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Link to original content: https://pubmed.ncbi.nlm.nih.gov/20974965
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. 2010 Nov 9;107(45):19332-7.
doi: 10.1073/pnas.1008248107. Epub 2010 Oct 25.

Coordination of Fc receptor signaling regulates cellular commitment to phagocytosis

Youxin Zhang  1 , Adam D Hoppe, Joel A Swanson
Affiliations

Coordination of Fc receptor signaling regulates cellular commitment to phagocytosis

Youxin Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

During Fcγ receptor (FcR)-mediated phagocytosis by macrophages, cytoplasm advances over IgG-coated particles by the sequential ligation of FcR in plasma membranes. If FcR signaling was strictly autonomous, then the signals generated during phagocytosis should be proportional to the number of ligated receptors. By measuring FcR-dependent responses to beads coated with various densities of IgG, this study identified nonlinear signaling that organizes an all or none response during particle ingestion. Phagocytosis of beads with IgG at low density either stalled after making small, actin-rich cups or proceeded to completion at the same rate as phagocytosis of high-density IgG beads. Signals were measured by quantifying the recruitment of YFP-labeled probes to phagocytic cup membranes. Although the magnitude of early signals correlated with IgG density, later signals showed an all or none response, which was regulated by the concentrations of 3' phosphoinositides in phagocytic cup membranes. Thus, 3' phosphoinositides, shown previously to be required for phagocytosis, function in a feedback regulatory mechanism affecting late but not early signals. This indicates a mechanism for the coordination of cell movements initiated by receptor signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IgG density on beads controls the frequency but not rate of phagocytosis. (A) Phagocytic indexes of B0.3, B1, and B10 were measured from six coverslips. Data represent mean ± SEM. The relationship between ligand density and phagocytic index was nonlinear. (B) The rates of phagosome formation were measured as the time from the first movement of cytoplasm to phagosome closure (n = 5).
Fig. 2.
Fig. 2.
Quantitation of fluorescent chimera recruitment during phagocytosis. (A) In cells expressing YFP chimeras and CFP, the ratio image was calculated by dividing corresponding pixels of the YFP and CFP images. Multiplying the CFP image by the average YFP/CFP ratio in cytosol,formula image(a single number determined for each cell) generated a YFP path-length image representing how that cell's quantity of YFP chimera would appear if it did not contain any localization information. Subtracting this image from the YFP image created a difference image, Re, which represented the quantity of YFP displaced by localization information. (B–D) Phase contrast and Re images of Syk-YFP–expressing RAWs internalizing B10 (B) and B1 (C) and Syk(R194A)-YFP–expressing RAW 264.7 macrophages internalizing B10 (D). Arrowheads indicate regions with increased recruitment of Syk-YFP. (Scale bar: 5 μm.) The average recruitments of Syk-YFP (E), YFP-p85 (G), YFP-actin (I), and YFP-AktPH (K) per pixel in phagosomes of B10 (red) and B1 (blue) were plotted as a function of time, aligned by the start of membrane movement for phagocytosis. (F) The maximum Syk-YFP recruitment averaged from single phagocytic events for B10, B6, B3, and B1 (solid circles; n = 8) as well as Syk(R194A)-YFP recruitment for B10 and B1 (open triangles; n = 4). B10 phagosomes recruited more YFP-p85 (H); *P = 0.024) and YFP-actin (J; *P = 0.029) than B1 phagosomes. Stalled phagocytic cups recruited slightly less YFP-actin than did completed phagosomes (open circles; n = 5; **P = 0.129). (L) The maximum YFP-AktPH recruitment during completed phagocytic events (solid circles; n = 8) showed no difference between B10 and B1 (*P = 0.223). In contrast, YFP-AktPH recruitment in stalled phagocytic cups (open circle; n = 5) increased slightly with ligand density but remained significantly lower than in finished phagosomes (**P = 0.001).
Fig. 3.
Fig. 3.
Late signals correlate inversely with IgG density. (A–C) Beads coated with higher IgG density recruited less PKCε-YFP to phagosomes during phagocytosis. Time lapse, phase contrast, and Re images of PKCε-YFP–transfected RAWs internalizing B10 (A) and B1 (B) are shown at 2-min intervals. The arrowheads show that B10 recruited less PKCε-YFP than B1. (Scale bar: 5 μm.) (C) Stalled phagocytic cups did not recruit PKCε-YFP. (D) The average recruitment of PKCε-YFP per pixel in phagosomes aligned by the timing of phagosome closure (n = 8). (E) The maximum PKCε-YFP recruitment of completed phagocytic events (solid circles) showed that B1 recruited more PKCε-YFP than B10 did (*P = 0.034). In addition, PKCε-YFP recruitment to stalled phagocytic cups (open circles; n = 10) was significantly lower than in finished phagosomes (**P < 0.0001). (F) Times for half-maximal recruitment during phagocytosis of B10. Phagocytosis of B1 showed similar half-maximal recruitment times with slightly longer values for Syk-YFP (1.8 ± 0.37 min) and YPF-p85 (2.9 ± 0.6 min). All values show mean ± SEM. (G) Ratios of maximal YFP chimera recruitment to B1 vs. B10.
Fig. 4.
Fig. 4.
3′PIs regulate commitment to phagocytosis with different effects on early and late signals. (A) Effect of Bpv(pic) on the phagocytic index of B0.3 (n = 6; *P < 0.0001). (B) Effects of inhibitors on the phagocytic indexes of B1. Bpv(pic) increased phagocytic index, whereas LY294002 and TGX-221 decreased phagocytic indexes (n = 6; *P < 0.01, **P < 0.001, ***P < 0.0001). (C) Effect of LY294002 on the phagocytic index of B10 (n = 6; *P < 0.0001). Percentages indicate actual phagocytic indexes. (D–F) Recruitment of YFP-actin (D), YFP-AktPH (E), and PKCε-YFP (F) to cups in LY294002-treated cells (LY cups) and stalled cups and early (early cup) and maximal values (max) for successfully formed B1 phagosomes. Early cups were similar in size and shape to stalled cups, using frames from early time points of successful phagocytic events. YFP-AktPH recruitment to early cups was greater than recruitment to stalled cups (n = 5; *P = 0.045). LY294002 did not inhibit the recruitment of YFP-actin (D; n = 3) but inhibited the recruitment of YFP-AktPH (E) and PKCε-YFP (F; n = 5).
Fig. 5.
Fig. 5.
A 3′PI concentration threshold regulates commitment to late stages of signaling. Signaling during internalization by phagocytosis was divided into early- and late-stage signals, depending on whether they appeared before or after PIP3 generation. Recruitment of the late-signal PKCε required 3′PIs to exceed a concentration threshold, whereas early-stage signals, such as Syk, P85, and actin, did not. However, optimal recruitment of actin and p85 may require suprathreshold 3′PIs.
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