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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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Moesin regulates MLC localization. (A) HL60 cells expressing both moesin-YFP and MLC-CFP were left untreated (left) or were treated with blebbistatin (middle) or Y27632 (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized by DIC (top) and fluorescence microscopy (middle and bottom). (B) Expression of moesin in control cells and moesin RNAi–treated cells was analyzed by immunoblot. Two moesin RNAi–treated cell lines are shown. Ezrin was used as a loading control. (C) Control and moesin RNAi–treated cells expressing MLC-DsRed were stimulated with 100 nM fMLF for 2 min, and cells were visualized by DIC (top) and fluorescence microscopy (bottom). (A and C) Arrowheads indicate the trailing edges. (D) Cells expressing ezrin-YFP were stimulated with 100 nM fMLF and imaged by fluorescence (top) and DIC microscopy (bottom) at the indicated times. Arrowheads point to the leading edges. (E) Expression of ezrin was assessed in control and ezrin RNAi–treated cells by immunoblot. Moesin and GAPDH were used as loading controls. (F) HL60 cells were left untreated (Ctrl) or were treated with ezrin RNAi in an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. Bars: (A, C, and D) 10 µm; (F) 100 µm. (G and H) Cells were treated as in F, and CI (G) and migration speed (H) were calculated. Data are representative of (A–F) or are compiled from three independent experiments (G and H; mean and SEM in G and H).
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fig3: Moesin regulates MLC localization. (A) HL60 cells expressing both moesin-YFP and MLC-CFP were left untreated (left) or were treated with blebbistatin (middle) or Y27632 (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized by DIC (top) and fluorescence microscopy (middle and bottom). (B) Expression of moesin in control cells and moesin RNAi–treated cells was analyzed by immunoblot. Two moesin RNAi–treated cell lines are shown. Ezrin was used as a loading control. (C) Control and moesin RNAi–treated cells expressing MLC-DsRed were stimulated with 100 nM fMLF for 2 min, and cells were visualized by DIC (top) and fluorescence microscopy (bottom). (A and C) Arrowheads indicate the trailing edges. (D) Cells expressing ezrin-YFP were stimulated with 100 nM fMLF and imaged by fluorescence (top) and DIC microscopy (bottom) at the indicated times. Arrowheads point to the leading edges. (E) Expression of ezrin was assessed in control and ezrin RNAi–treated cells by immunoblot. Moesin and GAPDH were used as loading controls. (F) HL60 cells were left untreated (Ctrl) or were treated with ezrin RNAi in an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. Bars: (A, C, and D) 10 µm; (F) 100 µm. (G and H) Cells were treated as in F, and CI (G) and migration speed (H) were calculated. Data are representative of (A–F) or are compiled from three independent experiments (G and H; mean and SEM in G and H).

Mentions: Next, we addressed the potential regulatory relationship between moesin and MLC by coexpressing YFP-tagged moesin and CFP-tagged MLC in HL60 cells. We again demonstrated that both moesin-YFP and MLC-CFP were localized to the trailing edges of polarized cells stimulated with fMLF (Fig. 3 A, left). To inhibit MLC activity, we treated cells with either the myosin inhibitor blebbistatin (100 µM, 30 min) or the Rho kinase inhibitor Y27632 (10 µM, 30 min). Upon uniform stimulation of fMLF, these inhibitor-treated cells formed multiple pseudopods in the earlier stage and gradually formed a single prominent pseudopod and a long tail, as reported previously (Xu et al., 2003). As expected, MLC-CFP showed reduced membrane localization and was more uniformly distributed in cells treated with either of the inhibitors (Fig. 3 A, middle and right panels in the third row). However, moesin-YFP remained associated with the cell membrane in these long tails (Fig. 3 A, middle and right panels in the second row; the distributions of both MLC and moesin over time are shown in Table S3). Thus, inhibiting MLC activity caused MLC to be uniformly distributed but did not alter the attractant-induced localization of moesin at the trailing edge.


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)

Moesin regulates MLC localization. (A) HL60 cells expressing both moesin-YFP and MLC-CFP were left untreated (left) or were treated with blebbistatin (middle) or Y27632 (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized by DIC (top) and fluorescence microscopy (middle and bottom). (B) Expression of moesin in control cells and moesin RNAi–treated cells was analyzed by immunoblot. Two moesin RNAi–treated cell lines are shown. Ezrin was used as a loading control. (C) Control and moesin RNAi–treated cells expressing MLC-DsRed were stimulated with 100 nM fMLF for 2 min, and cells were visualized by DIC (top) and fluorescence microscopy (bottom). (A and C) Arrowheads indicate the trailing edges. (D) Cells expressing ezrin-YFP were stimulated with 100 nM fMLF and imaged by fluorescence (top) and DIC microscopy (bottom) at the indicated times. Arrowheads point to the leading edges. (E) Expression of ezrin was assessed in control and ezrin RNAi–treated cells by immunoblot. Moesin and GAPDH were used as loading controls. (F) HL60 cells were left untreated (Ctrl) or were treated with ezrin RNAi in an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. Bars: (A, C, and D) 10 µm; (F) 100 µm. (G and H) Cells were treated as in F, and CI (G) and migration speed (H) were calculated. Data are representative of (A–F) or are compiled from three independent experiments (G and H; mean and SEM in G and H).
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fig3: Moesin regulates MLC localization. (A) HL60 cells expressing both moesin-YFP and MLC-CFP were left untreated (left) or were treated with blebbistatin (middle) or Y27632 (right) in the presence of 100 nM fMLF for 2 min. Cells were visualized by DIC (top) and fluorescence microscopy (middle and bottom). (B) Expression of moesin in control cells and moesin RNAi–treated cells was analyzed by immunoblot. Two moesin RNAi–treated cell lines are shown. Ezrin was used as a loading control. (C) Control and moesin RNAi–treated cells expressing MLC-DsRed were stimulated with 100 nM fMLF for 2 min, and cells were visualized by DIC (top) and fluorescence microscopy (bottom). (A and C) Arrowheads indicate the trailing edges. (D) Cells expressing ezrin-YFP were stimulated with 100 nM fMLF and imaged by fluorescence (top) and DIC microscopy (bottom) at the indicated times. Arrowheads point to the leading edges. (E) Expression of ezrin was assessed in control and ezrin RNAi–treated cells by immunoblot. Moesin and GAPDH were used as loading controls. (F) HL60 cells were left untreated (Ctrl) or were treated with ezrin RNAi in an fMLF gradient of 100 nM (>30 cells per condition). Each trace represents the trajectory of one cell. Bars: (A, C, and D) 10 µm; (F) 100 µm. (G and H) Cells were treated as in F, and CI (G) and migration speed (H) were calculated. Data are representative of (A–F) or are compiled from three independent experiments (G and H; mean and SEM in G and H).
Mentions: Next, we addressed the potential regulatory relationship between moesin and MLC by coexpressing YFP-tagged moesin and CFP-tagged MLC in HL60 cells. We again demonstrated that both moesin-YFP and MLC-CFP were localized to the trailing edges of polarized cells stimulated with fMLF (Fig. 3 A, left). To inhibit MLC activity, we treated cells with either the myosin inhibitor blebbistatin (100 µM, 30 min) or the Rho kinase inhibitor Y27632 (10 µM, 30 min). Upon uniform stimulation of fMLF, these inhibitor-treated cells formed multiple pseudopods in the earlier stage and gradually formed a single prominent pseudopod and a long tail, as reported previously (Xu et al., 2003). As expected, MLC-CFP showed reduced membrane localization and was more uniformly distributed in cells treated with either of the inhibitors (Fig. 3 A, middle and right panels in the third row). However, moesin-YFP remained associated with the cell membrane in these long tails (Fig. 3 A, middle and right panels in the second row; the distributions of both MLC and moesin over time are shown in Table S3). Thus, inhibiting MLC activity caused MLC to be uniformly distributed but did not alter the attractant-induced localization of moesin at the trailing edge.

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