e) Maximum projections of confocal stacks of live fibroblastmigration expressing a RhoA biosensor on fibronectin (FN) or fibrillar collagen(FIB COL). Knockdown of βPix on collagen results in overall elevation ofRhoA activity accompanied by a loss of front-back segregation of RhoA activity.Pseudocolor intensity scales were identical for each matrix condition; scalebars, 25 μm. White arrows designate direction of leading edgeprotrusions. (f) Average integrated whole cell RhoA FRET intensityon 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). (g) QuantificationRhoA 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 assessedacross three independent experiments (mean ± s.e.m.,t-test). (h) Phase contrast timelapse images(Supplementary Movie4) of an HFF expressing low levels of GFP-RhoAQ63L in 3D collagenreveal rounded morphology, spatially and temporally deregulated protrusions(white arrowheads) and loss of persistent migration. Scale bars, 25 μm.(i) Quantification of cell elliptical factor (maximallength/width) in cells low-expressing GFP-RhoAQ63L in 3D collagen. n = 30, 35,and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (j) Quantification of cell protrusions incells with low-level GFP-RhoAQ63L expression in 3D collagen. n = 36, 36, and 29cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (k) Quantification of cell velocity incells with low GFP-RhoAQ63L expression in 3D collagen. n = 25, 24, and 21 cellsfor NS, βPix sh#2, and RhoQ63L were assessed across three independentexperiments. (l) βPix shRNA fibroblasts were culturedovernight in 3D collagen gels in the presence of cell-permeable C3 transferase(2 μg/mL) or blebbistatin (25 μM). n = 25, 24, 20, and 20 cellsfor NS, βPix sh#2, βPix+C3, and βPix+Blebb were assessedacross three independent experiments. For (i-l), data given as mean± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction.Statistical source data can be found in Supplementary Table 2, *** P <0.001, * P < 0.05.
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Figure 3: βPix acts through Cdc42 to suppress and localize RhoA activity duringmigration in 3D collagen. (a, b) RhoA activity determined usingGST-RBD binding from NS and βPix shRNA-expressing HFFs migrating infibronectin or fibrillar collagen environments; collagen-specific increases(40-60%) in RhoA activity with loss of βPix (mean ± s.e.m, n = 3independent western blots, t-tests). (c, d)Similarly, knockdown of Cdc42, but not Rac1, during migration on fibrillarcollagen leads to increased intracellular RhoA activity (mean ± s.e.m, n= 3 independent western blots, one-way ANOVA with Bonferroni correction).(e) Maximum projections of confocal stacks of live fibroblastmigration expressing a RhoA biosensor on fibronectin (FN) or fibrillar collagen(FIB COL). Knockdown of βPix on collagen results in overall elevation ofRhoA activity accompanied by a loss of front-back segregation of RhoA activity.Pseudocolor intensity scales were identical for each matrix condition; scalebars, 25 μm. White arrows designate direction of leading edgeprotrusions. (f) Average integrated whole cell RhoA FRET intensityon 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). (g) QuantificationRhoA 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 assessedacross three independent experiments (mean ± s.e.m.,t-test). (h) Phase contrast timelapse images(Supplementary Movie4) of an HFF expressing low levels of GFP-RhoAQ63L in 3D collagenreveal rounded morphology, spatially and temporally deregulated protrusions(white arrowheads) and loss of persistent migration. Scale bars, 25 μm.(i) Quantification of cell elliptical factor (maximallength/width) in cells low-expressing GFP-RhoAQ63L in 3D collagen. n = 30, 35,and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (j) Quantification of cell protrusions incells with low-level GFP-RhoAQ63L expression in 3D collagen. n = 36, 36, and 29cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (k) Quantification of cell velocity incells with low GFP-RhoAQ63L expression in 3D collagen. n = 25, 24, and 21 cellsfor NS, βPix sh#2, and RhoQ63L were assessed across three independentexperiments. (l) βPix shRNA fibroblasts were culturedovernight in 3D collagen gels in the presence of cell-permeable C3 transferase(2 μg/mL) or blebbistatin (25 μM). n = 25, 24, 20, and 20 cellsfor NS, βPix sh#2, βPix+C3, and βPix+Blebb were assessedacross three independent experiments. For (i-l), data given as mean± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction.Statistical source data can be found in Supplementary Table 2, *** P <0.001, * P < 0.05.

Mentions: Because of the strong collagen contraction phenotype associated with loss ofβPix, we speculated that βPix/Cdc42 knockdown might increase RhoA activityduring migration in fibrillar collagen environments. We assayed intracellular RhoAactivity during fibronectin or fibrillar collagen migration in the presence and absenceof βPix. Knockdown of βPix resulted in 40-60% increased intracellular RhoAactivity in fibrillar collagen, but not fibronectin (Fig.3a,b), with similar increases during 3D collagen migration (Supplementary Fig. 3e).Importantly, knockdown of Cdc42, but not Rac1, also increased intracellular RhoAactivity levels on fibrillar collagen (Fig. 3c,d).We next used a single chain RhoA FRET biosensor23, 24 to determine RhoAactivity and localization during live-cell migration. Cells migrating on fibronectindisplayed a gradient of RhoA activity that was highest at the rear of the cell anddecreased toward the leading edge (Fig. 3e). Thislocalization pattern was also observed during migration on fibrillar collagen; however,after βPix knockdown, we observed a striking loss of this RhoA gradient with ageneral elevation of RhoA activity (Fig. 3e-g).Again, the loss of front-back RhoA FRET segregation and elevation in activity wasobserved in 3D collagen, but not 3D cell-derived matrix (Supplementary Fig. 3b),confirming the suppressive crosstalk mechanism between βPix/Cdc42 and RhoA incollagen microenvironments. We examined whether artificial increases in RhoA activityalone could mimic βPix knockdown in 3D collagen. Low-level overexpression ofconstitutively active RhoAQ63L, as determined by fluorescence intensity, not onlymimicked the rounded morphology (Fig. 3h,i) androbust collagen contraction (Supplementary Fig. 3c), but notably also the deregulated, hyper-protrusivebehavior (Fig. 3h,j); expressing RhoAQ63L atcomparable levels in HFFs migrating in cell-derived matrix did not perturb morphology orlead to hyper-protrusive behavior. Migration in fibrillar collagen environments (Supplementary Movie 4, Fig. 3k) was also significantly inhibited with lowRhoAQ63L expression. Finally, to test directly whether inhibiting RhoA could partiallyrescue the βPix knockdown phenotype, we treated βPix knockdown cells withthe RhoA inhibitor C3 transferase, or with blebbistatin to inhibit the RhoA effectormyosin II. Treating βPix knockdown cells in 3D collagen with C3 transferasesignificantly rescued both morphology and migration, while blebbistatin rescued themorphology with slight increases in motility (Fig.3l, Supplementary Fig.3d). We conclude that βPix acts through Cdc42 to suppress and localizeRhoA activity 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)

βPix acts through Cdc42 to suppress and localize RhoA activity duringmigration in 3D collagen. (a, b) RhoA activity determined usingGST-RBD binding from NS and βPix shRNA-expressing HFFs migrating infibronectin or fibrillar collagen environments; collagen-specific increases(40-60%) in RhoA activity with loss of βPix (mean ± s.e.m, n = 3independent western blots, t-tests). (c, d)Similarly, knockdown of Cdc42, but not Rac1, during migration on fibrillarcollagen leads to increased intracellular RhoA activity (mean ± s.e.m, n= 3 independent western blots, one-way ANOVA with Bonferroni correction).(e) Maximum projections of confocal stacks of live fibroblastmigration expressing a RhoA biosensor on fibronectin (FN) or fibrillar collagen(FIB COL). Knockdown of βPix on collagen results in overall elevation ofRhoA activity accompanied by a loss of front-back segregation of RhoA activity.Pseudocolor intensity scales were identical for each matrix condition; scalebars, 25 μm. White arrows designate direction of leading edgeprotrusions. (f) Average integrated whole cell RhoA FRET intensityon 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). (g) QuantificationRhoA 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 assessedacross three independent experiments (mean ± s.e.m.,t-test). (h) Phase contrast timelapse images(Supplementary Movie4) of an HFF expressing low levels of GFP-RhoAQ63L in 3D collagenreveal rounded morphology, spatially and temporally deregulated protrusions(white arrowheads) and loss of persistent migration. Scale bars, 25 μm.(i) Quantification of cell elliptical factor (maximallength/width) in cells low-expressing GFP-RhoAQ63L in 3D collagen. n = 30, 35,and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (j) Quantification of cell protrusions incells with low-level GFP-RhoAQ63L expression in 3D collagen. n = 36, 36, and 29cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (k) Quantification of cell velocity incells with low GFP-RhoAQ63L expression in 3D collagen. n = 25, 24, and 21 cellsfor NS, βPix sh#2, and RhoQ63L were assessed across three independentexperiments. (l) βPix shRNA fibroblasts were culturedovernight in 3D collagen gels in the presence of cell-permeable C3 transferase(2 μg/mL) or blebbistatin (25 μM). n = 25, 24, 20, and 20 cellsfor NS, βPix sh#2, βPix+C3, and βPix+Blebb were assessedacross three independent experiments. For (i-l), data given as mean± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction.Statistical source data can be found in Supplementary Table 2, *** P <0.001, * P < 0.05.
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Figure 3: βPix acts through Cdc42 to suppress and localize RhoA activity duringmigration in 3D collagen. (a, b) RhoA activity determined usingGST-RBD binding from NS and βPix shRNA-expressing HFFs migrating infibronectin or fibrillar collagen environments; collagen-specific increases(40-60%) in RhoA activity with loss of βPix (mean ± s.e.m, n = 3independent western blots, t-tests). (c, d)Similarly, knockdown of Cdc42, but not Rac1, during migration on fibrillarcollagen leads to increased intracellular RhoA activity (mean ± s.e.m, n= 3 independent western blots, one-way ANOVA with Bonferroni correction).(e) Maximum projections of confocal stacks of live fibroblastmigration expressing a RhoA biosensor on fibronectin (FN) or fibrillar collagen(FIB COL). Knockdown of βPix on collagen results in overall elevation ofRhoA activity accompanied by a loss of front-back segregation of RhoA activity.Pseudocolor intensity scales were identical for each matrix condition; scalebars, 25 μm. White arrows designate direction of leading edgeprotrusions. (f) Average integrated whole cell RhoA FRET intensityon 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). (g) QuantificationRhoA 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 assessedacross three independent experiments (mean ± s.e.m.,t-test). (h) Phase contrast timelapse images(Supplementary Movie4) of an HFF expressing low levels of GFP-RhoAQ63L in 3D collagenreveal rounded morphology, spatially and temporally deregulated protrusions(white arrowheads) and loss of persistent migration. Scale bars, 25 μm.(i) Quantification of cell elliptical factor (maximallength/width) in cells low-expressing GFP-RhoAQ63L in 3D collagen. n = 30, 35,and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (j) Quantification of cell protrusions incells with low-level GFP-RhoAQ63L expression in 3D collagen. n = 36, 36, and 29cells for NS, βPix sh#2, and RhoQ63L were assessed across threeindependent experiments. (k) Quantification of cell velocity incells with low GFP-RhoAQ63L expression in 3D collagen. n = 25, 24, and 21 cellsfor NS, βPix sh#2, and RhoQ63L were assessed across three independentexperiments. (l) βPix shRNA fibroblasts were culturedovernight in 3D collagen gels in the presence of cell-permeable C3 transferase(2 μg/mL) or blebbistatin (25 μM). n = 25, 24, 20, and 20 cellsfor NS, βPix sh#2, βPix+C3, and βPix+Blebb were assessedacross three independent experiments. For (i-l), data given as mean± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction.Statistical source data can be found in Supplementary Table 2, *** P <0.001, * P < 0.05.
Mentions: Because of the strong collagen contraction phenotype associated with loss ofβPix, we speculated that βPix/Cdc42 knockdown might increase RhoA activityduring migration in fibrillar collagen environments. We assayed intracellular RhoAactivity during fibronectin or fibrillar collagen migration in the presence and absenceof βPix. Knockdown of βPix resulted in 40-60% increased intracellular RhoAactivity in fibrillar collagen, but not fibronectin (Fig.3a,b), with similar increases during 3D collagen migration (Supplementary Fig. 3e).Importantly, knockdown of Cdc42, but not Rac1, also increased intracellular RhoAactivity levels on fibrillar collagen (Fig. 3c,d).We next used a single chain RhoA FRET biosensor23, 24 to determine RhoAactivity and localization during live-cell migration. Cells migrating on fibronectindisplayed a gradient of RhoA activity that was highest at the rear of the cell anddecreased toward the leading edge (Fig. 3e). Thislocalization pattern was also observed during migration on fibrillar collagen; however,after βPix knockdown, we observed a striking loss of this RhoA gradient with ageneral elevation of RhoA activity (Fig. 3e-g).Again, the loss of front-back RhoA FRET segregation and elevation in activity wasobserved in 3D collagen, but not 3D cell-derived matrix (Supplementary Fig. 3b),confirming the suppressive crosstalk mechanism between βPix/Cdc42 and RhoA incollagen microenvironments. We examined whether artificial increases in RhoA activityalone could mimic βPix knockdown in 3D collagen. Low-level overexpression ofconstitutively active RhoAQ63L, as determined by fluorescence intensity, not onlymimicked the rounded morphology (Fig. 3h,i) androbust collagen contraction (Supplementary Fig. 3c), but notably also the deregulated, hyper-protrusivebehavior (Fig. 3h,j); expressing RhoAQ63L atcomparable levels in HFFs migrating in cell-derived matrix did not perturb morphology orlead to hyper-protrusive behavior. Migration in fibrillar collagen environments (Supplementary Movie 4, Fig. 3k) was also significantly inhibited with lowRhoAQ63L expression. Finally, to test directly whether inhibiting RhoA could partiallyrescue the βPix knockdown phenotype, we treated βPix knockdown cells withthe RhoA inhibitor C3 transferase, or with blebbistatin to inhibit the RhoA effectormyosin II. Treating βPix knockdown cells in 3D collagen with C3 transferasesignificantly rescued both morphology and migration, while blebbistatin rescued themorphology with slight increases in motility (Fig.3l, Supplementary Fig.3d). We conclude that βPix acts through Cdc42 to suppress and localizeRhoA activity 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.

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Related in: MedlinePlus