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Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Liu X, Yang T, Suzuki K, Tsukita S, Ishii M, Zhou S, Wang G, Cao L, Qian F, Taylor S, Oh MJ, Levitan I, Ye RD, Carnegie GK, Zhao Y, Malik AB, Xu J - J. Exp. Med. (2015)

Bottom Line: Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria.Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity.Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions.

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Affiliation: Department of Pharmacology and Department of Medicine, University of Illinois, Chicago, IL 60612.

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Myosin phosphatase is recruited to the leading edges by front signals. (A) Control, PP1c RNAi, or MBS RNAi cells were exposed to an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. (B and C) Cells were treated as in A. CI (B) and migration speed (C) were calculated. **, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (D and E) Control, PP1c RNAi, PP1c RNAi + WT-moesin, and PP1c RNAi + mosein-T558A cells were exposed to an fMLF gradient. CI (D) and migration speed (E) were calculated. *, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (F) PP1c was pulled down with MBS in the presence or absence of fMLF in HL60 cells. (G) HL60 cells (n > 30 per group) expressing PP1c-YFP were left untreated (left) or treated with Hem1 RNAi (middle) or PTX (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized with fluorescence (top) and DIC microscopy (bottom). Bars: (A) 100 µm; (G) 10 µm. Data are representative of (A, F, and G) or are compiled from three independent experiments (B–E; mean and SEM in B–E).
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fig9: Myosin phosphatase is recruited to the leading edges by front signals. (A) Control, PP1c RNAi, or MBS RNAi cells were exposed to an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. (B and C) Cells were treated as in A. CI (B) and migration speed (C) were calculated. **, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (D and E) Control, PP1c RNAi, PP1c RNAi + WT-moesin, and PP1c RNAi + mosein-T558A cells were exposed to an fMLF gradient. CI (D) and migration speed (E) were calculated. *, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (F) PP1c was pulled down with MBS in the presence or absence of fMLF in HL60 cells. (G) HL60 cells (n > 30 per group) expressing PP1c-YFP were left untreated (left) or treated with Hem1 RNAi (middle) or PTX (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized with fluorescence (top) and DIC microscopy (bottom). Bars: (A) 100 µm; (G) 10 µm. Data are representative of (A, F, and G) or are compiled from three independent experiments (B–E; mean and SEM in B–E).

Mentions: When control cells were exposed to an fMLF gradient, they migrated up the entire gradient (Fig. 9 A). PP1c RNAi–treated cells also migrated, but with poor directionality (Fig. 9 A), and the CI was significantly lower in PP1 RNAi–treated cells compared with controls (0.26 vs. 0.72, P < 0.01; Fig. 9 B). The migration speed of PP1c RNAi–treated cells was also significantly decreased (11.5 vs. 18.4 µm/min, P < 0.01; Fig. 9 C). Similar results were obtained in MBS RNAi cells (Fig. 9, A–C). Expression of the moesin-T558A mutant, but not WT moesin, partially restored cell migration in PP1c RNAi cells (Fig. 9, D and E). Collectively, our data indicate that inhibition of myosin phosphatase prevented moesin dephosphorylation and dissociation from cell membrane, thus causing unstable cell polarity and impaired cell migration.


Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Liu X, Yang T, Suzuki K, Tsukita S, Ishii M, Zhou S, Wang G, Cao L, Qian F, Taylor S, Oh MJ, Levitan I, Ye RD, Carnegie GK, Zhao Y, Malik AB, Xu J - J. Exp. Med. (2015)

Myosin phosphatase is recruited to the leading edges by front signals. (A) Control, PP1c RNAi, or MBS RNAi cells were exposed to an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. (B and C) Cells were treated as in A. CI (B) and migration speed (C) were calculated. **, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (D and E) Control, PP1c RNAi, PP1c RNAi + WT-moesin, and PP1c RNAi + mosein-T558A cells were exposed to an fMLF gradient. CI (D) and migration speed (E) were calculated. *, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (F) PP1c was pulled down with MBS in the presence or absence of fMLF in HL60 cells. (G) HL60 cells (n > 30 per group) expressing PP1c-YFP were left untreated (left) or treated with Hem1 RNAi (middle) or PTX (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized with fluorescence (top) and DIC microscopy (bottom). Bars: (A) 100 µm; (G) 10 µm. Data are representative of (A, F, and G) or are compiled from three independent experiments (B–E; mean and SEM in B–E).
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fig9: Myosin phosphatase is recruited to the leading edges by front signals. (A) Control, PP1c RNAi, or MBS RNAi cells were exposed to an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. (B and C) Cells were treated as in A. CI (B) and migration speed (C) were calculated. **, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (D and E) Control, PP1c RNAi, PP1c RNAi + WT-moesin, and PP1c RNAi + mosein-T558A cells were exposed to an fMLF gradient. CI (D) and migration speed (E) were calculated. *, P < 0.01; ***, P < 0.001 compared with control (Student’s t test). (F) PP1c was pulled down with MBS in the presence or absence of fMLF in HL60 cells. (G) HL60 cells (n > 30 per group) expressing PP1c-YFP were left untreated (left) or treated with Hem1 RNAi (middle) or PTX (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized with fluorescence (top) and DIC microscopy (bottom). Bars: (A) 100 µm; (G) 10 µm. Data are representative of (A, F, and G) or are compiled from three independent experiments (B–E; mean and SEM in B–E).
Mentions: When control cells were exposed to an fMLF gradient, they migrated up the entire gradient (Fig. 9 A). PP1c RNAi–treated cells also migrated, but with poor directionality (Fig. 9 A), and the CI was significantly lower in PP1 RNAi–treated cells compared with controls (0.26 vs. 0.72, P < 0.01; Fig. 9 B). The migration speed of PP1c RNAi–treated cells was also significantly decreased (11.5 vs. 18.4 µm/min, P < 0.01; Fig. 9 C). Similar results were obtained in MBS RNAi cells (Fig. 9, A–C). Expression of the moesin-T558A mutant, but not WT moesin, partially restored cell migration in PP1c RNAi cells (Fig. 9, D and E). Collectively, our data indicate that inhibition of myosin phosphatase prevented moesin dephosphorylation and dissociation from cell membrane, thus causing unstable cell polarity and impaired cell migration.

Bottom Line: Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria.Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity.Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Department of Medicine, University of Illinois, Chicago, IL 60612.

Show MeSH
Related in: MedlinePlus