Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis

Abstract

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Fig. 1. CD45 is depleted from regions of contact between macrophages and IgG-opsonized targets by a diffusional barrier

A) Human macrophages incubated with polystyrene beads opsonized with 0.5 mg IgG/10⁷ particles. Cells were fixed after 45 s and stained for CD45 (green), pY (red) and IgG (cyan, inset). Scale bar=10 µm. B–F) Single CD45 particles were visualized in macrophages using anti-CD45 Fab fragments labeled with Qdots (B, inset). B) Macrophages were seeded onto either BSA-coated or IgG-coated coverslips and particles tracked for 20 s at 33 Hz. CD45 trajectories were analyzed by MSS; the motion type for each trajectory is color-coded: confined (blue), free (cyan), or linear (red). Scale bar =5 µm. C–E) CD45 motion type (C), median confinement diameter (D) and median diffusion coefficient (E) for cells seeded on BSA (20 min), IgG (20 min), or BSA + LatA (15+5 min) were determined from 10 s recordings. Horizontal lines are means of ≥17 cells from 3 independent experiments; >1000 trajectories analyzed/condition. F) Three trajectories from (B) shown with color-coded time course. Depletion zones drawn in B and F delineate the area depleted of CD45. They were drawn to approximate the contour of the F-actin ring.

Fig. 2. Quantifying CD45 exclusion from sites of receptor engagement

A) Initial and final positions of simulated particles undergoing Brownian diffusion in the presence of a barrier. Particles were initially dispersed randomly outside a circular region of radius 1.6 µm centered in a square with 6 µm sides (inset). The circular region is enclosed by a barrier (shown in red) characterized by p_exclusion –the probability that a particle colliding with the barrier will be unable to breach it. Particle positions at 20 s are shown for different exclusion probabilities. B) Ratio of density of particles inside/outside the barrier as a function of time for the 20 s simulation period (left), and the values calculated for the last 1 s of simulated and observed tracks (right). C) Single CD45 molecules were tracked for 20 s in the vicinity of micropatterned IgG. Image shows the superposition of 10 micropatterns from a single experiment. 300 trajectories are shown, averaging 86 frames each. D) Time course and asymptotic values of particle density ratios for bootstrap trajectories generated using different exclusion probabilities (color-coded) and for observed CD45 tracks (gray trace + black trend line). >300 observed CD45 tracks from the vicinity of 18 antigen spots were pooled to generate the experimental time course. E) Observed and bootstrap distributions of the fraction of time a random trajectory spends inside the barrier for different exclusion probabilities.

Fig. 3. CD45 depletion extends beyond regions of engaged Fcγ receptors and correlates with integrin adhesions

A) CD45 was labeled in RAW 264.7 cells expressing FcγRIIA-GFP using Qdots bound to anti-CD45 Fab fragments. CD45 trajectories from 20 s videos were classified as confined (blue), free (cyan), or linear (red) and overlaid on a single image of clustered FcγRIIA-GFP taken when SMT started. Scale bar =5 µm. B–G) Human macrophages seeded onto IgG (red in B–E; blue in F–G) micropatterned coverslips for 10 min. Cells were stained for F-actin (green in B,D,F,G), FcγRIIA or CD45 (cyan in B,D), pY (green in C), vinculin (red in F) or active β2 integrin (red in G). E) Cells transfected with Akt (PH)-GFP (green). Scale bars =2 µm. H) Maximum diameter of all signals from B–G determined for 3–5 micropatterned regions of IgG per cell for >30 cells from 3 independent experiments. Bars are mean ± SEM. I–J) xz sections of human macrophages incubated with IgG-opsonized beads. Cells were fixed after 45 s and stained for vinculin or CD45 (green) and F-actin (red). Beads indicated by dotted lines. Scale bar =5 µm.

Fig. 4. Integrin activation and linkage to F-actin, mediated by CalDAG-GEF1 signaling, are required for CD45 exclusion

A–C) Human macrophages seeded onto micropatterned IgG (red) for 10 min before adding vehicle (top), 1 µM latrunculin A (middle), or 1.5 mM EDTA (bottom) for 3 min. Cells were fixed and stained for CD45 (cyan) and F-actin (green). In B, depletion was determined as a ratio of average CD45 signal intensity outside/inside micropatterned IgG regions. In C the pY signal was measured and normalized. B and C, means ± SEM of >30 cells from 3 independent experiments, each measuring 3–5 micropatterned IgG regions. D) Single CD45 molecules were labeled in macrophages using Fab fragments and Qdots. Cells were laid onto micropatterned IgG coverslips for 5 min before recording for 10 s at 33 Hz. Trajectories (cyan) are overlaid on a single image of the micropattern. E) BMDMs from wildtype (WT) or CalDAG-GEF1^−/− mice were lysed and probed with indicated antibodies. F) Single CD45 molecules were labeled in WT or CalDAG-GEF1^−/− BMDMs using Fab fragments and Qdots. Cells were laid onto IgG coverslips 5 min before recording for 10 s at 33 Hz. G) BMDMs were laid onto micropatterned IgG (blue) coverslips 5 min before staining for F-actin (green) and vinculin (red). H–I) Cells were incubated with vehicle, 10 µM Arp2/3 inhibitor (CK-666), 10 µM formin inhibitor (SMIFH2), 10 µM blebbistatin, or 1 µM nocodazole in suspension at 37°C in HBSS. Cells were then seeded onto coverslips, stained and quantified as in A–B. J) Model for an integrin-dependent diffusional barrier initiated by FcγR engagement in macrophages. Scale bars =2 µm.

Fig. 5. Integrins exclude CD45 via an ectodomain size-based process required for phagocytosis

A) Comparison of the size of the ectodomain of an integrin heterodimer, short (3 nm) and long (80 nm) glycomimetic polymers, and CD43 and CD2 chimeric constructs used. B) Human macrophages incubated with 500 nM of either short or long Alexa488-conjugated glycomimetics for 20 min at 25°C before seeding onto micropatterned IgG (red) coverslips. Cells were imaged after 10 min. Scale bar =5 µm. C) Human macrophages were incubated with 10 nM of long biotinylated glycomimetic polymer for 20 min at 25°C then labeled with Qdots at 4°C. Cells were warmed to 37°C and seeded onto micropatterned IgG (white) and CD45 trajectories (cyan) recorded for 10 s at 33 Hz. Scale bar=2 µm. D) HeLa cells expressing the indicated GFP-tagged CD45 chimeric construct (green) fixed and stained for vinculin (red) and F-actin (blue). Mean ± SEM of exclusion determined as GFP signal intensity outside/inside 3–5 adhesions for >30 cells from 2 experiments. Scale bar =3 µm. E) RAW 264.7 cells expressing indicated fusion proteins, incubated with IgG-opsonized beads. Cells were first stained for accessible IgG (external beads, red), then permeabilized and stained for total IgG to determine internalized beads (cyan). Scale bar =10 µm. F) Phagocytic index determined as the mean number of internalized particles per cell for >100 cells from 3 independent experiments. Means ± SEM.

Fig. 6. Integrins facilitate engagement of distant points of opsonization

A) Human macrophages seeded onto micropatterned IgG spots (2 µm diameter) separated by 6 µm. The area of glass not covered by IgG was either left uncoated or was blocked with PEG. Cells were fixed after 2 min, stained for FcγRIIA and imaged by SIM. B–C) Cells were seeded as in A for 10 min on coverslips micropatterned with IgG spots separated by 2 or 6 µm before fixing and staining for F-actin. Representative experiment with 6 µm spacing is shown in B, with inverse coloring of the F-actin channel in black and white at bottom. C) Number of engaged opsonized spots. Means ± SD for >50 cells from 3 experiments. Scale bars =10 µm.

Fig. 7. Integrins coordinate points of opsonization for a uniform phagocytic response

A) CD45 was labeled for SMT in RAW 264.7 cells expressing Fcγ IIA-GFP. Cells were laid over IgG-coated coverslips for 5 min before treating with 1.5 mM EDTA (right). CD45 trajectories from 20 s videos were classified as confined (blue), free (cyan), or linear (red) and overlaid on a single image of clustered FcγRIIA-GFP. Scale bar =5 µm. B) Human macrophages were seeded onto IgG micropatterns separated by 6, 4, or 2 µm and after 10 min, fixed and stained for CD45 (cyan). Scale bar =10 µm. C) F-actin distribution on homogenously coated IgG (left) or on micropatterns separated by 2 µm of uncoated glass (middle) or PEG-treated glass (right). Scale bar =10 µm. D) Human macrophages were incubated with a non-invasive strain of Salmonella that was unopsonized or opsonized with O antiserum. Bars represent mean phagocytic index or number of internalized bacteria per cell ± SEM for >50 cells from 3 independent experiments. E) Human macrophages pretreated with EDTA for 2 min or integrin blocking antibodies for 10 min were incubated with bacteria opsonized with H, O, or H + O antisera. Non-internalized regions of bacteria were stained with H + O antisera and secondary antibodies before cells were permeabilized and stained for F-actin. Bacteria were detected by expression of dsRed. The number of completely engulfed bacteria, expressed as a percentage of the total bound, was then determined. Bars are means ± SEM of data from >100 cells from 3 independent experiments. F) BMDMs from WT or CalDAG-GEF1^−/− mice were incubated with bacteria opsonized with H or H + O antisera. G) Representative images from (E). Scale bar =5 µm. H) Human macrophages incubated with beads opsonized with the indicated concentrations of IgG in the presence of either vehicle, PMA or EDTA for 10 min. Data represent mean phagocytic efficiency (beads internalized/beads bound) from 30 cells. I) RAW 264.7 cells expressing full-length talin-GFP or talin head domain-GFP were incubated with opsonized microspheres for 10 min and the phagocytic index determined. Bars are means ± SEM of data from >50 cells from 3 independent experiments.