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

Abstract

α_Mβ₂ 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 α_Mβ₂ 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.

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. Scale bars are 5 μm, elapsed times are in seconds. Numerical source data are provided in Statistical Source Extended Data Figure 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. Scale bars are 5 μm, 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. Scale bars are 5 μm, elapsed time are in seconds.

Figure 1.. CR-mediated phagocytosis involves an actin-based reaching mechanism to engulf target particles.

Time-lapse spinning disc confocal microscopy images of complement mediated particle uptake by RAW 264.7 (a-d) or bone-marrow-derived (e) murine macrophages. a, two examples (upper row and lower row) of RAW macrophages expressing EGFP-F-tractin during phagocytosis of iC3b-opsonized 4.19 μm polystyrene Flash Red microspheres, from more than 10 independent experiments. b, Phagocytosis of complement-opsonized Zymosan A labelled with Texas Red by RAW macrophages expressing EGFP-F-tractin, from 3 independent experiments. c, Images (left) and kymographs (right) from time-lapse movies of phagocytosis of complement-opsonized Zymosan A labelled with Texas Red by RAW EGFP-F-tractin-expressing macrophages. Kymographs were generated by scanning mean intensities within a 6 μm wide line (blue box) along the direction of particle internalization into the cell. d, Three-dimensional time-lapse confocal of a RAW macrophage expressing EGFP-CAAX during phagocytosis of iC3b-opsonized 4.19 μm polystyrene Flash Red microspheres, from 4 independent experiments. Planar view (X-Y, bottom) and axial view (X-Z, top) corresponding to the center of particle are displayed. e, Bone marrow-derived macrophage expressing EGFP-Lifeact, during phagocytosis of an iC3b-opsonized 4.19 μm polystyrene Flash Red microsphere, from 3 independent experiments. Elapsed time shown in seconds. Scale bars are 5 μm, elapsed times are in seconds.

Figure 2.. Arp2/3 and mDia1 contribute to specific aspects of actin dynamics during CR-mediated phagocytosis.

a, Percent of bound particles that were internalized after perturbation of Arp2/3, formins, myosin II, and ROCK. RAW 264.7 macrophages were incubated in cytoskeletal drugs (Control 0.1%, DMSO (n= 79 fields from 9 independent experiments), CK-666 100 μM (n= 51 fields from 8 independent experiments), SMIFH2 20 μM (n= 35 fields from 7 independent experiments), CK-666 100 μM + SMIFH2 20 μM (n= 30 fields from 3 independent experiments) for 15 minutes prior to addition of iC3b-opsonized 5.15 μm polystyrene beads and internalization assayed one hour later. b, Confocal images from time-lapse movies of RAW macrophages expressing EGFP-F-tractin during the intermediate stage of iC3b-opsonized polystyrene bead engulfment, under the following treatment conditions: control 0.1% DMSO, CK-666 100 μM, SMIFH2 20 μM, from 3 independent experiments. c, Time-lapse confocal microscopy (elapsed time in seconds) of RAW 264.7 macrophage co-expressing mEmerald-ArpC2 (top row, green) or mEmerald-mDia1 (middle row, green) and mCherry-F-tractin (red) during phagocytosis of iC3b-opsonized 5.15 μm polystyrene beads. Fluorescence intensities were measured in a 6 μm diameter circular ROI, centered on the phagocytosed bead, and normalized to the maximal intensity and plotted over time relative to the peak of F-tractin recruitment (bottom row, left, center). Correlation between actin nucleator and F-tractin fluorescence intensities were calculated individually for each experiment, n=4 experiments. d, TIRF microscopy of a RAW macrophage co-expressing mEmerald-mDia1 (top and green) and mCherry-ArpC2 (middle and red) during formation of a frustrated phagocytic cup on an anti-αMβ2-coated coverslip, from 3 independent experiments. White box shows location of the inset. Blue bar shows location of the line scan of fluorescence intensities presented in the right panel. Scale bars are 5 μm, except d left panel is 2 μm. Error bars represent SEM, p values are from two tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 2.

Figure 3.. Coupling of the actin cytoskeleton to the target enables fast protrusion at the forming phagosome.

a, Fluorescent speckle microscopy during phagocytosis of iC3b-opsonized 5.15 μm polystyrene beads by RAW macrophages expressing Actin-mEos3.2, photoswitched with 405 nm light, and imaged with 561 nm light by time-lapse spinning disk confocal microscopy, from 4 independent experiments. Images were aligned with respect to the ingested particle. Red circles represent detected speckles in the corresponding frame, red lines represent tracks of the speckles from the first time point of their detection to the current frame, as analyzed using automated speckle-tracking software. b, Time lapse TIRF-SIM images of a RAW macrophage expressing F-tractin-mNeonGreen during formation of a frustrated phagocytic cup on anti-αMβ2 coated coverslip, from 3 independent experiments. c, Examples of kymographs generated by line scans perpendicular to the leading edge taken from time-lapse movies during frustrated phagocytosis by F-tractin-EGFP-expressing RAW macrophages spreading on anti-αMβ2-coated coverslips (left) or poly-L-lysine (PLL)-coated coverslips in the presence of 2 mM EDTA to inhibit integrin-ligand engagement (center). Initial leading edge protrusion and actin retrograde flow velocities (lamellipodium= 0-3μm from the leading edge, lamellum= 3-8μm from the leading edge) were quantified from such kymographs (n=5 experiments). Scale bars are 5 μm, except b left panel is 2 μm. Error bars represent SEM, p values are from two tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 3.

Figure 4.. β2 integrins mediate the formation of focal complex-like signaling platforms at the phagosome.

a, Immunofluorescence localization of vinculin, a-actinin, zyxin, phospho-tyrosine, FAK phosphorylated on tyrosine 397 (Y397), paxillin phosphorylated on tyrosine 31 (Y31), syk phosphorylated on tyrosine 342 (Y342) (top panels, and green) in RAW macrophages phagocytosing iC3b-opsonized 5.15 μm polystyrene beads, imaged by confocal microscopy. Actin filaments were labeled with fluorescent phalloidin (middle panels, and red). b, Immunofluorescence of paxillin phosphorylated on tyrosine 118 (Y118, middle right and red) and labeling of actin filaments with fluorescent phalloidin (middle left and green), at the frustrated phagocytic cup of a RAW macrophage on an anti-αMβ2 coated coverslips, imaged by TIRF-SIM. c, Time-lapse TIRF microscopy of the leading edge of a RAW macrophage expressing mEmerald-Paxillin (green) and mCherry-F-tractin (red) during formation of a frustrated phagocytic cup on an anti-αMβ2 coated coverslip. d, Time lapse TIRF microscopy of a RAW macrophage co-expressing αM, β2-EYFP (green) and mApple-Paxillin (red) during formation of a frustrated phagocytic cup on an anti-αMβ2 coated coverslip. Images are representative examples from 3 independent experiments. Scale bars are 5 μm, except b left panel is 2 μm. Elapsed times are in seconds.

Figure 5.. Mechanical coupling of integrins to the actin cytoskeleton by talin enhances particle engulfment.

a, Examples from time-lapse traction force microscopy of RAW macrophages expressing F-tractin-EGFP (left) or talin head domain-mEmerald (right), during frustrated phagocytosis on anti-αMβ2 coated 4 kPa polyacrylamide gels. Top: spinning disk confocal images, bottom: stress vectors calculated from the displacement of fluorescent 40nm particles incorporated in the gel, pseudocolor scale and vector lengths represent traction stress magnitude. b, Quantification of the total traction force generated within the phagocytic cup over time by RAW macrophages (green: mock-transfected control, brown: expressing talin head domain-mEmerald) from a representative experiment. c, Quantification of traction stress within the phagocytic cup during spreading of control RAW macrophages (n= 29 cells) or macrophages expressing Talin Head-mEmerald (n=8 cells). Maximal (red) and mean (blue) stress within the phagocytic cup were measured at the time point when the cup reached its full size. d, Binding index (relative to EGFP-expressing control) and (e) phagocytosis efficiency (percentage of internalized relative to contacted beads) after 1 hour incubation of iC3b-opsonized 5.15 μm polystyrene beads with RAW macrophages expressing EGFP, talin head domain-mEmerald, or full-length (FL) talin-mEmerald (n= 30 fields). Experiments were repeated 3 times independently. Scale bars are 5 μm, error bars represent SEM, p values are from two tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 5.

Figure 6.. Syk kinase activity is required for vinculin-mediated clutch reinforcement and optimal particle uptake.

a, b, e, Phagocytosis efficiency in RAW macrophages upon treatment with DMSO (n= 60 fields, from 6 independent experiments) PP2 (20 μM, n= 30 fields, form 3 experiments), piceatannol (50 μM, n= 40 fields, from 4 experiments), TAE226 (5 μM, n= 44 fields, from 5 experiments), or the three inhibitors combined (3YKi) (n= 29 fields, from 3 experiments) (a), upon treatment with DMSO (n= 43 fields), blebbistatin (50 μM, n= 36 fields) or Y-27632 (10 μM, n= 40 fields), from 5 independent experiments (b), or after siRNA knockdown of Syk or vinculin (n= 30 fields from 3 experiments) (f). f upper panels show representative western blots from 3 independent experiments of Syk, vinculin and GAPDH protein levels, . c, Time-lapse of vinculin concentration relative to a soluble marker during phagocytosis of iC3b-opsonized microspheres by macrophages treated with DMSO (control, upper row), Y-27632 (10 μM), or piceatannol (50 μM). d, Quantitation of vinculin-mEmerald relative concentration at the phagosome (control: n= 10 cells; piceatannol: n= 6 cells, Y-27632: n= 12 cells). Time expressed relative to the peak of vinculin-mEmerald recruitment. e, Percentage of phagosome showing a recruitment of vinculin-mEmerald during phagocytosis of iC3b-opsonized microspheres, imaged by confocal microscopy with a single plan 2.5 μm above the the coverslip, every 30 seconds. Macrophages were imaged in the presence of 0.1% DMSO (n= 15 fields), 50 μM piceatannol (n= 14 fields) 20 μM PP2 (n= 10 fields), 10 μM TAE226 (n= 10 fields), or 10 μM Y-27632 (n= 6 fields). g, Time-lapse traction force microscopy of untreated (control) or piceatannol-treated (50 μM) macrophages expressing F-tractin-EGFP during frustrated phagocytosis. Top: confocal images of F-tractin-EGFP, bottom: stress vectors, pseudocolor scale and vector lengths represent traction stress magnitude. Experiments were repeated 3 times independently. h, Quantification of traction stress within the phagocytic cup in control (n= 29 cells) or piceatannol-treated macrophages (n=12 cells). Maximal (red) and mean (blue) stress measured when the cup reached its full size. Scale bars are 5 μm,error bars represent SEM, p values from two-tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 6.

Figure 7.. Molecular clutch-mediated mechanosensing regulates phagocytosis.

a, Representative spinning disk confocal images of RAW macrophages expressing F-tractin-EGFP, from time-lapse movies of frustrated phagocytosis on anti-αMβ2-coated 0.4 kPa, 4 kPa, and 30 kPa polyacrylamide gel substrates. b, Quantification of the velocity of the edge of the phagocytic cup determined from movies of frustrated phagocytosis of RAW macrophages on anti-αMβ2-coated 0.4 kPa (n= 10 cells), 4 kPa (n= 20 cells), and 30 kPa (n= 20 cells) polyacrylamide gel substrates. c, Quantification of the phagocytic cup maximum surface area determined from movies of frustrated phagocytosis of RAW macrophages on anti-αMβ2-coated 0.4 kPa (n= 30 cells), 4 kPa (n= 30 cells), and 30 kPa (n= 14 cells) polyacrylamide gel substrates. d, Elastic moduli determined by atomic force microscopy of complement-opsonized untreated sheep red blood cells (n = 66 cells) or sheep red blood cells fixed with 0.05% glutaraldehyde for 1 minute (n= 29 cells). e, Percent of bound complement-opsonized untreated sheep red blood cells (circles) or sheep red blood cells fixed with 0.05% glutaraldehyde (triangles) that were internalized by RAW macrophages in absence (orange) or presence of 50 μM blebbistatin (green) (n= 30 fields from 3 independent experiments). Scale bars are 5 μm, error bars represent SEM, p values are from two tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 7.