Polarization of chemoattractant receptor signaling during neutrophil chemotaxis

Abstract

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.

Fig. 1

Translocation of PHAKT-GFP to the plasma membrane of neutrophil-differentiated HL-60 cells. (A) PHAKT-GFP–expressing cells (I through III), C5aR-GFP–expressing cells (IV), and GFP-expressing cells (V) were differentiated to neutrophil-like cells (7) and plated on glass cover slips as described (4). Cells were stimulated either with a uniform increase in fMLP concentration, from 0 (I) to 100 nM (I′: 60 s post-stimulation) or with a point source of fMLP (1 μM) delivered by a Femtotip micropipette (10) (II′, III′, IV, and V), whose position is indicated by an asterisk. Panels II and III correspond, respectively, to cells shown in panels II′ and III′, but before stimulation with the micropipette. Images were recorded in the fluorescein isothiocyanate (FITC) channel every 5 s as described (4). Each result was reproduced in at least 10 experiments (11). Bars, 10 μm. Videos of a time course and asymmetric translocation of PHAKT-GFP are available at (9). (B) Extracellular gradient generated with the micropipette (I), relative to the intracellular gradient of PHAKT-GFP recruitment (II). For the experiment in (I), a Femtotips micropipette filled with sulforhodamine, a fluorescent dye, was lowered onto a cover slip overlaid with the buffer used for cell stimulation (4, 12); the image was taken in the rhodamine channel (4) 2 min after repositioning the micropipette in a field lacking dye fluorescence. The micropipette tip was located just outside the illumination field, at the left of the hexagon. The solid white line in (I) indicates the intensity of dye fluorescence ( y-axis) along the x-axis at the level of the black horizontal bar shown to the left of the figure; this curve presumably reflects the shape of a gradient of chemoattractant of similar molecular size, such as fMLP. The x-axis indicates distance across the microscopic field (solid white scale bar, 50 μm). Fluorescence intensities at the ends of this solid scale bar in (I) were 1900 and 300 (arbitrary units, after subtracting a background value of 400, obtained outside the hexagon). The dashed white curve in (I) indicates the intensity of PHAKT-GFP fluorescence measured across a diameter of the neutrophil shown in (II), depicted at the same scale as in (I). This cell was exposed to fMLP supplied by a micropipette; the diameter across which PHAKT-GFP fluorescence was measured is positioned at the level of the black horizontal bar in (II). The 15-μm dashed white line in (I) represents the intensity of PHAKT-GFP recruitment across this diameter; fluorescence intensities at the two ends of this line were 900 versus 200 (arbitrary units, after subtracting a background of 100 units measured outside the cell boundary). Maximum fluorescence intensity for rhodamine decreases to half its value over a distance of 30 μm, while maximum fluorescence intensity for PHAKT-GFP decreases to half its value over 5 μm.

Fig. 2

(A through D) Asymmetric translocation of PHAKT-GFP at the plasma membrane of neutrophil-differentiated HL-60 cells during polarization in response to a uniform increase in chemoattractant concentration. Differentiated cells (7) were plated on glass cover slips as described (4). Cells were stimulated with 100 nM fMLP at time 0 and images were then recorded as described in the legend of Fig. 1. The arrow in (D) points to the advancing leading edge of the cell. Uniform stimulation was assessed in 13 different sessions, recording the behavior of 68 cells (13). Bar, 10 μm. A video of this experiment is available at (9).

Fig. 3

Asymmetric translocation of PHAKT-GFP in latrunculin-B–treated neutrophil-differentiated HL-60 cells. Differentiated cells (7) were plated on glass cover slips as described (4). Cells were then pretreated with 20 μg/ml latrunculin-B for 10 min (A) (14) and then stimulated, in the continued presence of the toxin, with a point source of fMLP (10 μM) delivered from a micropipette (10) [(B), asterisk]. Images were recorded as described in the legend of Fig. 1. Cells showed asymmetric recruitment biased by the micropipette’s position in five of nine stimulation sessions performed under these conditions. Bar, 10 μm.

Fig. 4

PHAKT-GFP translocation in cells treated with (A) PTX or C. difficile toxin-B and (B) a PI3K inhibitor, LY 294002 (14). (A) Neutrophil-differentiated cells (7), plated on glass cover slips (4), were stimulated sequentially with fMLP and insulin (panels II and III and panels V and VI, respectively) or C5a and insulin (panels VIII and IX, respectively). fMLP or C5a was added first, removed 2 min later, and replaced with insulin, then cells were stimulated for a further 3 min. Panel I through III: treatment with PTX (1 μg/ml). Panels IV through IX: treatment with C. difficile toxin-B (90 μg/ml). Panels I, IV, and VII: cells before stimulation with agonists (zero time). Images were recorded as described in the legend of Fig. 1. Stimulation times with the indicated agonists were as follows: II: 65 s; III: 115 s; V: 67 s; VI: 178 s; VIII: 39 s; IX: 161 s. Bars, 10 μm. (B) Effect of LY 294002 on chemoattractant- and insulin-induced plasma membrane translocation of PHAKT-GFP in neutrophil-differentiated HL-60 cells. Panels I, IV, and VII: untreated cells stimulated with insulin, fMLP, and C5a, respectively. Panels II, V, and VIII: unstimulated cells treated with LY 294002 (100 μM in panel II; 300 μM in panels V and VIII). Panels III, VI, and IX show responses of the same cells to insulin, fMLP, or C5a, respectively, in the presence of LY 294002 (100 μM in panel III; 300 μM in panels VI and IX). Images were recorded as described in the legend of Fig. 1. Stimulation times with agonists were as follows: I: 183 s; IV: 46 s; VII: 39 s; III: 199 s; VI: 61 s; IX: 41 s. For each treatment, the result shown is representative of at least eight different stimulation sessions performed on at least two different batches of neutrophil-differentiated HL-60 cells. Bars, 10 μm. Two additional panels of this figure (9) show the effect of 100 μM LY 294002 on chemoattractant-induced plasma membrane translocation of PHAKT-GFP (Web figure 4C) and a histogram (Web figure 4D) of percent cells responding to all three agonists tested in the presence of C. difficile toxin or LY 294002 at 100 or 300 μM.