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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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Loss of βPix leads to collagen-specific morphological and migratorydefects. (a) Quantification of western blot band intensities ofselect GEFs isolated from the RacG15A ECM-GEF screen. Values are fold intensityincrease above No ECM condition; (n=3 independent western blots, mean ±s.e.m, one-way ANOVA with Bonferroni multiple comparisons correction).(b) Western blot validation of βPix binding to RacG15Aduring migration on collagen. (c) Composite images of the leadingedge of HFFs show loss of βPix localization to focal adhesions duringmigration on fibrillar collagen (FIB COL) but not fibronectin (FN). HFFs wereimmunostained for endogenous paxillin (red) and βPix (green); yellowindicates co-localization. See Supplementary Fig. 1d for the whole-cell images. Scale bars, 15μm. (d) Triton X-100 fractionation of HFFs migrating onfibronectin or fibrillar collagen reveals a shift of βPix from soluble(GAPDH) to the insoluble (vimentin) fraction during migration on collagen,observed in three independent experiments. (e) Morphologicalanalysis of βPix knockdown in 3D fibrillar collagen (red, reflectionmicroscopy) versus 3D cell-derived matrix (red, fibronectin) reveals defects incell elongation after loss of βPix specific to 3D collagen. Scale bars,25 μm. (f) Representative phase timelapse of nonspecific(NS) and βPix shRNA fibroblasts migrating in 3D collagen. Whitearrowheads indicate cellular protrusions; scale bars, 25 μm.(g) Migratory tracks of three NS (red) and βPix (green)shRNA fibroblasts in 3D collagen reveal loss of persistent, directional motilityafter βPix knockdown. (h) Analysis of collagen fibers (red,reflection microscopy) adjacent to NS and βPix shRNA cells reveal robustcollagen contraction and remodeling with βPix knockdown (physical holes,asterisks). Scale bars, 25 μm. (i) Quantification of cellelliptical factor (maximal length/width) in 3D collagen versus 3D cell-derivedmatrix after loss of βPix. n = 44, 46, 30, and 35 cells for NS CDM,βPix sh#2 CDM, NS COL, and βPix sh#2 COL were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(j) Quantification of cell protrusions (e, white arrowheads)after fixation and phalloidin staining of βPix knockdown cells in 3Dcollagen. n = 36 cells for both NS and βPix shRNA were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(k) Quantification of cell velocities after βPixknockdown in different ECM conditions. n = 20-25 cells for NS and βPixshRNA in each matrix condition were assessed across three independentexperiments (mean ± s.e.m., t-tests). Statistical sourcedata can be found in Supplementary Table 2. *** P < 0.001, *P < 0.05.
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Figure 1: Loss of βPix leads to collagen-specific morphological and migratorydefects. (a) Quantification of western blot band intensities ofselect GEFs isolated from the RacG15A ECM-GEF screen. Values are fold intensityincrease above No ECM condition; (n=3 independent western blots, mean ±s.e.m, one-way ANOVA with Bonferroni multiple comparisons correction).(b) Western blot validation of βPix binding to RacG15Aduring migration on collagen. (c) Composite images of the leadingedge of HFFs show loss of βPix localization to focal adhesions duringmigration on fibrillar collagen (FIB COL) but not fibronectin (FN). HFFs wereimmunostained for endogenous paxillin (red) and βPix (green); yellowindicates co-localization. See Supplementary Fig. 1d for the whole-cell images. Scale bars, 15μm. (d) Triton X-100 fractionation of HFFs migrating onfibronectin or fibrillar collagen reveals a shift of βPix from soluble(GAPDH) to the insoluble (vimentin) fraction during migration on collagen,observed in three independent experiments. (e) Morphologicalanalysis of βPix knockdown in 3D fibrillar collagen (red, reflectionmicroscopy) versus 3D cell-derived matrix (red, fibronectin) reveals defects incell elongation after loss of βPix specific to 3D collagen. Scale bars,25 μm. (f) Representative phase timelapse of nonspecific(NS) and βPix shRNA fibroblasts migrating in 3D collagen. Whitearrowheads indicate cellular protrusions; scale bars, 25 μm.(g) Migratory tracks of three NS (red) and βPix (green)shRNA fibroblasts in 3D collagen reveal loss of persistent, directional motilityafter βPix knockdown. (h) Analysis of collagen fibers (red,reflection microscopy) adjacent to NS and βPix shRNA cells reveal robustcollagen contraction and remodeling with βPix knockdown (physical holes,asterisks). Scale bars, 25 μm. (i) Quantification of cellelliptical factor (maximal length/width) in 3D collagen versus 3D cell-derivedmatrix after loss of βPix. n = 44, 46, 30, and 35 cells for NS CDM,βPix sh#2 CDM, NS COL, and βPix sh#2 COL were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(j) Quantification of cell protrusions (e, white arrowheads)after fixation and phalloidin staining of βPix knockdown cells in 3Dcollagen. n = 36 cells for both NS and βPix shRNA were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(k) Quantification of cell velocities after βPixknockdown in different ECM conditions. n = 20-25 cells for NS and βPixshRNA in each matrix condition were assessed across three independentexperiments (mean ± s.e.m., t-tests). Statistical sourcedata can be found in Supplementary Table 2. *** P < 0.001, *P < 0.05.

Mentions: Nucleotide-free, dominant-negative Rho GTPase mutants can be used foraffinity-isolation of activated GEFs14,15. We initially focused on GEFsfor Rac1 because of its well-established role in driving 2D and 3D motility throughcoordination of lamellipodial dynamics16. In an ECM-based screen, we identified active GEFs binding torecombinant RacG15A, a Rac1 nucleotide-free mutant, from lysates of primary humanforeskin fibroblasts (HFFs) undergoing steady-state migration in collagen, fibronectin,or ECM-free environments. We utilized an unbiased screening approach14, 15 for identification of active GEFs in cells migrating in specificECM environments by identifying protein bands in Coomassie-stained polyacrylamide gelsthat bound selectively to RacG15A in different ECM environments (Supplementary Fig. 1a). MultipleGEFs were isolated that showed increased activity on both fibronectin and collagen(Fig. 1a, Supplementary Fig. 1b), but theRac1/Cdc42 GEF βPix was activated robustly and specifically only during migrationon collagen (Fig. 1b). βPix exists atmultiple subcellular sites, including focal adhesions and plasma membrane, consistentwith differential functions17-19. We therefore examined for alteredlocalization of βPix during fibroblast migration on fibronectin versus fibrillarcollagen. As expected, both immunofluorescence staining for endogenous βPix andlive-cell imaging of GFP-βPix showed strong localization to focal adhesionsduring migration on fibronectin and 3D cell-derived matrix (CDM), where the primary ECMligand is fibronectin20 (Fig. 1c, Supplementary Fig. 1d-g). Surprisingly, we found a dramaticdecrease in both endogenous and GFP-βPix focal adhesion localization infibroblasts migrating on both fibrillar collagen and 3D collagen (Fig. 1c, Supplementary Fig. 1d-g). Subcellular fractionation revealed that onfibrillar collagen, endogenous βPix transitioned from detergent-soluble to-insoluble fractions (Fig. 1d), and live cellGFP-βPix imaging displayed a patchwork localization on ventral cell membranes inamorphous, persistent aggregates of variable size that, while polarized to leading-edgeprotrusions, did not co-localize with paxillin (Supplementary Fig. 1g). These data demonstrate that theintracellular location of βPix changes dramatically when cells migrate oncollagen compared to fibronectin, supporting the existence of ECM-specific functionsobserved in the initial GEF screen.


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

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

Loss of βPix leads to collagen-specific morphological and migratorydefects. (a) Quantification of western blot band intensities ofselect GEFs isolated from the RacG15A ECM-GEF screen. Values are fold intensityincrease above No ECM condition; (n=3 independent western blots, mean ±s.e.m, one-way ANOVA with Bonferroni multiple comparisons correction).(b) Western blot validation of βPix binding to RacG15Aduring migration on collagen. (c) Composite images of the leadingedge of HFFs show loss of βPix localization to focal adhesions duringmigration on fibrillar collagen (FIB COL) but not fibronectin (FN). HFFs wereimmunostained for endogenous paxillin (red) and βPix (green); yellowindicates co-localization. See Supplementary Fig. 1d for the whole-cell images. Scale bars, 15μm. (d) Triton X-100 fractionation of HFFs migrating onfibronectin or fibrillar collagen reveals a shift of βPix from soluble(GAPDH) to the insoluble (vimentin) fraction during migration on collagen,observed in three independent experiments. (e) Morphologicalanalysis of βPix knockdown in 3D fibrillar collagen (red, reflectionmicroscopy) versus 3D cell-derived matrix (red, fibronectin) reveals defects incell elongation after loss of βPix specific to 3D collagen. Scale bars,25 μm. (f) Representative phase timelapse of nonspecific(NS) and βPix shRNA fibroblasts migrating in 3D collagen. Whitearrowheads indicate cellular protrusions; scale bars, 25 μm.(g) Migratory tracks of three NS (red) and βPix (green)shRNA fibroblasts in 3D collagen reveal loss of persistent, directional motilityafter βPix knockdown. (h) Analysis of collagen fibers (red,reflection microscopy) adjacent to NS and βPix shRNA cells reveal robustcollagen contraction and remodeling with βPix knockdown (physical holes,asterisks). Scale bars, 25 μm. (i) Quantification of cellelliptical factor (maximal length/width) in 3D collagen versus 3D cell-derivedmatrix after loss of βPix. n = 44, 46, 30, and 35 cells for NS CDM,βPix sh#2 CDM, NS COL, and βPix sh#2 COL were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(j) Quantification of cell protrusions (e, white arrowheads)after fixation and phalloidin staining of βPix knockdown cells in 3Dcollagen. n = 36 cells for both NS and βPix shRNA were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(k) Quantification of cell velocities after βPixknockdown in different ECM conditions. n = 20-25 cells for NS and βPixshRNA in each matrix condition were assessed across three independentexperiments (mean ± s.e.m., t-tests). Statistical sourcedata can be found in Supplementary Table 2. *** P < 0.001, *P < 0.05.
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Figure 1: Loss of βPix leads to collagen-specific morphological and migratorydefects. (a) Quantification of western blot band intensities ofselect GEFs isolated from the RacG15A ECM-GEF screen. Values are fold intensityincrease above No ECM condition; (n=3 independent western blots, mean ±s.e.m, one-way ANOVA with Bonferroni multiple comparisons correction).(b) Western blot validation of βPix binding to RacG15Aduring migration on collagen. (c) Composite images of the leadingedge of HFFs show loss of βPix localization to focal adhesions duringmigration on fibrillar collagen (FIB COL) but not fibronectin (FN). HFFs wereimmunostained for endogenous paxillin (red) and βPix (green); yellowindicates co-localization. See Supplementary Fig. 1d for the whole-cell images. Scale bars, 15μm. (d) Triton X-100 fractionation of HFFs migrating onfibronectin or fibrillar collagen reveals a shift of βPix from soluble(GAPDH) to the insoluble (vimentin) fraction during migration on collagen,observed in three independent experiments. (e) Morphologicalanalysis of βPix knockdown in 3D fibrillar collagen (red, reflectionmicroscopy) versus 3D cell-derived matrix (red, fibronectin) reveals defects incell elongation after loss of βPix specific to 3D collagen. Scale bars,25 μm. (f) Representative phase timelapse of nonspecific(NS) and βPix shRNA fibroblasts migrating in 3D collagen. Whitearrowheads indicate cellular protrusions; scale bars, 25 μm.(g) Migratory tracks of three NS (red) and βPix (green)shRNA fibroblasts in 3D collagen reveal loss of persistent, directional motilityafter βPix knockdown. (h) Analysis of collagen fibers (red,reflection microscopy) adjacent to NS and βPix shRNA cells reveal robustcollagen contraction and remodeling with βPix knockdown (physical holes,asterisks). Scale bars, 25 μm. (i) Quantification of cellelliptical factor (maximal length/width) in 3D collagen versus 3D cell-derivedmatrix after loss of βPix. n = 44, 46, 30, and 35 cells for NS CDM,βPix sh#2 CDM, NS COL, and βPix sh#2 COL were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(j) Quantification of cell protrusions (e, white arrowheads)after fixation and phalloidin staining of βPix knockdown cells in 3Dcollagen. n = 36 cells for both NS and βPix shRNA were assessed acrossthree independent experiments (mean ± s.e.m., t-tests).(k) Quantification of cell velocities after βPixknockdown in different ECM conditions. n = 20-25 cells for NS and βPixshRNA in each matrix condition were assessed across three independentexperiments (mean ± s.e.m., t-tests). Statistical sourcedata can be found in Supplementary Table 2. *** P < 0.001, *P < 0.05.
Mentions: Nucleotide-free, dominant-negative Rho GTPase mutants can be used foraffinity-isolation of activated GEFs14,15. We initially focused on GEFsfor Rac1 because of its well-established role in driving 2D and 3D motility throughcoordination of lamellipodial dynamics16. In an ECM-based screen, we identified active GEFs binding torecombinant RacG15A, a Rac1 nucleotide-free mutant, from lysates of primary humanforeskin fibroblasts (HFFs) undergoing steady-state migration in collagen, fibronectin,or ECM-free environments. We utilized an unbiased screening approach14, 15 for identification of active GEFs in cells migrating in specificECM environments by identifying protein bands in Coomassie-stained polyacrylamide gelsthat bound selectively to RacG15A in different ECM environments (Supplementary Fig. 1a). MultipleGEFs were isolated that showed increased activity on both fibronectin and collagen(Fig. 1a, Supplementary Fig. 1b), but theRac1/Cdc42 GEF βPix was activated robustly and specifically only during migrationon collagen (Fig. 1b). βPix exists atmultiple subcellular sites, including focal adhesions and plasma membrane, consistentwith differential functions17-19. We therefore examined for alteredlocalization of βPix during fibroblast migration on fibronectin versus fibrillarcollagen. As expected, both immunofluorescence staining for endogenous βPix andlive-cell imaging of GFP-βPix showed strong localization to focal adhesionsduring migration on fibronectin and 3D cell-derived matrix (CDM), where the primary ECMligand is fibronectin20 (Fig. 1c, Supplementary Fig. 1d-g). Surprisingly, we found a dramaticdecrease in both endogenous and GFP-βPix focal adhesion localization infibroblasts migrating on both fibrillar collagen and 3D collagen (Fig. 1c, Supplementary Fig. 1d-g). Subcellular fractionation revealed that onfibrillar collagen, endogenous βPix transitioned from detergent-soluble to-insoluble fractions (Fig. 1d), and live cellGFP-βPix imaging displayed a patchwork localization on ventral cell membranes inamorphous, persistent aggregates of variable size that, while polarized to leading-edgeprotrusions, did not co-localize with paxillin (Supplementary Fig. 1g). These data demonstrate that theintracellular location of βPix changes dramatically when cells migrate oncollagen compared to fibronectin, supporting the existence of ECM-specific functionsobserved in the initial GEF screen.

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