Journal: The Journal of Cell Biology

Article Title: A Rac switch regulates random versus directionally persistent cell migration

doi: 10.1083/jcb.200503152

Figure Lengend Snippet: Integrin mutation inhibits Rac signaling and suppresses random cell motility. (A) GD25 cells with a point mutation in the β 1 integrin cytoplasmic domain (W775A) have a selective defect in Rac activation. Activities in pull-down assays for Rac-GTP, Cdc42-GTP, and Rho-GTP and total amounts of each protein in lysates of cells cultured on 1 μg/ml fibronectin were analyzed using antibodies against Rac, Cdc42, and Rho. Densitometry for each GTP-bound protein was normalized to the amount of the total protein, and results are presented as fold change compared with cells expressing wild-type integrin. The reduction of active Rac was 75% (P < 0.0001), whereas active Cdc42 and active Rho were not significantly reduced (by 10%, P = 0.31 and 8%, P = 0.79, respectively). In this and subsequent figures, all adjacent gel lanes are from the same gel and blot, but their order may be rearranged for clarity as indicated by white lines between the lanes. (B) Representative examples of migration tracks of cells expressing wild-type β 1 (β 1 wt) or mutant (W775A) integrins cultured on fibronectin and tracked for 12 h. In these and subsequent composite migration figures, randomly selected individual migration tracks were copied and combined into a single figure to avoid empty spaces. Bar, 100 μm. (C) Quantification of the persistence of migratory directionality and velocity. Cell movements were recorded by time-lapse video microscopy and quantified by MetaMorph software. D/T ratios represent the ratio of the direct distance from start to end point [D] divided by the total track distance [T]. Motility was calculated as velocity (μm/h). Data were pooled from four independent experiments; error bars indicate SEM based on n = 42–45 cells. (D) Activated Akt can restore Rac activity. W775A cells were transfected with constitutively activated Akt, and stable clones were selected by serum starvation for 1 wk to induce apoptosis in cells with low Akt activity. The activity of Akt in the isolated clones (Akt clones 1–3) was assayed by immunoblotting with antibodies against phosphorylated Akt (p-Akt). Densitometry values for phospho-Akt were normalized to actin in the lysates, and the changes were calculated as fold increases compared with the parental W775A cells. The activity of Rac in the isolated Akt clones was calculated as the fold increase above W775A cell values after normalization of densitometry of the pull-down samples (Rac-GTP) to the total Rac in the lysates. (E) Activated Akt restores random motility. Quantification of cell movements of the Akt clones was performed as described in C. The decreases in D/T ratio after activated Akt expression were all significant (Akt clone 1, P < 0.01; clone 2, P < 0.001; and clone 3, P < 0.001), as were the decreases in velocity (clone 1, P < 0.01; and clones 2 and 3, P < 0.001). Error bars indicate SEM.

Article Snippet: Antibodies to total-, phospho (Ser 473)-, and phospho (Thr 308)-Akt as well as total, phospho (Thr 202), and (Tyr 204) p44/42 MAP kinase (ERK1/2) were purchased from Cell Signaling; anti-Cdc42 antibody was purchased from BD Biosciences; phalloidin conjugated with Alexa 488 or Alexa 594 was obtained from Molecular Probes; total, phospho (Thr 183), and phospho (Tyr 185) JNK antibodies, Cdc42 siRNA sc29256, and RhoA siRNA sc29471 were obtained from Santa Cruz Biotechnology, Inc.; anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cytoskeleton, Inc.; anti-VSV epitope and antiactin clone AC-40 were purchased from Sigma-Aldrich; anti-Rac antibody and Rhotekin-Rho binding domain were purchased from Upstate Biotechnology or Cytoskeleton, Inc.; anti-β 1 antibody 4080 was raised against a 50-mer cytoplasmic domain peptide in a rabbit; and anti-β 3 integrin hybridoma AP3 was purchased from American Type Culture Collection.

Techniques: Mutagenesis, Activation Assay, Cell Culture, Expressing, Migration, Microscopy, Software, Activity Assay, Transfection, Clone Assay, Isolation, Western Blot

Journal: The Journal of Cell Biology

Article Title: A Rac switch regulates random versus directionally persistent cell migration

doi: 10.1083/jcb.200503152

Figure Lengend Snippet: Suppression of Rac1 expression lowers Rac activity and inhibits random motility of fibroblasts. (A) Primary human fibroblasts were transfected with 1 nM Rac1 siRNA or 1 nM nonspecific control pool siRNA (Contr). Pull-down assays were performed as described for after 72 h of siRNA treatment, using an immunoblot of lysates for actin to verify equal protein loading. The amount of active Rac (Rac-GTP) was reduced to 67% of original levels and the total amount of Rac protein (total-Rac) was reduced to 52% after this Rac1 siRNA transfection. In contrast, levels of active Cdc42 and Rho did not change. The immunoblot for total Rac was stripped and reprobed for total Cdc42 protein, which was not altered by the Rac1 siRNA. (B and D) Composite collection of representative migration tracks of human fibroblasts starting 72 h after transfection with control compared with either 0.1 or 100 nM Rac1 siRNA, respectively, tracked at 15-min intervals over a span of 10 h. Bars, 100 μm. (C and E) Quantification of persistence of migratory directionality (D/T) and velocity of the transfected cells at the two different siRNA concentrations: (C) 0.1 nM and (E) 100 nM. Error bars indicate SEM; the differences between control and 0.1 nM or 100 nM Rac1 siRNA D/T values were significant at the P < 0.001 level. (F) Increases in D/T ratios by reduction of active Rac using Rac siRNA were observed in all (11 out of 11) independent experiments, each testing three siRNA concentrations against the control. All data were pooled together for this composite graph based on video time-lapse microscopy on a total of 933 cells.

Article Snippet: Antibodies to total-, phospho (Ser 473)-, and phospho (Thr 308)-Akt as well as total, phospho (Thr 202), and (Tyr 204) p44/42 MAP kinase (ERK1/2) were purchased from Cell Signaling; anti-Cdc42 antibody was purchased from BD Biosciences; phalloidin conjugated with Alexa 488 or Alexa 594 was obtained from Molecular Probes; total, phospho (Thr 183), and phospho (Tyr 185) JNK antibodies, Cdc42 siRNA sc29256, and RhoA siRNA sc29471 were obtained from Santa Cruz Biotechnology, Inc.; anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cytoskeleton, Inc.; anti-VSV epitope and antiactin clone AC-40 were purchased from Sigma-Aldrich; anti-Rac antibody and Rhotekin-Rho binding domain were purchased from Upstate Biotechnology or Cytoskeleton, Inc.; anti-β 1 antibody 4080 was raised against a 50-mer cytoplasmic domain peptide in a rabbit; and anti-β 3 integrin hybridoma AP3 was purchased from American Type Culture Collection.

Techniques: Expressing, Activity Assay, Transfection, Control, Western Blot, Migration, Time-lapse Microscopy

Journal: The Journal of Cell Biology

Article Title: A Rac switch regulates random versus directionally persistent cell migration

doi: 10.1083/jcb.200503152

Figure Lengend Snippet: Suppression of Cdc42 or RhoA expression does not affect directional persistence of cell migration. (A and B) Specificity of Cdc42 and RhoA knockdowns. (A) 3 d after transfection of primary human fibroblasts with 100 nM Cdc42 siRNA, total Cdc42 protein levels were assayed by Western blotting with antibodies against Cdc42 in lysates of the transfected (Cdc42) and control cells treated with nonspecific siRNA (Control). Cdc42 siRNA led to a substantial decrease of both Cdc42 protein levels (total-Cdc42) and Cdc42 activity (Cdc42-GTP) by pull-down assay, whereas the activity and total amount of Rac in the same lysates (Rac-GTP and total-Rac) were not affected. (B) Primary human fibroblasts were transfected with 100 nM RhoA siRNA (RhoA) or nonspecific siRNA (Control). 3 d later, Rho protein levels were determined by Western blotting with antibodies against Rho. Although the levels of other Rho family members were not affected, the amount and activity of RhoA were substantially decreased. (C) After knockdown of Cdc42 by >80%, there were no significant changes in the D/T directionality ratio (D/T Control = 0.46 ± 0.03 and D/T Cdc42 = 0.46 ± 0.05; P D/T = 0.89) or the velocity (V Contr = 44.4 ± 3.9 μm/h and V Cdc42 = 38.6 ± 2.1 μm/h; P V = 0.18). (C and D) Error bars represent the SEM. (D) Suppression of RhoA expression affects the velocity of migration but not directionality. After reducing RhoA levels by at least 60% using siRNA, velocity of cell migration was reduced by 40% (V Contr = 34.4 ± 2.4 μm/h and V RhoA = 20.8 ± 1.4 μm/h; P < 0.0001), but directional persistence of migration was unaffected (D/T Control = 0.44 ± 0.03 and D/T RhoA = 0.42 ± 0.03; P = 0.74).

Article Snippet: Antibodies to total-, phospho (Ser 473)-, and phospho (Thr 308)-Akt as well as total, phospho (Thr 202), and (Tyr 204) p44/42 MAP kinase (ERK1/2) were purchased from Cell Signaling; anti-Cdc42 antibody was purchased from BD Biosciences; phalloidin conjugated with Alexa 488 or Alexa 594 was obtained from Molecular Probes; total, phospho (Thr 183), and phospho (Tyr 185) JNK antibodies, Cdc42 siRNA sc29256, and RhoA siRNA sc29471 were obtained from Santa Cruz Biotechnology, Inc.; anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cytoskeleton, Inc.; anti-VSV epitope and antiactin clone AC-40 were purchased from Sigma-Aldrich; anti-Rac antibody and Rhotekin-Rho binding domain were purchased from Upstate Biotechnology or Cytoskeleton, Inc.; anti-β 1 antibody 4080 was raised against a 50-mer cytoplasmic domain peptide in a rabbit; and anti-β 3 integrin hybridoma AP3 was purchased from American Type Culture Collection.

Techniques: Expressing, Migration, Transfection, Western Blot, Control, Activity Assay, Pull Down Assay, Knockdown

Journal: The Journal of Cell Biology

Article Title: A Rac switch regulates random versus directionally persistent cell migration

doi: 10.1083/jcb.200503152

Figure Lengend Snippet: Inhibition of PI 3-kinase or Cdc42 inhibits chemotaxis but not directionally persistent cell migration. (A) Inhibition of PI3K by 50 μM LY 294002 blocks primary human fibroblast chemotaxis toward 300 nM fMLP whether or not Rac is suppressed by 1 nM Rac1 siRNA. (B) Localization of GFP-tagged Akt PH domain to lamellae is suppressed by treatment with LY 294002. Insets show lower magnification views of the entire cell. Bars, 20 μm. (C) Inhibition of Cdc42 by 100 nM Cdc42 siRNA (>80% of both total Cdc42 protein and activity) blocks fMLP-stimulated chemotaxis of human fibroblasts. (D) Inhibition of PI3K by 50 μM LY 294002 does not suppress the increased directional persistence of migration induced by reduction of active Rac using 1 nM Rac1 siRNA. (A, C, and D) Error bars represent SEM.

Article Snippet: Antibodies to total-, phospho (Ser 473)-, and phospho (Thr 308)-Akt as well as total, phospho (Thr 202), and (Tyr 204) p44/42 MAP kinase (ERK1/2) were purchased from Cell Signaling; anti-Cdc42 antibody was purchased from BD Biosciences; phalloidin conjugated with Alexa 488 or Alexa 594 was obtained from Molecular Probes; total, phospho (Thr 183), and phospho (Tyr 185) JNK antibodies, Cdc42 siRNA sc29256, and RhoA siRNA sc29471 were obtained from Santa Cruz Biotechnology, Inc.; anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cytoskeleton, Inc.; anti-VSV epitope and antiactin clone AC-40 were purchased from Sigma-Aldrich; anti-Rac antibody and Rhotekin-Rho binding domain were purchased from Upstate Biotechnology or Cytoskeleton, Inc.; anti-β 1 antibody 4080 was raised against a 50-mer cytoplasmic domain peptide in a rabbit; and anti-β 3 integrin hybridoma AP3 was purchased from American Type Culture Collection.

Techniques: Inhibition, Chemotaxis Assay, Migration, Activity Assay

Journal: The Journal of Cell Biology

Article Title: A Rac switch regulates random versus directionally persistent cell migration

doi: 10.1083/jcb.200503152

Figure Lengend Snippet: 3D fibronectin matrix reduces both Rac activity and random migration. (A) Primary human fibroblasts were plated on 2D substrates coated with fibronectin or 3D matrices rich in fibronectin and assayed for activity as in . The reduction of active Rac in cells plated in a 3D matrix compared with 2D was 31% (P = 0.017), whereas Cdc42 and Rho changes were not significant (Cdc42 4.5% reduction, P = 0.66; Rho 25% increase, P = 0.65). Error bars indicate SEM. (B) Cells in 2D versus 3D environments have different morphologies and locations of lamellae (arrowheads), as shown after staining for F-actin with phalloidin-Alexa 594 (left and red color in the right) and anti-fibronectin antibody (green). Bar, 20 μm. (C) Migration of cells in 2D versus 3D environments was recorded, and directionality (D/T) and velocity were calculated. (C and D) Error bars represent SEM. (D) Decreased random motility of cells within three-dimensional (3D) matrix. Primary human fibroblasts were cultured overnight within intact 3D matrices, on mechanically flattened 3D matrix (2D matrix), or on surfaces coated with solubilized 3D matrix (2D mix). Cell movements were recorded for 10 h, and the D/T ratio and velocity were calculated as described above. (E) Activated Rac in cells in 3D matrix restores random motility. Primary human fibroblasts were cotransfected with Rac Q61L VSV and Rac Q61L GFP or GFP alone (Control), sorted for low levels of GFP expression (1–50% from the peak of positive cells), and plated on 3D matrices. Cell movements were recorded for 10 h, and the D/T ratio and velocity were quantified. The bars represent the means of data pooled from two experiments, and error bars indicate SEM ( n = 32–34 cells).

Article Snippet: Antibodies to total-, phospho (Ser 473)-, and phospho (Thr 308)-Akt as well as total, phospho (Thr 202), and (Tyr 204) p44/42 MAP kinase (ERK1/2) were purchased from Cell Signaling; anti-Cdc42 antibody was purchased from BD Biosciences; phalloidin conjugated with Alexa 488 or Alexa 594 was obtained from Molecular Probes; total, phospho (Thr 183), and phospho (Tyr 185) JNK antibodies, Cdc42 siRNA sc29256, and RhoA siRNA sc29471 were obtained from Santa Cruz Biotechnology, Inc.; anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cytoskeleton, Inc.; anti-VSV epitope and antiactin clone AC-40 were purchased from Sigma-Aldrich; anti-Rac antibody and Rhotekin-Rho binding domain were purchased from Upstate Biotechnology or Cytoskeleton, Inc.; anti-β 1 antibody 4080 was raised against a 50-mer cytoplasmic domain peptide in a rabbit; and anti-β 3 integrin hybridoma AP3 was purchased from American Type Culture Collection.

Techniques: Activity Assay, Migration, Staining, Cell Culture, Control, Expressing

Journal: Oncology reports

Article Title: CCL19-induced chemokine receptor 7 activates the phosphoinositide-3 kinase-mediated invasive pathway through Cdc42 in metastatic squamous cell carcinoma of the head and neck.

doi: 10.3892/or.2010.1109

Figure Lengend Snippet: Figure 3. Activation of Cdc42 is involved in CCL19-induced actin polymerization. (a) Characteristic organization of filamentous actin (F-actin) in PCI-4B and intense F-actin stain is visible in the leading edge after CCL19 stimulation (arrow). The change was blocked by pretreatment with inhibitors and Cdc42 siRNA. (A), control; (B), CCL19; (C), pretreated with CCR7 mAb and incubated with CCL19; (D), pretreated with LY294002 and incubated with CCL19; (E), pretreated with Toxin B and incubated with CCL19; (F), Cdc42 siRNA and incubated with CCL19. F-actin was stained with TRITC-labeled phalloidin and visualized using CLSM. (b) Characteristic organization of filamentous actin (F-actin) in PCI-37B. Intense F-actin stain is visible in the leading edge after CCL19 stimulation (arrow). The change was blocked by pretreatment with inhibitors and Cdc42 siRNA. (A), control; (B), CCL19; (C), pretreated with CCR7 mAb and incubated with CCL19; (D), pretreated with LY294002 and incubated with CCL19; (E), pretreated with Toxin B and incubated with CCL19; (F), Cdc42 siRNA and incubated with CCL19. F-actin was stained with TRITC-labeled phalloidin and visualized using CLSM. (c) SiRNA resulted in over 90% knock down of Cdc42 in PCI-37B. SCCHN cells were transfected using 100 pmol Cdc42-specific or controled siRNA for 48 h. Levels of total Cdc42 were determined for whole cell lysates (WCL) were detected by Western blotting. Western blot analysis of ß-actin was done to confirm equal protein loading.

Article Snippet: Goat polyclone Cdc42 anti-body (C-20), rabbit polyclone Cdc42 antibody (P1), goat polyclone Rac antibody (S-18) rabbit polyclone Rac antibody (C-11), siRNA targeting human CCR7 and Cdc42, and non-specific control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Control, Incubation, Labeling, Knockdown, Transfection, Western Blot

Journal: Oncology reports

Article Title: CCL19-induced chemokine receptor 7 activates the phosphoinositide-3 kinase-mediated invasive pathway through Cdc42 in metastatic squamous cell carcinoma of the head and neck.

doi: 10.3892/or.2010.1109

Figure Lengend Snippet: Figure 4. Cdc42 is required for activation of Rac induced by CCL19 stimulation. Rac activated by CCL19 sitmulation and the activation were blocked by Cdc42 siRNA. Levels of total Rac were determined for whole cell lysates (WCL) by Western blotting. Western blot analysis of ß-actin was done to confirm equal protein loading. Levels of GTP-bound Rac were deter- mined using a GTPase activity pull-down assay (PBD pull-down) followed by Western blotting. GST-p21-PAK was used for GST pull-down of activated Rac.

Article Snippet: Goat polyclone Cdc42 anti-body (C-20), rabbit polyclone Cdc42 antibody (P1), goat polyclone Rac antibody (S-18) rabbit polyclone Rac antibody (C-11), siRNA targeting human CCR7 and Cdc42, and non-specific control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, Activity Assay, Pull Down Assay

Journal: Oncology reports

Article Title: CCL19-induced chemokine receptor 7 activates the phosphoinositide-3 kinase-mediated invasive pathway through Cdc42 in metastatic squamous cell carcinoma of the head and neck.

doi: 10.3892/or.2010.1109

Figure Lengend Snippet: Figure 5. CCR7 induced PI3K-mediated migration of metastatic SCCHN cell lines through Cdc42. Cell migration was significantly increased by CCL19 stimulation and blocked by CCR7 siRNA (P<0.01). PCI-4B and PCI-37B were pretreated with/without CCR7 mAb, PI3K inhibitor LY294002, Toxin B and Cdc42 siRNA. CCL19 was the chemoattractant. Transwell inserts were used to analyze migration at 24 h post seeding. The chemotaxis index was expressed as percentage of control sample which was not exposed to CCL19.

Article Snippet: Goat polyclone Cdc42 anti-body (C-20), rabbit polyclone Cdc42 antibody (P1), goat polyclone Rac antibody (S-18) rabbit polyclone Rac antibody (C-11), siRNA targeting human CCR7 and Cdc42, and non-specific control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Chemotaxis Assay, Control

Journal: Oncology reports

Article Title: CCL19-induced chemokine receptor 7 activates the phosphoinositide-3 kinase-mediated invasive pathway through Cdc42 in metastatic squamous cell carcinoma of the head and neck.

doi: 10.3892/or.2010.1109

Figure Lengend Snippet: Figure 6. CCR7 induces PI3K-mediated invasion of metastatic SCCHN cell lines through Cdc42. Cell invasion was significantly increased by CCL19 stimulation and blocked by CCR7 siRNA (P<0.01). PCI-4B and PCI-37B were pretreated with/without CCR7 mAb, PI3K inhibitor LY294002, Toxin B and Cdc42 siRNA. CCL19 was the chemoattractant. Matrigel-coated inserts were used to analyze invasion at 48 h post seeding. The invasion index was expressed as percentage of control sample which was not exposed to CCL19.

Article Snippet: Goat polyclone Cdc42 anti-body (C-20), rabbit polyclone Cdc42 antibody (P1), goat polyclone Rac antibody (S-18) rabbit polyclone Rac antibody (C-11), siRNA targeting human CCR7 and Cdc42, and non-specific control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control