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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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Differential activation of moesin and MLC. (A) HL60 cells (n > 30 per group) expressing moesin-YFP or MLC-YFP were stimulated for the indicated times with 100 nM fMLF and visualized by fluorescence (top rows) and differential interference contrast (DIC) microscopy (bottom rows). Arrows indicate leading edges. Bars, 10 µm. (B and C) HL60 cells were left untreated or were stimulated with 100 nM fMLF, and quantification of membrane-bound (m-) moesin (B) or m-MLC (C) was evaluated by immunoblot. Levels were quantitated and presented relative to the maximum value. *, P < 0.05 compared with the value at basal (Student’s t test). (D and E) HL60 cells were stimulated for the indicated times with 100 nM fMLF, and phosphorylated (p-) moesin (D) or p-MLC (E) versus total protein levels were assessed by immunoblot. Graph shows quantification of p-moesin and p-MLC, presented relative to maximum activation of p-moesin at 0 min or p-MLC at 2 min. **, P < 0.01; ***, P < 0.001 compared with the value at 0 min (Student’s t test). Data are representative of (A and blots in B–E) or are compiled from three independent experiments (graphs in B–E; mean and SEM in B–E).
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fig2: Differential activation of moesin and MLC. (A) HL60 cells (n > 30 per group) expressing moesin-YFP or MLC-YFP were stimulated for the indicated times with 100 nM fMLF and visualized by fluorescence (top rows) and differential interference contrast (DIC) microscopy (bottom rows). Arrows indicate leading edges. Bars, 10 µm. (B and C) HL60 cells were left untreated or were stimulated with 100 nM fMLF, and quantification of membrane-bound (m-) moesin (B) or m-MLC (C) was evaluated by immunoblot. Levels were quantitated and presented relative to the maximum value. *, P < 0.05 compared with the value at basal (Student’s t test). (D and E) HL60 cells were stimulated for the indicated times with 100 nM fMLF, and phosphorylated (p-) moesin (D) or p-MLC (E) versus total protein levels were assessed by immunoblot. Graph shows quantification of p-moesin and p-MLC, presented relative to maximum activation of p-moesin at 0 min or p-MLC at 2 min. **, P < 0.01; ***, P < 0.001 compared with the value at 0 min (Student’s t test). Data are representative of (A and blots in B–E) or are compiled from three independent experiments (graphs in B–E; mean and SEM in B–E).

Mentions: We continued to explore how moesin regulates neutrophil migration and sequestration. Although both moesin and the myosin II light chain (MLC) are localized at the trailing edges of migrating neutrophils (Yoshinaga-Ohara et al., 2002; Xu et al., 2003), we found that they exhibited different translocation and activation patterns. Moesin-YFP (stably expressed in differentiated human promyelocytic leukemia [HL60] cells) localized uniformly around the cell membrane in the resting state and dissociated from the membrane on the side of the cell where the leading edge started to form after fMLF stimulation (Fig. 2 A, top; >100 cells were examined). The remaining membrane-bound moesin localized to the trailing edge and was almost absent from the leading edge (Fig. 2 A, top; the time course of moesin translocation to the uropod is quantified in Table S2). Consistent with the dissociation of moesin-YFP from the cell membrane, the levels of membrane-bound moesin and moesin phosphorylation (p-moesin, active form) were also decreased upon attractant stimulation (Fig. 2, B and D).


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)

Differential activation of moesin and MLC. (A) HL60 cells (n > 30 per group) expressing moesin-YFP or MLC-YFP were stimulated for the indicated times with 100 nM fMLF and visualized by fluorescence (top rows) and differential interference contrast (DIC) microscopy (bottom rows). Arrows indicate leading edges. Bars, 10 µm. (B and C) HL60 cells were left untreated or were stimulated with 100 nM fMLF, and quantification of membrane-bound (m-) moesin (B) or m-MLC (C) was evaluated by immunoblot. Levels were quantitated and presented relative to the maximum value. *, P < 0.05 compared with the value at basal (Student’s t test). (D and E) HL60 cells were stimulated for the indicated times with 100 nM fMLF, and phosphorylated (p-) moesin (D) or p-MLC (E) versus total protein levels were assessed by immunoblot. Graph shows quantification of p-moesin and p-MLC, presented relative to maximum activation of p-moesin at 0 min or p-MLC at 2 min. **, P < 0.01; ***, P < 0.001 compared with the value at 0 min (Student’s t test). Data are representative of (A and blots in B–E) or are compiled from three independent experiments (graphs in B–E; mean and SEM in B–E).
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Related In: Results  -  Collection

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fig2: Differential activation of moesin and MLC. (A) HL60 cells (n > 30 per group) expressing moesin-YFP or MLC-YFP were stimulated for the indicated times with 100 nM fMLF and visualized by fluorescence (top rows) and differential interference contrast (DIC) microscopy (bottom rows). Arrows indicate leading edges. Bars, 10 µm. (B and C) HL60 cells were left untreated or were stimulated with 100 nM fMLF, and quantification of membrane-bound (m-) moesin (B) or m-MLC (C) was evaluated by immunoblot. Levels were quantitated and presented relative to the maximum value. *, P < 0.05 compared with the value at basal (Student’s t test). (D and E) HL60 cells were stimulated for the indicated times with 100 nM fMLF, and phosphorylated (p-) moesin (D) or p-MLC (E) versus total protein levels were assessed by immunoblot. Graph shows quantification of p-moesin and p-MLC, presented relative to maximum activation of p-moesin at 0 min or p-MLC at 2 min. **, P < 0.01; ***, P < 0.001 compared with the value at 0 min (Student’s t test). Data are representative of (A and blots in B–E) or are compiled from three independent experiments (graphs in B–E; mean and SEM in B–E).
Mentions: We continued to explore how moesin regulates neutrophil migration and sequestration. Although both moesin and the myosin II light chain (MLC) are localized at the trailing edges of migrating neutrophils (Yoshinaga-Ohara et al., 2002; Xu et al., 2003), we found that they exhibited different translocation and activation patterns. Moesin-YFP (stably expressed in differentiated human promyelocytic leukemia [HL60] cells) localized uniformly around the cell membrane in the resting state and dissociated from the membrane on the side of the cell where the leading edge started to form after fMLF stimulation (Fig. 2 A, top; >100 cells were examined). The remaining membrane-bound moesin localized to the trailing edge and was almost absent from the leading edge (Fig. 2 A, top; the time course of moesin translocation to the uropod is quantified in Table S2). Consistent with the dissociation of moesin-YFP from the cell membrane, the levels of membrane-bound moesin and moesin phosphorylation (p-moesin, active form) were also decreased upon attractant stimulation (Fig. 2, B and D).

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