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. Author manuscript; available in PMC: 2017 Jan 14.
Published in final edited form as: Cell. 2016 Jan 14;164(0):128–140. doi: 10.1016/j.cell.2015.11.048

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

Fig. 4

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.

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