Limits...
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.

View Article: PubMed Central - HTML - PubMed

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

Show MeSH
Myosin phosphatase releases moesin-mediated inhibition. (A) HL60 cells (n > 30) expressing PP1c-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrow indicates the leading edge. (B) Expression of PP1c (top) or MBS (bottom) in control and PP1c RNAi cells or in control and MBS RNAi cells was measured by immunoblot. Two PP1c or MBS RNAi cell lines are shown. GAPDH was used as a loading control. (C) Control, PP1c, or MBS RNAi cells were stimulated with 100 nM fMLF. Expression of p-moesin and total moesin was measured with immunoblot. Graph shows quantification of immunoblot data. Results are presented relative to maximum activation of p-moesin at 0 min. ***, P < 0.001 compared with control without fMLF. (D) PP1c RNAi cells or MBS RNAi cells (n > 30 per group) expressing moesin-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrowheads indicate transient leading edges. (A and D) Bars, 10 µm. Data are representative of (A, B, D, and blots in C) or are compiled from three independent experiments (graph in C; mean and SEM in C).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4322047&req=5

fig8: Myosin phosphatase releases moesin-mediated inhibition. (A) HL60 cells (n > 30) expressing PP1c-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrow indicates the leading edge. (B) Expression of PP1c (top) or MBS (bottom) in control and PP1c RNAi cells or in control and MBS RNAi cells was measured by immunoblot. Two PP1c or MBS RNAi cell lines are shown. GAPDH was used as a loading control. (C) Control, PP1c, or MBS RNAi cells were stimulated with 100 nM fMLF. Expression of p-moesin and total moesin was measured with immunoblot. Graph shows quantification of immunoblot data. Results are presented relative to maximum activation of p-moesin at 0 min. ***, P < 0.001 compared with control without fMLF. (D) PP1c RNAi cells or MBS RNAi cells (n > 30 per group) expressing moesin-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrowheads indicate transient leading edges. (A and D) Bars, 10 µm. Data are representative of (A, B, D, and blots in C) or are compiled from three independent experiments (graph in C; mean and SEM in C).

Mentions: Moesin inactivates both frontness and backness signals in resting neutrophils through the inhibition of Rho GTPases, as described above. Hence, to undergo polarization and migration, cells must deactivate moesin-mediated inhibition and thereby initiate cell migration. We next addressed the mechanism responsible for breaking the symmetry and initiating cell migration. We focused on the role of myosin phosphatase, which dephosphorylates both moesin and MLC (Fukata et al., 1998). Both the catalytic subunit (PP1c) and the MBS of myosin phosphatase are coimmunoprecipitated with the Hem-1 complex, which organizes the neutrophil’s leading edge (Weiner et al., 2006). We first confirmed PP1c localization to the leading edge by expressing YFP-tagged PP1c in HL60 cells. PP1c-YFP localized to both the cytosol and the nucleus in the basal stage. A uniform concentration of fMLF (100 nM) induced the recruitment of PP1c-YFP to the cell periphery and subsequently to the leading edge in polarized cells (Fig. 8 A; a time course of PP1 localization is shown in Table S2).


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 releases moesin-mediated inhibition. (A) HL60 cells (n > 30) expressing PP1c-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrow indicates the leading edge. (B) Expression of PP1c (top) or MBS (bottom) in control and PP1c RNAi cells or in control and MBS RNAi cells was measured by immunoblot. Two PP1c or MBS RNAi cell lines are shown. GAPDH was used as a loading control. (C) Control, PP1c, or MBS RNAi cells were stimulated with 100 nM fMLF. Expression of p-moesin and total moesin was measured with immunoblot. Graph shows quantification of immunoblot data. Results are presented relative to maximum activation of p-moesin at 0 min. ***, P < 0.001 compared with control without fMLF. (D) PP1c RNAi cells or MBS RNAi cells (n > 30 per group) expressing moesin-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrowheads indicate transient leading edges. (A and D) Bars, 10 µm. Data are representative of (A, B, D, and blots in C) or are compiled from three independent experiments (graph in C; mean and SEM in C).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4322047&req=5

fig8: Myosin phosphatase releases moesin-mediated inhibition. (A) HL60 cells (n > 30) expressing PP1c-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrow indicates the leading edge. (B) Expression of PP1c (top) or MBS (bottom) in control and PP1c RNAi cells or in control and MBS RNAi cells was measured by immunoblot. Two PP1c or MBS RNAi cell lines are shown. GAPDH was used as a loading control. (C) Control, PP1c, or MBS RNAi cells were stimulated with 100 nM fMLF. Expression of p-moesin and total moesin was measured with immunoblot. Graph shows quantification of immunoblot data. Results are presented relative to maximum activation of p-moesin at 0 min. ***, P < 0.001 compared with control without fMLF. (D) PP1c RNAi cells or MBS RNAi cells (n > 30 per group) expressing moesin-YFP were stimulated for the indicated times with 100 nM fMLF and were visualized with fluorescence (top) and DIC microscopy (bottom). Arrowheads indicate transient leading edges. (A and D) Bars, 10 µm. Data are representative of (A, B, D, and blots in C) or are compiled from three independent experiments (graph in C; mean and SEM in C).
Mentions: Moesin inactivates both frontness and backness signals in resting neutrophils through the inhibition of Rho GTPases, as described above. Hence, to undergo polarization and migration, cells must deactivate moesin-mediated inhibition and thereby initiate cell migration. We next addressed the mechanism responsible for breaking the symmetry and initiating cell migration. We focused on the role of myosin phosphatase, which dephosphorylates both moesin and MLC (Fukata et al., 1998). Both the catalytic subunit (PP1c) and the MBS of myosin phosphatase are coimmunoprecipitated with the Hem-1 complex, which organizes the neutrophil’s leading edge (Weiner et al., 2006). We first confirmed PP1c localization to the leading edge by expressing YFP-tagged PP1c in HL60 cells. PP1c-YFP localized to both the cytosol and the nucleus in the basal stage. A uniform concentration of fMLF (100 nM) induced the recruitment of PP1c-YFP to the cell periphery and subsequently to the leading edge in polarized cells (Fig. 8 A; a time course of PP1 localization is shown in Table S2).

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