Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis
- Department of Biology and Howard Hughes Medical Institute, University of Washington, United States
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, United States
- Department of Mechanical Engineering, Stanford University, United States
- IFOM, FIRC Institute of Molecular Oncology, Italy
Figures
Figure 1 with 3 supplements
Combined microparticle traction force microscopy (MP-TFM) and lattice light-sheet microscopy (LLSM) reveals phagocytic force-induced deformations in real time.
RAW macrophages transfected with mEmerald-Lifeact were fed soft deformable acrylamide-co-acrylic acid micro (DAAM)-particles (9 μm,1.4 kPa) functionalized with IgG and AF647-Cadaverineand imaged …
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Figure 1—source data 1
Numeric data for Figure 1—figure supplement 1a–c,e,f and Figure 1—figure supplement 2c–f.
- https://cdn.elifesciences.org/articles/68627/elife-68627-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Constriction and bulk compressive stresses during phagosome formation and maturation.
(a,b,c) Violin plot shows individual phagocytic events (colored markers, n = 23), mean (black cross), and median (dashed line). (a) Engulfment time for individual phagocytic events. (b) Maximum …
Figure 1—figure supplement 2
Phagocytosis of stiffer targets follows a qualitatively similar mechanical progression as for softer targets.
(a) Time lapse montage (min:s) of RAW macrophage transfected with mEmerald-Lifeact internalizing a deformable acrylamide-co-acrylic acid-microparticle (DAAMP) (9 μm, Young’s modulus 6.5 kPa) …
Figure 1—figure supplement 3
Murine bone marrow-derived macrophage (BMDM) phagocytosis is mechanically qualitatively similar to RAW macrophage phagocytosis.
(a) Time lapse montage (min:s) of a BMDM transfected with mEmerald-Lifeact internalizing a soft deformable acrylamide-co-acrylic acid-microparticle (DAAMP) (9 μm, Young’s modulus 0.3 kPa) …
Figure 2 with 1 supplement
Phagocytic forces include strong actin-mediated constriction and increase with phagocytic progression.
(a) Confocal images of fixed RAW macrophages phagocytosing soft deformable acrylamide-co-acrylic acid micro (DAAM)-particles functionalized with IgG, and AF488-Cadaverine for visualization. Cells …
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Figure 2—source data 1
Numeric data for Figure 2d,f–i.
- https://cdn.elifesciences.org/articles/68627/elife-68627-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Automated image analysis reveals deformation, F-actin localization, and inside/outside (I/O) stain for 68 phagocytic events.
(a) Determination of contact area between cell and target was calculated using both I/O signal (immunostain of exposed particle surface) and the F-actin signal for confocal microscopy data. First, …
Figure 3 with 5 supplements
Arp2/3-mediated actin polymerization and myosin-II have distinct roles in phagocytic force generation and progression.
(a) Confocal images of drug-treated fixed RAW cells phagocytosing deformable acrylamide-co-acrylic acid micro (DAAM)-particles functionalized with IgG and AF488-Cadaverine for visualization. Cells …
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Figure 3—source data 1
Numeric data for Figure 3d–j and Figure 3—figure supplement 1a–e.
- https://cdn.elifesciences.org/articles/68627/elife-68627-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Actin organization and target deformation along the phagocytic axis are affected by perturbation of actomyosin activity.
(a) Determination of F-actin and contractile ring localization, peak magnitude, and peak width. Particles were aligned with the centroid of the cell-target contact area (base of the cup) at the …
Figure 3—figure supplement 2
Automated image analysis reveals deformation, F-actin localization, and inside/outside (I/O) stain for 63 phagocytic events in CK666-treated cells.
Visualization of deformations, F-actin intensity, and I/O stain for all 63 particles being phagocytosed by CK666-treated cells used in this manuscript shown using Mollweide projections. Particles …
Figure 3—figure supplement 3
Automated image analysis reveals deformation, F-actin localization, and inside/outside (I/O) stain for 75 phagocytic events in Blebbistatin-treated cells.
Visualization of deformations, F-actin intensity, and I/O stain for all 75 particles being phagocytosed by Blebbistatin-treated cells used in this manuscript shown using Mollweide projections. …
Figure 3—figure supplement 4
Automated image analysis reveals deformation, F-actin localization, and inside/outside (I/O) stain for 55 phagocytic events in SMIFH2-treated cells.
Visualization of deformations, F-actin intensity, and I/O stain for all 55 particles being phagocytosed by SMIFH2-treated cells used in this manuscript shown using Mollweide projections. Particles …
Figure 3—figure supplement 5
SMIFH2 treatment has modest effect on force generation and F-actin distribution during phagocytosis.
(a) Confocal images of representative SMIFH2-treated fixed RAW cell phagocytosing deformable acrylamide-co-acrylic acid-micro (DAAM)-particle functionalized with IgG and AF488-Cadaverine for …
Figure 4 with 2 supplements
Actin-based teeth are dynamic, and likely interconnected, structures whose protrusive activity contributes to target constriction.
(a,b) RAW macrophages were fed 1.4 kPa deformable acrylamide-co-acrylic acid-microparticles (DAAMPs) and stained for F-actin. Images represent maximum intensity projections of confocal z-stacks. …
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Figure 4—source data 1
Numeric data for Figure 4d–j, Figure 4—figure supplement 1d–g and Figure 4—figure supplement 2c.
- https://cdn.elifesciences.org/articles/68627/elife-68627-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Automated teeth identification reveals how teeth number, size, and indentation correlate with overall target constriction.
(a) Example of teeth identification method. Teeth were defined as areas of high-actin intensity and inward protrusion. For the mean curvature, the average value of mean curvature for edge …
Figure 4—figure supplement 2
Indentation simulations of phagocytic teeth allow comparison of teeth number, size, and indentation with and total target constriction.
(a) Parametrization of the model. Ten rigid teeth indent a target around the equator of the particle, with Rteeth the tooth radius, Rtarget the target radius (3.7 μm), d the absolute indentation …
Figure 5 with 1 supplement
Multiple actin regulatory proteins localize to phagocytic teeth.
(a) RAW macrophages were transfected with fluorescently tagged actin-binding proteins and challenged to ingest deformable acrylamide-co-acrylic acid-microparticles (DAAMPs) (11 μm, 1.4 kPa) …
Figure 5—figure supplement 1
Multiple actin-binding proteins localize to phagocytic teeth.
RAW macrophages were transfected with fluorescently tagged actin binding proteins and challenged to ingest deformable acrylamide-co-acrylic acid-micro (DAAM)-particles (11 μm, 1.4 kPa) …
Figure 6 with 1 supplement
Contractile activity may enable resolving conflicts by partial target eating and a popping release mechanism.
(a) Maximum intensity projections (MIP) of lattice light-sheet microscopy (LLSM) stacks showing failed internalization attempt of RAW macrophage with IgG-functionalized 1.4 kPa deformable …
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Figure 6—source data 1
Numeric data for Figure 6d–f and Figure 6—figure supplement 1b.
- https://cdn.elifesciences.org/articles/68627/elife-68627-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Target squeezing can result in the target popping into the phagocytic cup.
(a) Top, maximum intensity projections (MIP) of lattice light-sheet microscopy (LLSM) time lapse (min:s) showing successful internalization attempt of RAW macrophage with IgG-functionalized 1.4 kPa …
Figure 7
Model of the molecular players and force balance during phagocytosis.
(a) Graphical model: Arp2/3-mediated actin teeth, guided by myosin-I motors, and organized in a ring drive phagocytic progression and inward target deformation. Teeth are potentially interconnected …
Author response image 1
Videos
Video 1
RAW macrophage ingesting deformable acrylamide-co-acrylic acid-microparticle (DAAMP) imaged by lattice light-sheet microscopy.
RAW macrophages transfected with mEmerald-Lifeact (gray) were fed DAAM-particles (9 μm,1.4 kPa) (blue) functionalized with AF647-Cadaverine, BSA, and anti-BSA IgG. Maximum intensity projections in …
Video 2
3D microparticle shape reconstruction shows phagocytic force-induced deformations in real time.
Video 3
RAW macrophage ingesting stiffer deformable acrylamide-co-acrylic acid-microparticle (DAAMP) shows similar phagocytic force-induced deformation patterns by lattice light-sheet microscopy.
Maximum intensity projection of RAW macrophage transfected with mEmerald-Lifeact (gray) ingesting DAAM-particles (9 μm, 6.5 kPa) (blue) functionalized with AF647-Cadaverine, BSA, and anti-BSA IgG. …
Video 4
Deformable acrylamide-co-acrylic acid-microparticle (DAAMP) phagosome briefly accumulates F-actin, imaged by lattice light-sheet microscopy.
Maximum intensity projection of RAW macrophage transfected with mEmerald-Lifeact (gray) ingesting DAAM-particles (9 μm, 6.5 kPa) (blue) functionalized with AF647-Cadaverine, BSA, and anti-BSA IgG. …
Video 5
Failed deformable acrylamide-co-acrylic acid-microparticle (DAAMP) phagocytosis imaged by lattice light-sheet microscopy.
Maximum intensity projections of RAW macrophages transfected with mEmerald-Lifeact (gray) challenged with DAAM-particles (9 μm,1.4 kPa) (blue). Merged images (left) with single DAAMP channel in gray …
Video 6
Primary bone marrow-derived macrophage (BMDM) partakes in deformable acrylamide-co-acrylic acid-microparticle (DAAMP) meal sharing imaged by lattice light-sheet microscopy.
Murine BMDMs transfected with mEmerald-Lifeact (gray) were fed DAAM-particles (11 μm,1.4 kPa) (blue) functionalized with TRITC-Cadaverine, BSA, and anti-BSA IgG. Merged maximum intensity projections …
Video 7
Contractile activity involved in phagocytic conflict marked by myosin-II.
Maximum intensity projections of RAW macrophages transfected with EGFP-NMMIIA (non-muscle myosin-IIa) (gray) challenged with deformable acrylamide-co-acrylic acid-micro (DAAM)-particles (9 μm,1.4 …
Video 8
Contractile activity leads to sudden forfeit of phagocytic target.
Lattice light-sheet microscopy (LLSM) maximum intensity projections of RAW macrophage expressing mEmerald-Lifeact (gray) challenged with deformable acrylamide-co-acrylic acid-micro (DAAM)-particles …
Video 9
Contractile activity leads to sudden completion of phagocytic internalization.
Lattice light-sheet microscopy (LLSM) maximum intensity projections of RAW macrophage expressing mEmerald-Lifeact (gray) ingesting deformable acrylamide-co-acrylic acid-micro (DAAM)-particles …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | RAW264.7 | ATCC | TIB-71RRID:CVCL_0493 | Macrophage-like cell line |
| Cell line (Homo sapiens) | HL-60 | ATCC | CCL-240RRID:CVCL_0002 | Differentiated into neutrophil-like cells |
| Biological sample (mouse) | Primary bone marrow cells | Barger et al., 2019 | ||
| Antibody | Goat polyclonal anti-rabbit-Alexa Fluor-405 | Invitrogen | A31556RRID:AB_221605 | (1:400) |
| Antibody | Rabbit polyclonal anti-mouse BSA | MP Bio | 0865111RRID: AB_2335061 | (3 mg/mL) |
| Recombinant DNA reagent | EGFP-myo1e (plasmid) | Krendel et al., 2007 | Plasmid construct to transfect and examine myo1e localization | |
| Recombinant DNA reagent | EGFP-myo1f (plasmid) | Barger et al., 2019 | Plasmid construct to transfect and examine myo1f localization | |
| Recombinant DNA reagent | mEmerald-Lifeact (plasmid) | Addgene (a gift from Michael Davidson) | #54148 | Plasmid construct to transfect and monitor F-actin dynamics |
| Recombinant DNA reagent | EGFP-RLC (plasmid) | Plasmid construct to transfect and examine myosin-II localization | ||
| Recombinant DNA reagent | pUB-Halo-WASP (plasmid) | This paper | Plasmid construct to transfect and examine WASP localization | |
| Recombinant DNA reagent | CMV-GFP-NMHCII-A (plasmid) | Addgene | #11347 | Plasmid construct to transfect and examine myosin-II localization |
| Recombinant DNA reagent | EGFP-ARP3 (plasmid) | This paper | Plasmid construct to transfect and examine Arp2/3 complex localization | |
| Recombinant DNA reagent | mEmerald-cortactin (plasmid) | This paper | Plasmid construct to transfect and examine cortactin localization | |
| Recombinant DNA reagent | Cofilin-EGFP(plasmid) | Addgene | #50859 | Plasmid construct to transfect and examine cofilin localization |
| Recombinant DNA reagent | mScarlet-i-paxillin(plasmid) | This paper | Plasmid construct to transfect and examine paxillin localization | |
| Recombinant DNA reagent | pUB-mEmerald-vinculin(plasmid) | This paper | Plasmid construct to transfect and examine vinculin localization | |
| Peptide, recombinant protein | BSA | Sigma | A3059 | 20 mg/mL |
| Peptide, recombinant protein | Alexa Fluor-488 conjugated phalloidin | Life Technologies | A12379 | 1:300 |
| Peptide, recombinant protein | Alexa Fluor-568 conjugated phalloidin | Life Technologies | A12380 | 1:300 |
| Peptide, recombinant protein | Alexa Fluor-488 Cadaverine | Thermo Fisher Scientific | A30679 | 0.2 mM final concentration |
| Peptide, recombinant protein | Alexa Fluor-647 Cadaverine | Thermo Fisher Scientific | A30676 | 0.2 mM final concentration |
| Commercial assay or kit | Neon Transfection System 100 µL Kit | Thermo Fisher Scientific | MPK10096 | |
| Chemical compound, drug | CK666 | EMD Millipore | SML0006 | 150 μM |
| Chemical compound, drug | SMIFH2 | EMD Millipore | S4826 | 10 μM |
| Chemical compound, drug | Blebbistatin | EMD Millipore | B0560 | 15 μM |
| Chemical compound, drug | DAAM-particles | Vorselen et al., 2020b | Can be obtained by contacting Julie A Theriot | |
| Software, algorithm | Fiji (ImageJ) | NIH | RRID:SCR_002285 | |
| Software, algorithm | Imaris | Bitplane | RRID:SCR_007370 | |
| Software, algorithm | MATLAB | Mathworks | RRID:SCR_001622 | |
| Software, algorithm | Python | Python Software foundation | RRID:SCR_008394 | |
| Software, algorithm | Custom MATLAB code | Other | https://gitlab.com/dvorselen/DAAMparticle_Shape_Analysis | |
| Software, algorithm | Custom Python code | Other | https://gitlab.com/micronano_public/ShElastic | |
| Other | VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000 |
