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Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis

Nature Cell Biology volume 21, pages 1357–1369 (2019)Cite this article

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Abstract

αMβ2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins’ activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMβ2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained β2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

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Fig. 1: CR-mediated phagocytosis involves an actin-based reaching mechanism to engulf target particles.
Fig. 2: Arp2/3 and mDia1 contribute to specific aspects of actin dynamics during CR-mediated phagocytosis.
Fig. 3: Coupling of the actin cytoskeleton to the target enables fast protrusion at the forming phagosome.
Fig. 4: β2 integrins mediate the formation of focal complex-like signalling platforms at the phagosome.
Fig. 5: Mechanical coupling of integrins to the actin cytoskeleton by talin enhances particle engulfment.
Fig. 6: Syk kinase activity is required for vinculin-mediated clutch reinforcement and optimal particle uptake.
Fig. 7: Molecular clutch-mediated mechanosensing regulates phagocytosis.

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Data availability

Statistical source data supporting Figs. 2, 3, 5–7 and Extended Data Fig. 1 are provided in Supplementary Information. All data supporting the findings of this study are available from the corresponding author on reasonable request.

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Author information

Authors and Affiliations

  1. Cell and Developmental Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Valentin Jaumouillé & Clare M. Waterman

  2. National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA

    Alexander X. Cartagena-Rivera

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V.J. designed the research, performed experiments, analysed the data and wrote the manuscript. A.X.C.-R. performed the experiments and analysed the data. C.M.W. designed and supervised the research and wrote the manuscript.

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Correspondence to Clare M. Waterman.

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Extended data

Extended Data Fig. 1 CR-mediated phagocytosis is blocked by anti-integrin antibodies and divalent cation chelation.

a, time-lapse spinning disc confocal microscopy images of a human THP-1 macrophage expressing F-tractin-mCherry during phagocytosis of iC3b-opsonized 5.15 µm polystyrene microspheres, representative of three independent experiments. Elapsed time shown in seconds. b-e, binding index (fraction of cell-associated particles relative to PMA control), phagocytosis index (fraction of internalized particles relative to PMA control) and phagocytosis efficiency (percentage of internalized particles relative to all cell-associated particles) after 1 hour incubation of iC3b-opsonized or BSA-coated 5.15 µm polystyrene beads (b), complement-opsonized or unopsonized sheep red blood cells (c), complement-opsonized or unopsonized sheep red blood cells previously fixed with glutaraldehyde (d) and complement-opsonized or unopsonized zymosan A (e). RAW 264.7 macrophages were preincubated with 10 µg/mL LPS, or 150 ng/mL PMA, without or with blocking antibodies to αM or β2 integrins. Error bars represent SEM. P values were calculated for each individual condition compared the PMA control using two tailed Mann-Whitney test. b, iC3b-beads untreated n = 28 fields, LPS n = 30 fields, PMA n = 47 fields, PMA + anti αM n = 44 fields, PMA + anti β2 n = 30 fields; BSA-beads n = 25 fields, from 3 independent experiments. c, opsonized sRBCs n = 30 fields except, PMA + anti β2 n = 20 fields; unopsonized sRBCs n = 20 fields, from 3 independent experiments. d, opsonized fixed-sRBCs with LPS, PMA, or PMA + anti αM: n = 25 fields, others n = 20 fields, form 3 independent experiments. e, opsonized zymosan with PMA: n = 10 fields, others n = 5 fields. Elapsed times are in seconds. Numerical source data are provided in Statistical Source Extended Data Fig. 1.

Extended Data Fig. 2 The formin mDia1, but not mDia2, is transiently recruited to the forming phagosome.

Time-lapse spinning disc confocal microscopy images of RAW 264.7 macrophages expressing mDia1-mEmerald (top) or mDia2-mEmerald (bottom), during phagocytosis of iC3b-opsonized 5.15 µm polystyrene microspheres. Representative examples from three independent experiments. Elapsed time are in seconds.

Extended Data Fig. 3 Formation of a frustrated phagocytic cup requires integrin engagement.

Time-lapse TIRF microscopy of a RAW 264.7 macrophage expressing EGFP-F-tractin during formation of a frustrated phagocytic cup on an anti-αMβ2-coated coverslip (top) or on an isotype control antibody (bottom). Representative examples from three independent experiments. Elapsed time are in seconds.

Supplementary information

Reporting Summary

Supplementary Video 1

Actin dynamics during CR-mediated phagocytosis of diverse target particles. Time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages expressing EGFP-F-tractin to mark actin filaments during phagocytosis of iC3b-opsonized 4.19 µm polystyrene Flash Red microspheres (left, iC3b-bead), complement-opsonized zymosan A labelled with Texas Red (middle, Comp-zymosan), complement-opsonized sheep red blood cells labelled with Alexa Fluor 647 (right, Comp-sRBC). A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Representative examples from more than 10 phagocytosis events over at least 3 independent experiments.

Supplementary Video 2

Comparison of actin dynamics during CR-mediated phagocytosis upon CR activation by PMA or LPS. Time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages expressing EGFP-F-tractin to mark actin filaments during phagocytosis of iC3b-opsonized 4.19 µm polystyrene Flash Red microspheres (iC3b-bead), upon activation by PMA 150 ng/ml or LPS 10 µg/ml. A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Representative examples from 3 independent experiments.

Supplementary Video 3

Membrane dynamics in 3D during CR-mediated phagocytosis. Three-dimensional time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages expressing EGFP-CAAX to mark the plasma membrane during phagocytosis of iC3b-opsonized 4.19 µm polystyrene Flash Red microspheres. A single confocal image plane (X-Y, left) and a reconstructed axial image plane (X-Z, right) corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Representative examples from 4 independent experiments.

Supplementary Video 4

Actin dynamics during CR-mediated phagocytosis with perturbation of actin nucleators. Time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages expressing EGFP-F-tractin to mark actin filaments during phagocytosis of iC3b-opsonized 5.15 µm polystyrene microspheres: untreated (control, left), 100 µM CK-666 (CK-666, middle), 20 µM SMIFH2 (SMIFH2, right). A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Representative examples from 3 independent experiments.

Supplementary Video 5

Recruitment dynamics of ArpC2 and mDia1 during CR-mediated phagocytosis. Time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages co-expressing mEmerald-ArpC2 (top left and green) or mEmerald-mDia1 (bottom left and green) and mCherry-F-tractin (middle panels and red) during phagocytosis of iC3b-opsonized 5.15 µm polystyrene microspheres. A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Representative examples from 4 independent experiments.

Supplementary Video 6

Formation of a frustrated phagocytic cup requires integrin engagement. Time-lapse TIRF microscopy of a RAW 264.7 macrophage expressing EGFP-F-tractin during formation of a frustrated phagocytic cup on an anti-αMβ2-coated coverslip (Anti-Mac-1, left) or on an isotype control antibody (right). Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 7

Arp2/3 dynamics during CR-mediated frustrated phagocytosis. Time-lapse TIRF microscopy of a RAW 264.7 macrophage co-expressing and mCherry-ArpC2 (left and red) mEmerald-mDia1 (middle and green) during formation of a ‘frustrated phagocytic cup’ on anti-αMβ2 antibody-coated coverslip. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 8

Super-resolution microscopy of actin dynamics during CR-mediated frustrated phagocytosis. Time-lapse TIRF-SIM of a RAW 264.7 macrophage expressing mNeonGreen-F-tractin during formation of a ‘frustrated phagocytic cup’ on anti-αMβ2 antibody-coated coverslip. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 9

Fluorescent speckle microscopy of actin dynamics during CR-mediated phagocytosis. A RAW 264.7 macrophage expressing green-to-red photoconvertible Actin-mEos3.2 during phagocytosis of iC3b-opsonized 5.15 µm polystyrene microspheres was exposed to a low level of 405 nm light and photo-converted red fluorescent actin recorded by time-lapse spinning disc confocal microscopy. A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Images in the time-lapse were aligned relative to the negative image of the bead and individual fluorescent speckles were detected (red circles) and tracked (red lines) with qFSM automated image analysis software. Elapsed time shown in minutes: seconds. Representative example from 4 independent experiments.

Supplementary Video 10

Actin dynamics during frustrated phagocytosis in the absence of integrin engagement. Time-lapse TIRF microscopy of a RAW 264.7 macrophage expressing mCherry-F-tractin during formation of a ‘frustrated phagocytic cup’ on a poly-l-lysine-coated coverslip. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 11

Adhesion dynamics during CR-mediated frustrated phagocytosis. Time-lapse TIRF microscopy of a RAW 264.7 macrophage expressing mEmerald-Paxillin (green) and mCherry-F-tractin (red) during formation of a ‘frustrated phagocytic cup’ on anti-αMβ2 antibody-coated coverslip. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 12

β2 integrin dynamics during CR-mediated frustrated phagocytosis. Time-lapse TIRF microscopy of a RAW 264.7 macrophage expressing co-expressing αM, β2-YFP (green) and mApple-paxillin (red) during formation of a ‘frustrated phagocytic cup’ on anti-αMβ2 antibody-coated coverslip. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 13

Traction stress dynamics during CR-mediated frustrated phagocytosis with inhibition of Syk kinase or overexpression of talin head domain. Top panels: Time lapse spinning disc confocal microscopy of RAW 264.7 macrophages expressing EGFP-F-tractin with (middle) or without (left) treatment with 50 µM piceatannol, or expressing mEmerald-Talin Head (right), during frustrated phagocytosis on anti-αMβ2 antibody-coated 4 kPa polyacrylamide gels. A single confocal image plane at the gel surface is shown over time (minutes: seconds) in each panel. Bottom panels: Time-lapse of stress vectors computed from images of fluorescent beads embedded in the polyacrylamide gel substrate, pseudocolor scale and vector lengths represent the magnitude of traction stresses. Elapsed time shown in minutes: seconds. Representative examples from 3 independent experiments.

Supplementary Video 14

Effect of of talin head domain over-expression on CR-mediated phagocytosis. Time-lapse spinning disc confocal microscopy of RAW 264.7 macrophages co-expressing mEmerald-talin head domain (green) during phagocytosis of iC3b-opsonized 4.19 µm Flash Red polystyrene microspheres (red). A single confocal image plane corresponding to the center of particle is shown over time (minutes: seconds) in each panel. Elapsed time shown in minutes: seconds. Representative example from 3 independent experiments.

Supplementary Video 15

Vinculin localization to the phagosome is Syk kinase dependent. Time-lapse of vinculin concentration relative to a soluble marker from spinning-disk microscopy during phagocytosis of iC3b-opsonized 5.15 µm polystyrene beads by RAW 264.7 macrophages expressing vinculin-mEmerald and FusionRed, treated with DMSO (control), ROCK inhibitor (Y-27632, 10 µM), or Syk inhibitor (piceatannol, 50 µM). Heat maps were generated by subtracting mean-normalized fusion red intensity from mean-normalized vinculin-mEmerald intensity. Elapsed time in minutes:seconds. Representative examples from 3 independent experiments.

Supplementary Video 16

Formation of frustrated phagocytic cups is enhanced by target stiffness. Time-lapse spinning disk confocal microscopy of RAW 264.7 macrophages expressing F-tractin-EGFP during spreading on anti-αMβ2-coated 0.4 kPa, 4 kPa, and 30 kPa polyacrylamide gel substrates. Elapsed time shown in hours:minutes. Representative examples from 3 independent experiments.

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Jaumouillé, V., Cartagena-Rivera, A.X. & Waterman, C.M. Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis. Nat Cell Biol 21, 1357–1369 (2019). https://doi.org/10.1038/s41556-019-0414-2

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