Figure 1.
LPHN2 signals via cAMP/Rap1 and negatively regulates FA turnover, stress fiber formation, and ECM-elicited mechanosensing.(A) Confocal microscopy analysis of ECs indicates how endogenous LPHN2, as detected by an anti-LPHN2 Ab (green), colocalizes with vinculin (Vin; red) in ECM adhesions of ECs as shown in merge (right). Bottom: Magnifications of the corresponding top row panels. Scale bars, 20 µm. (B) Fluorescence confocal microcopy reveals that in ECs transfected with HA-Lphn2-EGFP, both the extracellular, as detected by an anti-HA Ab (red), and the EGFP-fused intracellular (green) moieties colocalize with vinculin (blue) at ECM adhesions, as shown in merge (right). Bottom: Magnifications of the corresponding upper row panels. Scale bars, 20 µm. (C) Real-time analysis of cell migration in siCTL or siLPHN2 ECs toward Coll I, assessed with an xCELLigence RTCA DP system. Results are the mean ± SD of five independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001. (D) LPHN2 silencing in ECs decreases basal amount of cAMP. Results are the mean ± SD of nine independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; ***, P ≤ 0.001. (E) Rap1-GTP was pulled down on a GST fusion protein carrying the Rap1-binding domain of human Ral guanine nucleotide dissociation stimulator. LPHN2 silencing in human ECs decreases basal GTP loading of Rap1 small GTPase, and pCCL lentivirus-mediated overexpression of silencing-resistant mouse Lphn2 rescues the decrease. Total Rap1 was employed to calculate the normalized OD (N.O.D.) levels of active Rap1-GTP. Results are the mean ± SD of five independent experiments (a representative one is shown). Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001 for CTL/siCTL versus CTL/siLPHN2, and **, P ≤ 0.01 for CTL/siLPHN2 versus Lphn2/siLPHN2. (F–J) Confocal microscopy analysis (F) of endogenous LPHN2 (green), vinculin (blue), and phalloidin-labeled F-actin (red; each high magnification images are on the right) reveals how, compared with siCTL ECs, LPHN2 silencing increases the number, normalized on cell area (G) and size (expressed by maximum Feret diameter, H) of vinculin-containing FAs and the number, normalized on cell area (I) and amount (evaluated as mean gray intensity, J) of F-actin stress fibers in siLPHN2 ECs. Scale bars, 20 µm. Results concerning vinculin-containing FAs and F-actin stress fibers are the mean ± SD of two independent experiments for a total of 13 siCTL and 13 siLPHN2 ECs and two independent experiments for a total of 19 siCTL and 20 siLPHN2 ECs, respectively. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; ***, P ≤ 0.001. (K and L) Confocal microscopy analysis (K) of endogenous YAP (green) and TAZ (red) reveals how, compared with siCTL ECs, LPHN2 silencing increases the nuclear (nucl)/cytoplasmic (cyto) ratio of both YAP (L, top) and TAZ (L, bottom). Nuclei were stained with TO-PRO3 (blue). Scale bar, 20 µm. Representative images of ECs plated on 10 kPa stiffness on FN (5 µg/ml) are shown. Results are the mean ± SD of two independent experiments for a total of 11 ECs for each condition (10 kPa and increasing FN 1, 3, and 5 µg/ml concentration). Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (M) Real-time quantitative PCR analysis of CTGF and CYR61 mRNA in siCTL or siLPHN2 human ECs relative to the housekeeping genes, GAPDH and TBP, and normalized on siCTL levels. Data of one of two independent assays are shown. Results are the mean ± SD of three technical replicates. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; **, P ≤ 0.01. Max., maximum; n., number.

LPHN2 signals via cAMP/Rap1 and negatively regulates FA turnover, stress fiber formation, and ECM-elicited mechanosensing. (A) Confocal microscopy analysis of ECs indicates how endogenous LPHN2, as detected by an anti-LPHN2 Ab (green), colocalizes with vinculin (Vin; red) in ECM adhesions of ECs as shown in merge (right). Bottom: Magnifications of the corresponding top row panels. Scale bars, 20 µm. (B) Fluorescence confocal microcopy reveals that in ECs transfected with HA-Lphn2-EGFP, both the extracellular, as detected by an anti-HA Ab (red), and the EGFP-fused intracellular (green) moieties colocalize with vinculin (blue) at ECM adhesions, as shown in merge (right). Bottom: Magnifications of the corresponding upper row panels. Scale bars, 20 µm. (C) Real-time analysis of cell migration in siCTL or siLPHN2 ECs toward Coll I, assessed with an xCELLigence RTCA DP system. Results are the mean ± SD of five independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001. (D) LPHN2 silencing in ECs decreases basal amount of cAMP. Results are the mean ± SD of nine independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; ***, P ≤ 0.001. (E) Rap1-GTP was pulled down on a GST fusion protein carrying the Rap1-binding domain of human Ral guanine nucleotide dissociation stimulator. LPHN2 silencing in human ECs decreases basal GTP loading of Rap1 small GTPase, and pCCL lentivirus-mediated overexpression of silencing-resistant mouse Lphn2 rescues the decrease. Total Rap1 was employed to calculate the normalized OD (N.O.D.) levels of active Rap1-GTP. Results are the mean ± SD of five independent experiments (a representative one is shown). Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001 for CTL/siCTL versus CTL/siLPHN2, and **, P ≤ 0.01 for CTL/siLPHN2 versus Lphn2/siLPHN2. (F–J) Confocal microscopy analysis (F) of endogenous LPHN2 (green), vinculin (blue), and phalloidin-labeled F-actin (red; each high magnification images are on the right) reveals how, compared with siCTL ECs, LPHN2 silencing increases the number, normalized on cell area (G) and size (expressed by maximum Feret diameter, H) of vinculin-containing FAs and the number, normalized on cell area (I) and amount (evaluated as mean gray intensity, J) of F-actin stress fibers in siLPHN2 ECs. Scale bars, 20 µm. Results concerning vinculin-containing FAs and F-actin stress fibers are the mean ± SD of two independent experiments for a total of 13 siCTL and 13 siLPHN2 ECs and two independent experiments for a total of 19 siCTL and 20 siLPHN2 ECs, respectively. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; ***, P ≤ 0.001. (K and L) Confocal microscopy analysis (K) of endogenous YAP (green) and TAZ (red) reveals how, compared with siCTL ECs, LPHN2 silencing increases the nuclear (nucl)/cytoplasmic (cyto) ratio of both YAP (L, top) and TAZ (L, bottom). Nuclei were stained with TO-PRO3 (blue). Scale bar, 20 µm. Representative images of ECs plated on 10 kPa stiffness on FN (5 µg/ml) are shown. Results are the mean ± SD of two independent experiments for a total of 11 ECs for each condition (10 kPa and increasing FN 1, 3, and 5 µg/ml concentration). Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (M) Real-time quantitative PCR analysis of CTGF and CYR61 mRNA in siCTL or siLPHN2 human ECs relative to the housekeeping genes, GAPDH and TBP, and normalized on siCTL levels. Data of one of two independent assays are shown. Results are the mean ± SD of three technical replicates. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; **, P ≤ 0.01. Max., maximum; n., number.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal