Structured Review

Stratagene vinculin
A <t>vinculin</t> mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P
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1) Product Images from "Recruitment of the Arp2/3 complex to vinculin"

Article Title: Recruitment of the Arp2/3 complex to vinculin

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200206043

A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P
Figure Legend Snippet: A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P

Techniques Used: Mutagenesis, Plasmid Preparation, Immunofluorescence, Transfection, Expressing

Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.
Figure Legend Snippet: Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.

Techniques Used: Migration, Plasmid Preparation, Mutagenesis, Expressing

PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .
Figure Legend Snippet: PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .

Techniques Used: Inhibition, Immunoprecipitation

Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.
Figure Legend Snippet: Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.

Techniques Used: Binding Assay, Incubation, Purification

Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.
Figure Legend Snippet: Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.

Techniques Used: Expressing, Immunofluorescence, Staining, Incubation

Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.
Figure Legend Snippet: Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.

Techniques Used: Dominant Negative Mutation, Transfection, Plasmid Preparation, Immunoprecipitation, Activation Assay, Activity Assay

PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).
Figure Legend Snippet: PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).

Techniques Used: Binding Assay, Incubation

Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.
Figure Legend Snippet: Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.

Techniques Used: Immunoprecipitation, Western Blot, Concentration Assay

Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.
Figure Legend Snippet: Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.

Techniques Used: Binding Assay, Incubation, Western Blot, In Vitro, Mutagenesis, Expressing, Plasmid Preparation

Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.
Figure Legend Snippet: Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.

Techniques Used: Mutagenesis, Binding Assay, Immunofluorescence, Microscopy

2) Product Images from "CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics"

Article Title: CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-013-1450-x

Model for regulation of CAS-dependent mechanosensing. CAS is anchored in focal adhesions by N-terminal SH3 and C-terminal CCH domains [ 22 , 24 ]. In quiescent cells, the substrate domain of CAS adopts a compact structure ( left ). Mechanical stretch leads to extension of the CAS substrate domain and subsequent phosphorylation by Src, which activates CAS-mediated mechanotransduction signals ( middle ). Src phosphorylation of CAS on tyrosine 12 blocks CAS–vinculin binding, and the substrate domain returns to a compact structure. CAS-mediated mechanotransduction is attenuated ( right ) either by a gradual loss of substrate domain phosphorylation or by making the phosphorylated tyrosines in the substrate domain inaccessible for downstream signaling proteins
Figure Legend Snippet: Model for regulation of CAS-dependent mechanosensing. CAS is anchored in focal adhesions by N-terminal SH3 and C-terminal CCH domains [ 22 , 24 ]. In quiescent cells, the substrate domain of CAS adopts a compact structure ( left ). Mechanical stretch leads to extension of the CAS substrate domain and subsequent phosphorylation by Src, which activates CAS-mediated mechanotransduction signals ( middle ). Src phosphorylation of CAS on tyrosine 12 blocks CAS–vinculin binding, and the substrate domain returns to a compact structure. CAS-mediated mechanotransduction is attenuated ( right ) either by a gradual loss of substrate domain phosphorylation or by making the phosphorylated tyrosines in the substrate domain inaccessible for downstream signaling proteins

Techniques Used: Binding Assay

Disruption of CAS–vinculin interaction results in impaired traction forces generation. a Upper row cell image represented by GFP fluorescence (Vin Wt, Vin PNNS) and bright field image (Vin−/−) traction field ( middle row ), and fluorescent ( bottom row ) images of Vin−/− MEFs re-expressing different vinculin variants. Scale bar 10 μm. b The bar plot shows the strain energy generated by single cells (mean ± SE). Numbers in columns indicate number of analyzed cells
Figure Legend Snippet: Disruption of CAS–vinculin interaction results in impaired traction forces generation. a Upper row cell image represented by GFP fluorescence (Vin Wt, Vin PNNS) and bright field image (Vin−/−) traction field ( middle row ), and fluorescent ( bottom row ) images of Vin−/− MEFs re-expressing different vinculin variants. Scale bar 10 μm. b The bar plot shows the strain energy generated by single cells (mean ± SE). Numbers in columns indicate number of analyzed cells

Techniques Used: Fluorescence, Expressing, Generated

Dependence of vinculin dynamics on CAS–vinculin interaction. a FRAP curves of GFP-vinculin WT ( left side ) or GFP-vinculin PNSS ( right side ) associated with focal adhesions in CAS−/− MEFs re-expressing indicated CAS variants. Numbers in plot indicate average half-maximum recovery times ( t 1/2). b The bar plot shows half-maximum recovery times of GFP-fused Vin WT ( left ) and GFP-vinculin PNSS ( right ) in CAS−/− MEFs re-expressing indicated CAS variants. Error bars represent standard errors
Figure Legend Snippet: Dependence of vinculin dynamics on CAS–vinculin interaction. a FRAP curves of GFP-vinculin WT ( left side ) or GFP-vinculin PNSS ( right side ) associated with focal adhesions in CAS−/− MEFs re-expressing indicated CAS variants. Numbers in plot indicate average half-maximum recovery times ( t 1/2). b The bar plot shows half-maximum recovery times of GFP-fused Vin WT ( left ) and GFP-vinculin PNSS ( right ) in CAS−/− MEFs re-expressing indicated CAS variants. Error bars represent standard errors

Techniques Used: Expressing

Stretch-mediated phosphorylation of CAS is dependent on CAS–vinculin interaction. a Vin−/− MEFs or Vin−/− MEFS re-expressing indicated vinculin variants, b CAS−/− MEFs re-expressing indicated CAS variants and c MEFs transformed by constitutively active Src (MEFs + Src527F) were seeded on fibronectin-coated flexible membrane, incubated for 24 h, and then subjected to 20 % static stretch for 10 min ( a , b ) or for indicated times ( c ). Subsequently, cells were lysed and analyzed by Western blotting against phosphorylated Y410 in the CAS substrate domain and Y12 in CAS SH3 domain. Numbers indicate fold-change (mean ± SD) in CAS Y410 and Y12 phosphorylation after stretching. The immunoblots are representative of three independent experiments
Figure Legend Snippet: Stretch-mediated phosphorylation of CAS is dependent on CAS–vinculin interaction. a Vin−/− MEFs or Vin−/− MEFS re-expressing indicated vinculin variants, b CAS−/− MEFs re-expressing indicated CAS variants and c MEFs transformed by constitutively active Src (MEFs + Src527F) were seeded on fibronectin-coated flexible membrane, incubated for 24 h, and then subjected to 20 % static stretch for 10 min ( a , b ) or for indicated times ( c ). Subsequently, cells were lysed and analyzed by Western blotting against phosphorylated Y410 in the CAS substrate domain and Y12 in CAS SH3 domain. Numbers indicate fold-change (mean ± SD) in CAS Y410 and Y12 phosphorylation after stretching. The immunoblots are representative of three independent experiments

Techniques Used: Expressing, Transformation Assay, Incubation, Western Blot

Effect of CAS–vinculin interaction on cell mechanical properties. Stiffness of MEFs analyzed at 6nN force using magnetic tweezers. The bar plots show stiffness of a CAS−/− MEFs re-expressing indicated CAS variants and b Vin−/− MEFs re-expressing indicated vinculin variants. Numbers in columns indicate number of analyzed cells, and error bars represent standard error
Figure Legend Snippet: Effect of CAS–vinculin interaction on cell mechanical properties. Stiffness of MEFs analyzed at 6nN force using magnetic tweezers. The bar plots show stiffness of a CAS−/− MEFs re-expressing indicated CAS variants and b Vin−/− MEFs re-expressing indicated vinculin variants. Numbers in columns indicate number of analyzed cells, and error bars represent standard error

Techniques Used: Expressing

The CAS SH3 domain interacts with the polyproline region of vinculin. a Binding of vinculin to SH3 domains of CAS WT, CAS Y12E, and CAS Y12F fused with GST was analyzed with pull-down assays by immunoblotting. Vinculin was detected with an anti vinculin antibody. GST fused SH3 domains were detected by Ponceau S staining. Aliquots of total cell lysates (total) were used as a control. b GFP CAS was immunoprecipitated from CAS−/− MEFs expressing CAS Y12 variants, and binding of vinculin and FAK (as a control) was analyzed using vinculin and FAK antibodies. c GFP CAS WT was immunoprecipitated from FAK−/− MEFs and binding of vinculin was analyzed using vinculin antibody. d In a far-Western experiment, Vin WT GFP or GFP immunoprecipitated from Vin−/− MEFs expressing the GFP constructs were transferred to nitrocellulose membranes and incubated with recombinant CAS–GST, followed by detection with anti-GST antibody. Loading controls of GFP constructs were analyzed by anti-GFP antibody. As a positive control for anti-GST reactivity, purified CAS–GST was run alongside. e GFP vinculin was immunoprecipitated from Vin−/− MEFs re-expressing GFP-fused Vin WT or Vin PNSS (PKPP sequence in the proline-rich region changed to PNSS), and binding of CAS and paxillin was detected with CAS and paxillin antibodies. f GFP vinculin was immunoprecipitated from Vin−/− MEFs re-expressing GFP-fused Vin WT or Vin PNSS (PKPP sequence in proline-rich region changed to PNSS), and binding of Arp2 was detected with Arp2 antibody
Figure Legend Snippet: The CAS SH3 domain interacts with the polyproline region of vinculin. a Binding of vinculin to SH3 domains of CAS WT, CAS Y12E, and CAS Y12F fused with GST was analyzed with pull-down assays by immunoblotting. Vinculin was detected with an anti vinculin antibody. GST fused SH3 domains were detected by Ponceau S staining. Aliquots of total cell lysates (total) were used as a control. b GFP CAS was immunoprecipitated from CAS−/− MEFs expressing CAS Y12 variants, and binding of vinculin and FAK (as a control) was analyzed using vinculin and FAK antibodies. c GFP CAS WT was immunoprecipitated from FAK−/− MEFs and binding of vinculin was analyzed using vinculin antibody. d In a far-Western experiment, Vin WT GFP or GFP immunoprecipitated from Vin−/− MEFs expressing the GFP constructs were transferred to nitrocellulose membranes and incubated with recombinant CAS–GST, followed by detection with anti-GST antibody. Loading controls of GFP constructs were analyzed by anti-GFP antibody. As a positive control for anti-GST reactivity, purified CAS–GST was run alongside. e GFP vinculin was immunoprecipitated from Vin−/− MEFs re-expressing GFP-fused Vin WT or Vin PNSS (PKPP sequence in the proline-rich region changed to PNSS), and binding of CAS and paxillin was detected with CAS and paxillin antibodies. f GFP vinculin was immunoprecipitated from Vin−/− MEFs re-expressing GFP-fused Vin WT or Vin PNSS (PKPP sequence in proline-rich region changed to PNSS), and binding of Arp2 was detected with Arp2 antibody

Techniques Used: Binding Assay, Staining, Immunoprecipitation, Expressing, Western Blot, Construct, Incubation, Recombinant, Positive Control, Purification, Sequencing

CAS localization in focal adhesions is dependent on FAK and vinculin. The bar graphs show the percentage of focal adhesions stained positive for GFP–CAS ( a ) or for mCherry-CAS ( b ). Focal adhesions were considered CAS-positive if the GFP–CAS or mCherry-CAS signal is at least double the signal in cytoplasm, adjacent to the focal adhesions, indicated by paxillin staining. Numbers in columns indicate number of analyzed focal adhesions and error bars represent standard deviation
Figure Legend Snippet: CAS localization in focal adhesions is dependent on FAK and vinculin. The bar graphs show the percentage of focal adhesions stained positive for GFP–CAS ( a ) or for mCherry-CAS ( b ). Focal adhesions were considered CAS-positive if the GFP–CAS or mCherry-CAS signal is at least double the signal in cytoplasm, adjacent to the focal adhesions, indicated by paxillin staining. Numbers in columns indicate number of analyzed focal adhesions and error bars represent standard deviation

Techniques Used: Staining, Standard Deviation

Dependence of CAS dynamics on FAK and vinculin within focal adhesions. a FRAP curves of CAS-Venus associated with focal adhesions in MEFs lacking FAK or vinculin, or re-expressing either Vin WT or mutated Vin PNSS. Numbers indicate average half-maximum recovery times ( t 1/2). b The bar plot shows average half-maximum recovery times of CAS-Venus in different MEFs. Error bars represent standard errors
Figure Legend Snippet: Dependence of CAS dynamics on FAK and vinculin within focal adhesions. a FRAP curves of CAS-Venus associated with focal adhesions in MEFs lacking FAK or vinculin, or re-expressing either Vin WT or mutated Vin PNSS. Numbers indicate average half-maximum recovery times ( t 1/2). b The bar plot shows average half-maximum recovery times of CAS-Venus in different MEFs. Error bars represent standard errors

Techniques Used: Expressing

CAS–vinculin interaction affects focal adhesion size. a Vin−/− MEFs re-expressing either Vin WT or Vin PNSS C-terminally fused with GFP were grown on fibronectin-coated coverslips and stained for paxillin (focal adhesion marker) and F-actin. Focal adhesion size was determined using ImageJ software. Scale bar 10 μm ( b ). The histogram bars represent average size of adhesion structures in cells deficient in vinculin, or re-expressing either Vin WT or Vin PNSS mutant. Numbers in columns indicate number of analyzed focal adhesions
Figure Legend Snippet: CAS–vinculin interaction affects focal adhesion size. a Vin−/− MEFs re-expressing either Vin WT or Vin PNSS C-terminally fused with GFP were grown on fibronectin-coated coverslips and stained for paxillin (focal adhesion marker) and F-actin. Focal adhesion size was determined using ImageJ software. Scale bar 10 μm ( b ). The histogram bars represent average size of adhesion structures in cells deficient in vinculin, or re-expressing either Vin WT or Vin PNSS mutant. Numbers in columns indicate number of analyzed focal adhesions

Techniques Used: Expressing, Staining, Marker, Software, Mutagenesis

Related Articles

Polymerase Chain Reaction:

Article Title: Recruitment of the Arp2/3 complex to vinculin
Article Snippet: .. All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene). .. These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y.

Mutagenesis:

Article Title: Recruitment of the Arp2/3 complex to vinculin
Article Snippet: .. All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene). .. These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y.

Subcloning:

Article Title: Recruitment of the Arp2/3 complex to vinculin
Article Snippet: .. All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene). .. These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y.

Amplification:

Article Title: Recruitment of the Arp2/3 complex to vinculin
Article Snippet: .. All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene). .. These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y.

Construct:

Article Title: Recruitment of the Arp2/3 complex to vinculin
Article Snippet: .. All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene). .. These mutants were subcloned into full-length vinculin in pCMV-myc (CLONTECH Laboratories, Inc.) with a modified multiple cloning site. pGEX-VCA domain was a gift from H-Y.

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  • 92
    Stratagene vinculin
    A <t>vinculin</t> mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P
    Vinculin, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vinculin/product/Stratagene
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    86
    Stratagene non phosphorylatable vinculin y1065f
    <t>Vinculin</t> phosphorylation at Tyr 1065 does not affect the recruitment of vinculin to the membrane in response to ACh. A , freshly dissociated smooth muscle cells from tissues expressing EGFP vinculin WT (unstimulated ( US ), n = 17; ACh, n = 5), vinculin Y1065E (unstimulated, n = 25; ACh, n = 17), or vinculin <t>Y1065F</t> (unstimulated, n = 28; ACh, n = 14) were plated onto coverslips and stimulated with 10 −4 m ACh or left unstimulated. The localization of EGFP-vinculin was visualized in live cells by confocal microscopy. EGFP-vinculin WT, EGFP-vinculin Y1065E, and EGFP-vinculin Y1065F localized throughout the cytoplasm in unstimulated cells and were more heavily concentrated at the cell membrane in ACh-stimulated cells. B , mean ratios of pixel intensity between the cell membrane and cytoplasm. The membrane/cytoplasm ratio was significantly higher in ACh-stimulated cells from all recombinant vinculin treatment groups ( p > 0.05). The localization of mutant and wild type vinculin EGFP-vinculin proteins did not differ significantly among different recombinant vinculin treatment groups. Error bars , S.E.
    Non Phosphorylatable Vinculin Y1065f, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    non phosphorylatable vinculin y1065f - by Bioz Stars, 2020-08
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    88
    Stratagene gfp vinculin
    Vt CT hairpin deletion affects cell adhesion. A , Vin −/− MEFs were transfected with WT or ΔC5 <t>vinculin</t> and plated on FN. After fixation, F-actin and FAs were stained using phalloidin and <t>GFP-tagged</t> vinculin variants, respectively. Scale bar is 25 μm. B–D , adhesion number per cell ( B ), cell area ( C ), and adhesion size ( D ) were analyzed ( n = 19; *, p
    Gfp Vinculin, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp vinculin - by Bioz Stars, 2020-08
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    86
    Stratagene full length chicken vinculin
    <t>Vinculin</t> constructs used in these studies. A , domain structure of EGFP-vinculin proteins, vinculin conformation-sensitive FRET probes, amber mutant, and Cerulean-5aa-Venus FRET probes. B , vinculin FRET probes in open and closed conformation. When vinculin is in an open conformation, there is maximum Cerulean probe emission at 470 nm. When vinculin assumes a closed conformation, Cerulean donor emission at 470 nm is quenched, and emission from the Venus acceptor at 530 nm is increased. C , emission spectrum of vinculin amber FRET mutant probe from a dissociated smooth muscle cell. Excitation at 405 nm on vinculin amber FRET mutant probe results in emission at 470 nm. D , emission spectrum of Cerulean-5aa-Venus FRET probe ( C5V ) from a dissociated smooth muscle cell. Excitation of Cerulean-5aa-Venus at 405 nm results in emission at 530 nm.
    Full Length Chicken Vinculin, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    full length chicken vinculin - by Bioz Stars, 2020-08
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    A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Mutagenesis, Plasmid Preparation, Immunofluorescence, Transfection, Expressing

    Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Migration, Plasmid Preparation, Mutagenesis, Expressing

    PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Inhibition, Immunoprecipitation

    Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation, Purification

    Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Expressing, Immunofluorescence, Staining, Incubation

    Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Dominant Negative Mutation, Transfection, Plasmid Preparation, Immunoprecipitation, Activation Assay, Activity Assay

    PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation

    Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Immunoprecipitation, Western Blot, Concentration Assay

    Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation, Western Blot, In Vitro, Mutagenesis, Expressing, Plasmid Preparation

    Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Mutagenesis, Binding Assay, Immunofluorescence, Microscopy

    Vinculin phosphorylation at Tyr 1065 does not affect the recruitment of vinculin to the membrane in response to ACh. A , freshly dissociated smooth muscle cells from tissues expressing EGFP vinculin WT (unstimulated ( US ), n = 17; ACh, n = 5), vinculin Y1065E (unstimulated, n = 25; ACh, n = 17), or vinculin Y1065F (unstimulated, n = 28; ACh, n = 14) were plated onto coverslips and stimulated with 10 −4 m ACh or left unstimulated. The localization of EGFP-vinculin was visualized in live cells by confocal microscopy. EGFP-vinculin WT, EGFP-vinculin Y1065E, and EGFP-vinculin Y1065F localized throughout the cytoplasm in unstimulated cells and were more heavily concentrated at the cell membrane in ACh-stimulated cells. B , mean ratios of pixel intensity between the cell membrane and cytoplasm. The membrane/cytoplasm ratio was significantly higher in ACh-stimulated cells from all recombinant vinculin treatment groups ( p > 0.05). The localization of mutant and wild type vinculin EGFP-vinculin proteins did not differ significantly among different recombinant vinculin treatment groups. Error bars , S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Vinculin phosphorylation at Tyr 1065 does not affect the recruitment of vinculin to the membrane in response to ACh. A , freshly dissociated smooth muscle cells from tissues expressing EGFP vinculin WT (unstimulated ( US ), n = 17; ACh, n = 5), vinculin Y1065E (unstimulated, n = 25; ACh, n = 17), or vinculin Y1065F (unstimulated, n = 28; ACh, n = 14) were plated onto coverslips and stimulated with 10 −4 m ACh or left unstimulated. The localization of EGFP-vinculin was visualized in live cells by confocal microscopy. EGFP-vinculin WT, EGFP-vinculin Y1065E, and EGFP-vinculin Y1065F localized throughout the cytoplasm in unstimulated cells and were more heavily concentrated at the cell membrane in ACh-stimulated cells. B , mean ratios of pixel intensity between the cell membrane and cytoplasm. The membrane/cytoplasm ratio was significantly higher in ACh-stimulated cells from all recombinant vinculin treatment groups ( p > 0.05). The localization of mutant and wild type vinculin EGFP-vinculin proteins did not differ significantly among different recombinant vinculin treatment groups. Error bars , S.E.

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, Confocal Microscopy, Recombinant, Mutagenesis

    Expression of vinculin Y1065F in tracheal smooth muscle tissues inhibited the ACh-stimulated increase in the interaction between vinculin and talin, as detected by in situ PLAs performed on freshly dissociated cells. A , fluorescence and phase-contrast images of unstimulated ( US ) and ACh-stimulated smooth muscle cells from tissues expressing vinculin Y1065E or vinculin Y1065F or sham-treated. Each fluorescent spot (PLA spot) indicates interaction between talin and vinculin. B , the number of PLA spots was significantly higher in ACh-stimulated smooth muscle cells than in unstimulated cells in sham-treated tissues (unstimulated, n = 44; ACh, n = 59) and tissues expressing vinculin Y1065E (unstimulated, n = 34; ACh, n = 40). ACh did not stimulate an increase in vinculin-talin interactions in cells dissociated from tissues expressing vinculin Y1065F (unstimulated, n = 40; ACh, n = 42). All of the values are the means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated cells ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Expression of vinculin Y1065F in tracheal smooth muscle tissues inhibited the ACh-stimulated increase in the interaction between vinculin and talin, as detected by in situ PLAs performed on freshly dissociated cells. A , fluorescence and phase-contrast images of unstimulated ( US ) and ACh-stimulated smooth muscle cells from tissues expressing vinculin Y1065E or vinculin Y1065F or sham-treated. Each fluorescent spot (PLA spot) indicates interaction between talin and vinculin. B , the number of PLA spots was significantly higher in ACh-stimulated smooth muscle cells than in unstimulated cells in sham-treated tissues (unstimulated, n = 44; ACh, n = 59) and tissues expressing vinculin Y1065E (unstimulated, n = 34; ACh, n = 40). ACh did not stimulate an increase in vinculin-talin interactions in cells dissociated from tissues expressing vinculin Y1065F (unstimulated, n = 40; ACh, n = 42). All of the values are the means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated cells ( p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, In Situ, Fluorescence, Proximity Ligation Assay

    Expression of Y1065F inhibits vinculin phosphorylation at Tyr 1065 in response to ACh. A , immunoblots against phosphovinculin (pY1065), vinculin, and EGFP in protein extracts from unstimulated ( US ) and ACh-stimulated tissues that were sham-treated or expressing WT vinculin, vinculin Y1065E, or vinculin Y1065F. Vinculin Ab reacts with both vinculin (116 kDa) and metavinculin (145 kDa). The recombinant EGFP vinculin proteins have a similar mobility to metavinculin (145 kDa). Vinculin phosphorylation increased in ACh-stimulated tissues from Sham-treated, WT-transfected, and Y1065E-transfected tissues but not in those from Y1065F-transfected tissues. B , mean ratios of phosphovinculin normalized to total vinculin/metavinculin from each tissue. The phosphovinculin in ACh-stimulated tissues from sham-treated tissues and tissues expressing WT and Y1065E was significantly higher than that in unstimulated tissues; there was no significant difference between unstimulated and ACh-stimulated tissues in the Y1065F-treated group. Shown are means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated tissues ( n = 7, p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Expression of Y1065F inhibits vinculin phosphorylation at Tyr 1065 in response to ACh. A , immunoblots against phosphovinculin (pY1065), vinculin, and EGFP in protein extracts from unstimulated ( US ) and ACh-stimulated tissues that were sham-treated or expressing WT vinculin, vinculin Y1065E, or vinculin Y1065F. Vinculin Ab reacts with both vinculin (116 kDa) and metavinculin (145 kDa). The recombinant EGFP vinculin proteins have a similar mobility to metavinculin (145 kDa). Vinculin phosphorylation increased in ACh-stimulated tissues from Sham-treated, WT-transfected, and Y1065E-transfected tissues but not in those from Y1065F-transfected tissues. B , mean ratios of phosphovinculin normalized to total vinculin/metavinculin from each tissue. The phosphovinculin in ACh-stimulated tissues from sham-treated tissues and tissues expressing WT and Y1065E was significantly higher than that in unstimulated tissues; there was no significant difference between unstimulated and ACh-stimulated tissues in the Y1065F-treated group. Shown are means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated tissues ( n = 7, p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, Western Blot, Recombinant, Transfection

    Phosphorylation at Tyr 1065 does not affect the recruitment of vinculin FRET probes to the membrane in response to ACh. A , the average fluorescence intensity at the plasma membrane and in the central cytoplasmic regions of cells dissociated from smooth muscle tissues expressing vinculin FRET probes was analyzed by averaging emissions in six 1-μm 2 regions along the membrane and six 1-μm 2 regions in the cytoplasm. B , to determine the cellular localization of the FRET probes, the total fluorescence emission of the probes over the entire spectrum from 420 to 660 nm was calculated for each region, and the mean fluorescence intensities of membrane regions and cytoplasmic regions were separately determined. In unstimulated cells ( US ), the ratio of fluorescence intensity of membrane/cytoplasm was less than 1 in all groups of cells. Stimulation with ACh significantly increased the fluorescence intensity at the cell membrane in all groups of cells, regardless of the phosphorylation status of the vinculin FRET probe. There were no significant differences in the ratio of fluorescence intensity of membrane/cytoplasm among ACh-stimulated or unstimulated cells expressing the different vinculin Tyr 1065 phosphorylation mutant FRET probes ( p > 0.05). Values are mean ± S.E. ( error bars ): vinculin WT (unstimulated, n = 7; ACh, n = 7), vinculin Y1065E (unstimulated, n = 3; ACh, n = 5), vinculin Y1065F (unstimulated, n = 7; ACh, n = 16).

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Phosphorylation at Tyr 1065 does not affect the recruitment of vinculin FRET probes to the membrane in response to ACh. A , the average fluorescence intensity at the plasma membrane and in the central cytoplasmic regions of cells dissociated from smooth muscle tissues expressing vinculin FRET probes was analyzed by averaging emissions in six 1-μm 2 regions along the membrane and six 1-μm 2 regions in the cytoplasm. B , to determine the cellular localization of the FRET probes, the total fluorescence emission of the probes over the entire spectrum from 420 to 660 nm was calculated for each region, and the mean fluorescence intensities of membrane regions and cytoplasmic regions were separately determined. In unstimulated cells ( US ), the ratio of fluorescence intensity of membrane/cytoplasm was less than 1 in all groups of cells. Stimulation with ACh significantly increased the fluorescence intensity at the cell membrane in all groups of cells, regardless of the phosphorylation status of the vinculin FRET probe. There were no significant differences in the ratio of fluorescence intensity of membrane/cytoplasm among ACh-stimulated or unstimulated cells expressing the different vinculin Tyr 1065 phosphorylation mutant FRET probes ( p > 0.05). Values are mean ± S.E. ( error bars ): vinculin WT (unstimulated, n = 7; ACh, n = 7), vinculin Y1065E (unstimulated, n = 3; ACh, n = 5), vinculin Y1065F (unstimulated, n = 7; ACh, n = 16).

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing, Mutagenesis

    Tyr 1065 phosphorylation is required for vinculin to assume an open conformation in dissociated smooth muscle cells. Shown are fluorescence images at 470 nm (donor) and 530 nm (acceptor) emissions from unstimulated ( US ) and ACh-stimulated cells dissociated from tissues expressing vinculin WT, 1065E, or 1065F FRET probes. An increase in donor emissions indicates that more vinculin is in an open conformation. A , WT vinculin FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin FRET WT probes were higher at membrane regions than that in cytoplasm in both unstimulated and ACh-stimulated cells (unstimulated, n = 7; ACh, n = 8). B , vinculin phosphomimetic Y1065E FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. ACh stimulation increased fluorescence intensity at 470 nm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin Y1065E FRET probes were higher at membrane regions than that in cytoplasm (unstimulated, n = 3; ACh, n = 5). C , vinculin non-phosphorylatable Y1065F FRET probes maintain a closed conformation at the cell membrane and in the cytoplasm. ACh stimulation did not cause an increase in fluorescence intensity at 470 nm. No significant difference was detected in the ratio of donor emission (470 nm)/acceptor emission (530 nm) at the membrane regions or in the cytoplasm (unstimulated, n = 7; ACh, n = 16). *, significant difference between membrane and cytoplasm ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Tyr 1065 phosphorylation is required for vinculin to assume an open conformation in dissociated smooth muscle cells. Shown are fluorescence images at 470 nm (donor) and 530 nm (acceptor) emissions from unstimulated ( US ) and ACh-stimulated cells dissociated from tissues expressing vinculin WT, 1065E, or 1065F FRET probes. An increase in donor emissions indicates that more vinculin is in an open conformation. A , WT vinculin FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin FRET WT probes were higher at membrane regions than that in cytoplasm in both unstimulated and ACh-stimulated cells (unstimulated, n = 7; ACh, n = 8). B , vinculin phosphomimetic Y1065E FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. ACh stimulation increased fluorescence intensity at 470 nm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin Y1065E FRET probes were higher at membrane regions than that in cytoplasm (unstimulated, n = 3; ACh, n = 5). C , vinculin non-phosphorylatable Y1065F FRET probes maintain a closed conformation at the cell membrane and in the cytoplasm. ACh stimulation did not cause an increase in fluorescence intensity at 470 nm. No significant difference was detected in the ratio of donor emission (470 nm)/acceptor emission (530 nm) at the membrane regions or in the cytoplasm (unstimulated, n = 7; ACh, n = 16). *, significant difference between membrane and cytoplasm ( p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing

    Non-phosphorylatable vinculin Y1065F FRET probes do not convert to an open conformation in response to ACh in smooth muscle tissues and cells. A , fluorescence emissions at 470 nm (donor) and 530 nm (acceptor) in extracts from smooth muscle tissues were measured using a MicroSpectrometer after excitation at 405 nm. The donor/acceptor emission ratio was significantly higher in tissues expressing the vinculin WT or Y1065E FRET probes, but the donor/acceptor emission ratio was not increased in tissues expressing the Y1065F FRET probe. *, significant difference between ACh and unstimulated ( US ) tissues ( n = 4–5, p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Non-phosphorylatable vinculin Y1065F FRET probes do not convert to an open conformation in response to ACh in smooth muscle tissues and cells. A , fluorescence emissions at 470 nm (donor) and 530 nm (acceptor) in extracts from smooth muscle tissues were measured using a MicroSpectrometer after excitation at 405 nm. The donor/acceptor emission ratio was significantly higher in tissues expressing the vinculin WT or Y1065E FRET probes, but the donor/acceptor emission ratio was not increased in tissues expressing the Y1065F FRET probe. *, significant difference between ACh and unstimulated ( US ) tissues ( n = 4–5, p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing

    Vt CT hairpin deletion affects cell adhesion. A , Vin −/− MEFs were transfected with WT or ΔC5 vinculin and plated on FN. After fixation, F-actin and FAs were stained using phalloidin and GFP-tagged vinculin variants, respectively. Scale bar is 25 μm. B–D , adhesion number per cell ( B ), cell area ( C ), and adhesion size ( D ) were analyzed ( n = 19; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Vinculin C-terminal Hairpin Mediates F-actin Bundle Formation, Focal Adhesion, and Cell Mechanical Properties *

    doi: 10.1074/jbc.M111.244293

    Figure Lengend Snippet: Vt CT hairpin deletion affects cell adhesion. A , Vin −/− MEFs were transfected with WT or ΔC5 vinculin and plated on FN. After fixation, F-actin and FAs were stained using phalloidin and GFP-tagged vinculin variants, respectively. Scale bar is 25 μm. B–D , adhesion number per cell ( B ), cell area ( C ), and adhesion size ( D ) were analyzed ( n = 19; *, p

    Article Snippet: The ΔC5 variant of GFP vinculin was generated using a QuikChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing.

    Techniques: Transfection, Staining

    Vinculin constructs used in these studies. A , domain structure of EGFP-vinculin proteins, vinculin conformation-sensitive FRET probes, amber mutant, and Cerulean-5aa-Venus FRET probes. B , vinculin FRET probes in open and closed conformation. When vinculin is in an open conformation, there is maximum Cerulean probe emission at 470 nm. When vinculin assumes a closed conformation, Cerulean donor emission at 470 nm is quenched, and emission from the Venus acceptor at 530 nm is increased. C , emission spectrum of vinculin amber FRET mutant probe from a dissociated smooth muscle cell. Excitation at 405 nm on vinculin amber FRET mutant probe results in emission at 470 nm. D , emission spectrum of Cerulean-5aa-Venus FRET probe ( C5V ) from a dissociated smooth muscle cell. Excitation of Cerulean-5aa-Venus at 405 nm results in emission at 530 nm.

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Vinculin constructs used in these studies. A , domain structure of EGFP-vinculin proteins, vinculin conformation-sensitive FRET probes, amber mutant, and Cerulean-5aa-Venus FRET probes. B , vinculin FRET probes in open and closed conformation. When vinculin is in an open conformation, there is maximum Cerulean probe emission at 470 nm. When vinculin assumes a closed conformation, Cerulean donor emission at 470 nm is quenched, and emission from the Venus acceptor at 530 nm is increased. C , emission spectrum of vinculin amber FRET mutant probe from a dissociated smooth muscle cell. Excitation at 405 nm on vinculin amber FRET mutant probe results in emission at 470 nm. D , emission spectrum of Cerulean-5aa-Venus FRET probe ( C5V ) from a dissociated smooth muscle cell. Excitation of Cerulean-5aa-Venus at 405 nm results in emission at 530 nm.

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Construct, Mutagenesis

    Vinculin phosphorylation at Tyr 1065 does not affect the recruitment of vinculin to the membrane in response to ACh. A , freshly dissociated smooth muscle cells from tissues expressing EGFP vinculin WT (unstimulated ( US ), n = 17; ACh, n = 5), vinculin Y1065E (unstimulated, n = 25; ACh, n = 17), or vinculin Y1065F (unstimulated, n = 28; ACh, n = 14) were plated onto coverslips and stimulated with 10 −4 m ACh or left unstimulated. The localization of EGFP-vinculin was visualized in live cells by confocal microscopy. EGFP-vinculin WT, EGFP-vinculin Y1065E, and EGFP-vinculin Y1065F localized throughout the cytoplasm in unstimulated cells and were more heavily concentrated at the cell membrane in ACh-stimulated cells. B , mean ratios of pixel intensity between the cell membrane and cytoplasm. The membrane/cytoplasm ratio was significantly higher in ACh-stimulated cells from all recombinant vinculin treatment groups ( p > 0.05). The localization of mutant and wild type vinculin EGFP-vinculin proteins did not differ significantly among different recombinant vinculin treatment groups. Error bars , S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Vinculin phosphorylation at Tyr 1065 does not affect the recruitment of vinculin to the membrane in response to ACh. A , freshly dissociated smooth muscle cells from tissues expressing EGFP vinculin WT (unstimulated ( US ), n = 17; ACh, n = 5), vinculin Y1065E (unstimulated, n = 25; ACh, n = 17), or vinculin Y1065F (unstimulated, n = 28; ACh, n = 14) were plated onto coverslips and stimulated with 10 −4 m ACh or left unstimulated. The localization of EGFP-vinculin was visualized in live cells by confocal microscopy. EGFP-vinculin WT, EGFP-vinculin Y1065E, and EGFP-vinculin Y1065F localized throughout the cytoplasm in unstimulated cells and were more heavily concentrated at the cell membrane in ACh-stimulated cells. B , mean ratios of pixel intensity between the cell membrane and cytoplasm. The membrane/cytoplasm ratio was significantly higher in ACh-stimulated cells from all recombinant vinculin treatment groups ( p > 0.05). The localization of mutant and wild type vinculin EGFP-vinculin proteins did not differ significantly among different recombinant vinculin treatment groups. Error bars , S.E.

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, Confocal Microscopy, Recombinant, Mutagenesis

    Expression of vinculin Y1065F in tracheal smooth muscle tissues inhibited the ACh-stimulated increase in the interaction between vinculin and talin, as detected by in situ PLAs performed on freshly dissociated cells. A , fluorescence and phase-contrast images of unstimulated ( US ) and ACh-stimulated smooth muscle cells from tissues expressing vinculin Y1065E or vinculin Y1065F or sham-treated. Each fluorescent spot (PLA spot) indicates interaction between talin and vinculin. B , the number of PLA spots was significantly higher in ACh-stimulated smooth muscle cells than in unstimulated cells in sham-treated tissues (unstimulated, n = 44; ACh, n = 59) and tissues expressing vinculin Y1065E (unstimulated, n = 34; ACh, n = 40). ACh did not stimulate an increase in vinculin-talin interactions in cells dissociated from tissues expressing vinculin Y1065F (unstimulated, n = 40; ACh, n = 42). All of the values are the means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated cells ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Expression of vinculin Y1065F in tracheal smooth muscle tissues inhibited the ACh-stimulated increase in the interaction between vinculin and talin, as detected by in situ PLAs performed on freshly dissociated cells. A , fluorescence and phase-contrast images of unstimulated ( US ) and ACh-stimulated smooth muscle cells from tissues expressing vinculin Y1065E or vinculin Y1065F or sham-treated. Each fluorescent spot (PLA spot) indicates interaction between talin and vinculin. B , the number of PLA spots was significantly higher in ACh-stimulated smooth muscle cells than in unstimulated cells in sham-treated tissues (unstimulated, n = 44; ACh, n = 59) and tissues expressing vinculin Y1065E (unstimulated, n = 34; ACh, n = 40). ACh did not stimulate an increase in vinculin-talin interactions in cells dissociated from tissues expressing vinculin Y1065F (unstimulated, n = 40; ACh, n = 42). All of the values are the means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated cells ( p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, In Situ, Fluorescence, Proximity Ligation Assay

    Expression of Y1065F inhibits vinculin phosphorylation at Tyr 1065 in response to ACh. A , immunoblots against phosphovinculin (pY1065), vinculin, and EGFP in protein extracts from unstimulated ( US ) and ACh-stimulated tissues that were sham-treated or expressing WT vinculin, vinculin Y1065E, or vinculin Y1065F. Vinculin Ab reacts with both vinculin (116 kDa) and metavinculin (145 kDa). The recombinant EGFP vinculin proteins have a similar mobility to metavinculin (145 kDa). Vinculin phosphorylation increased in ACh-stimulated tissues from Sham-treated, WT-transfected, and Y1065E-transfected tissues but not in those from Y1065F-transfected tissues. B , mean ratios of phosphovinculin normalized to total vinculin/metavinculin from each tissue. The phosphovinculin in ACh-stimulated tissues from sham-treated tissues and tissues expressing WT and Y1065E was significantly higher than that in unstimulated tissues; there was no significant difference between unstimulated and ACh-stimulated tissues in the Y1065F-treated group. Shown are means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated tissues ( n = 7, p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Expression of Y1065F inhibits vinculin phosphorylation at Tyr 1065 in response to ACh. A , immunoblots against phosphovinculin (pY1065), vinculin, and EGFP in protein extracts from unstimulated ( US ) and ACh-stimulated tissues that were sham-treated or expressing WT vinculin, vinculin Y1065E, or vinculin Y1065F. Vinculin Ab reacts with both vinculin (116 kDa) and metavinculin (145 kDa). The recombinant EGFP vinculin proteins have a similar mobility to metavinculin (145 kDa). Vinculin phosphorylation increased in ACh-stimulated tissues from Sham-treated, WT-transfected, and Y1065E-transfected tissues but not in those from Y1065F-transfected tissues. B , mean ratios of phosphovinculin normalized to total vinculin/metavinculin from each tissue. The phosphovinculin in ACh-stimulated tissues from sham-treated tissues and tissues expressing WT and Y1065E was significantly higher than that in unstimulated tissues; there was no significant difference between unstimulated and ACh-stimulated tissues in the Y1065F-treated group. Shown are means ± S.E. ( error bars ). *, significant difference between ACh and unstimulated tissues ( n = 7, p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Expressing, Western Blot, Recombinant, Transfection

    Phosphorylation at Tyr 1065 does not affect the recruitment of vinculin FRET probes to the membrane in response to ACh. A , the average fluorescence intensity at the plasma membrane and in the central cytoplasmic regions of cells dissociated from smooth muscle tissues expressing vinculin FRET probes was analyzed by averaging emissions in six 1-μm 2 regions along the membrane and six 1-μm 2 regions in the cytoplasm. B , to determine the cellular localization of the FRET probes, the total fluorescence emission of the probes over the entire spectrum from 420 to 660 nm was calculated for each region, and the mean fluorescence intensities of membrane regions and cytoplasmic regions were separately determined. In unstimulated cells ( US ), the ratio of fluorescence intensity of membrane/cytoplasm was less than 1 in all groups of cells. Stimulation with ACh significantly increased the fluorescence intensity at the cell membrane in all groups of cells, regardless of the phosphorylation status of the vinculin FRET probe. There were no significant differences in the ratio of fluorescence intensity of membrane/cytoplasm among ACh-stimulated or unstimulated cells expressing the different vinculin Tyr 1065 phosphorylation mutant FRET probes ( p > 0.05). Values are mean ± S.E. ( error bars ): vinculin WT (unstimulated, n = 7; ACh, n = 7), vinculin Y1065E (unstimulated, n = 3; ACh, n = 5), vinculin Y1065F (unstimulated, n = 7; ACh, n = 16).

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Phosphorylation at Tyr 1065 does not affect the recruitment of vinculin FRET probes to the membrane in response to ACh. A , the average fluorescence intensity at the plasma membrane and in the central cytoplasmic regions of cells dissociated from smooth muscle tissues expressing vinculin FRET probes was analyzed by averaging emissions in six 1-μm 2 regions along the membrane and six 1-μm 2 regions in the cytoplasm. B , to determine the cellular localization of the FRET probes, the total fluorescence emission of the probes over the entire spectrum from 420 to 660 nm was calculated for each region, and the mean fluorescence intensities of membrane regions and cytoplasmic regions were separately determined. In unstimulated cells ( US ), the ratio of fluorescence intensity of membrane/cytoplasm was less than 1 in all groups of cells. Stimulation with ACh significantly increased the fluorescence intensity at the cell membrane in all groups of cells, regardless of the phosphorylation status of the vinculin FRET probe. There were no significant differences in the ratio of fluorescence intensity of membrane/cytoplasm among ACh-stimulated or unstimulated cells expressing the different vinculin Tyr 1065 phosphorylation mutant FRET probes ( p > 0.05). Values are mean ± S.E. ( error bars ): vinculin WT (unstimulated, n = 7; ACh, n = 7), vinculin Y1065E (unstimulated, n = 3; ACh, n = 5), vinculin Y1065F (unstimulated, n = 7; ACh, n = 16).

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing, Mutagenesis

    Tyr 1065 phosphorylation is required for vinculin to assume an open conformation in dissociated smooth muscle cells. Shown are fluorescence images at 470 nm (donor) and 530 nm (acceptor) emissions from unstimulated ( US ) and ACh-stimulated cells dissociated from tissues expressing vinculin WT, 1065E, or 1065F FRET probes. An increase in donor emissions indicates that more vinculin is in an open conformation. A , WT vinculin FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin FRET WT probes were higher at membrane regions than that in cytoplasm in both unstimulated and ACh-stimulated cells (unstimulated, n = 7; ACh, n = 8). B , vinculin phosphomimetic Y1065E FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. ACh stimulation increased fluorescence intensity at 470 nm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin Y1065E FRET probes were higher at membrane regions than that in cytoplasm (unstimulated, n = 3; ACh, n = 5). C , vinculin non-phosphorylatable Y1065F FRET probes maintain a closed conformation at the cell membrane and in the cytoplasm. ACh stimulation did not cause an increase in fluorescence intensity at 470 nm. No significant difference was detected in the ratio of donor emission (470 nm)/acceptor emission (530 nm) at the membrane regions or in the cytoplasm (unstimulated, n = 7; ACh, n = 16). *, significant difference between membrane and cytoplasm ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Tyr 1065 phosphorylation is required for vinculin to assume an open conformation in dissociated smooth muscle cells. Shown are fluorescence images at 470 nm (donor) and 530 nm (acceptor) emissions from unstimulated ( US ) and ACh-stimulated cells dissociated from tissues expressing vinculin WT, 1065E, or 1065F FRET probes. An increase in donor emissions indicates that more vinculin is in an open conformation. A , WT vinculin FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin FRET WT probes were higher at membrane regions than that in cytoplasm in both unstimulated and ACh-stimulated cells (unstimulated, n = 7; ACh, n = 8). B , vinculin phosphomimetic Y1065E FRET probes maintain an open conformation at the cell membrane and a closed conformation in the cytoplasm. ACh stimulation increased fluorescence intensity at 470 nm. Mean ratios of donor emission (470 nm)/acceptor emission (530 nm) from cells expressing vinculin Y1065E FRET probes were higher at membrane regions than that in cytoplasm (unstimulated, n = 3; ACh, n = 5). C , vinculin non-phosphorylatable Y1065F FRET probes maintain a closed conformation at the cell membrane and in the cytoplasm. ACh stimulation did not cause an increase in fluorescence intensity at 470 nm. No significant difference was detected in the ratio of donor emission (470 nm)/acceptor emission (530 nm) at the membrane regions or in the cytoplasm (unstimulated, n = 7; ACh, n = 16). *, significant difference between membrane and cytoplasm ( p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing

    Non-phosphorylatable vinculin Y1065F FRET probes do not convert to an open conformation in response to ACh in smooth muscle tissues and cells. A , fluorescence emissions at 470 nm (donor) and 530 nm (acceptor) in extracts from smooth muscle tissues were measured using a MicroSpectrometer after excitation at 405 nm. The donor/acceptor emission ratio was significantly higher in tissues expressing the vinculin WT or Y1065E FRET probes, but the donor/acceptor emission ratio was not increased in tissues expressing the Y1065F FRET probe. *, significant difference between ACh and unstimulated ( US ) tissues ( n = 4–5, p

    Journal: The Journal of Biological Chemistry

    Article Title: Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues *

    doi: 10.1074/jbc.M113.508077

    Figure Lengend Snippet: Non-phosphorylatable vinculin Y1065F FRET probes do not convert to an open conformation in response to ACh in smooth muscle tissues and cells. A , fluorescence emissions at 470 nm (donor) and 530 nm (acceptor) in extracts from smooth muscle tissues were measured using a MicroSpectrometer after excitation at 405 nm. The donor/acceptor emission ratio was significantly higher in tissues expressing the vinculin WT or Y1065E FRET probes, but the donor/acceptor emission ratio was not increased in tissues expressing the Y1065F FRET probe. *, significant difference between ACh and unstimulated ( US ) tissues ( n = 4–5, p

    Article Snippet: Plasmid vectors used were pEGFP-vinculin plasmids encoding full-length chicken vinculin (residues 1–1066) provided by Dr. Susan Craig (Johns Hopkins University, Baltimore, MD) ( ); non-phosphorylatable vinculin Y1065F and phosphomimetic vinculin Y1065E, constructed by site mutation at Tyr1065 using a QuikChange II site-directed mutagenesis kit (Stratagene); and vinculin conformation-sensitive FRET probes, constructed as described below using plasmids pmCerulean3-N1.

    Techniques: Fluorescence, Expressing