Figure 3.
Figure 3. Chemoattractant-mediated phosphorylation of myosin light chain requires Rac1. (A) Effect of GTPase mutants on phosphorylation of myosin light chain in human neutrophils. Human neutrophils were transduced with the indicated GTPases using BioPorter reagent, globally stimulated with 1 μM fMLP, fixed, and simultaneously stained with antibodies to myosin heavy chain (green) and phosphorylated MLC (red). There is a significant reduction in myosin phosphorylation upon treatment with Rac1T17N compared with control. In contrast, phosphorylation is significantly restored by RhoAG14V. Inhibition of RhoA by RhoAT19N also suppresses MLC phosphorylation. Cells shown are representative of more than 20 cells screened per condition. (B) Suppression of myosin light chain phosphorylation by Rac1T17N. Human neutrophils transduced (using BioPorter reagent) with the dominant-negative Rac1T17N mutant (3 μg/mL), with dominant-negative RhoA (9 μg/mL), with Rac1T17N (3 μg/mL) plus constitutively active RhoAG14V (12 μg/mL), or with a combination of the drugs ML-7 and Y27632 (at 20 μM each) to simultaneously inactivate MLCK and Rho kinase, respectively, were stimulated with fMLP and stained with antibodies to MHC and pMLC. After normalization for total myosin, the relative intensity of pMLC, as determined from the immunofluorescent images, was compared with untreated control in the bar graph shown. Values represent an average of at least 100 cells. (C) Biochemical analysis of MLC Ser19 phosphorylation. In addition to single-cell measurements, overall changes in myosin phosphorylation were also measured by immunostaining of blots made from cell lysates. Lane a represents controls; b and c, cells treated with Rac1T17N at 3 and 30 μg/mL, respectively; and d, cells treated with a combination of ML-7 and Y27632 (20-μM each). (D) Myosin II phosphorylation requires Rac1 activity in mouse neutrophils. Quantitation of immunoblots of phospho-myosin II regulatory light chain (MLC) in bone marrow neutrophils exposed to fMLP (1 μM). FMLP-mediated phospho-MLC level increases in a similar pattern in wild-type and Rac2-null neutrophils over 120 seconds, but is severely perturbed in Rac1-null neutrophils.

Chemoattractant-mediated phosphorylation of myosin light chain requires Rac1. (A) Effect of GTPase mutants on phosphorylation of myosin light chain in human neutrophils. Human neutrophils were transduced with the indicated GTPases using BioPorter reagent, globally stimulated with 1 μM fMLP, fixed, and simultaneously stained with antibodies to myosin heavy chain (green) and phosphorylated MLC (red). There is a significant reduction in myosin phosphorylation upon treatment with Rac1T17N compared with control. In contrast, phosphorylation is significantly restored by RhoAG14V. Inhibition of RhoA by RhoAT19N also suppresses MLC phosphorylation. Cells shown are representative of more than 20 cells screened per condition. (B) Suppression of myosin light chain phosphorylation by Rac1T17N. Human neutrophils transduced (using BioPorter reagent) with the dominant-negative Rac1T17N mutant (3 μg/mL), with dominant-negative RhoA (9 μg/mL), with Rac1T17N (3 μg/mL) plus constitutively active RhoAG14V (12 μg/mL), or with a combination of the drugs ML-7 and Y27632 (at 20 μM each) to simultaneously inactivate MLCK and Rho kinase, respectively, were stimulated with fMLP and stained with antibodies to MHC and pMLC. After normalization for total myosin, the relative intensity of pMLC, as determined from the immunofluorescent images, was compared with untreated control in the bar graph shown. Values represent an average of at least 100 cells. (C) Biochemical analysis of MLC Ser19 phosphorylation. In addition to single-cell measurements, overall changes in myosin phosphorylation were also measured by immunostaining of blots made from cell lysates. Lane a represents controls; b and c, cells treated with Rac1T17N at 3 and 30 μg/mL, respectively; and d, cells treated with a combination of ML-7 and Y27632 (20-μM each). (D) Myosin II phosphorylation requires Rac1 activity in mouse neutrophils. Quantitation of immunoblots of phospho-myosin II regulatory light chain (MLC) in bone marrow neutrophils exposed to fMLP (1 μM). FMLP-mediated phospho-MLC level increases in a similar pattern in wild-type and Rac2-null neutrophils over 120 seconds, but is severely perturbed in Rac1-null neutrophils.

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