rabbit anti daam antibody  (Developmental Studies Hybridoma Bank)

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 xy (left) and xz (right). Lower left time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: Base, side, and front views of reconstructed deformable acrylamide- co -acrylic acid-microparticle (DAAMP) internalized in Figure 1 , Video 1 showing target deformations (above) and F-actin localization on particle surface (below). Color scales for radial deviation and F-actin intensity shown on right. Upper right time stamp: min:s. Scale bar, 3 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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. Lower left time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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. Lower left time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 (right) to highlight target deformations. Time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 (left) with single DAAMP channel in gray (right) to highlight target deformation. Transfected macrophage attempts to bite DAAMP in half with second untransfected macrophage on the other end, whose presence is implicated by local deformations on the side of the particle not in contact with the transfected cell. Time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 kPa) (blue). Time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 (DAAMPs) (9 μm,1.4 kPa) (blue). Transfected cell attempts to internalize second DAAMP target leading to meal sharing event with second, untransfected cell. Dramatic biting of the DAAMP in two leads to the sudden forfeit of the phagocytic target. Merged images (left) with single DAAMP channel in gray (right) to highlight target deformations. Time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: Lattice light-sheet microscopy (LLSM) maximum intensity projections of RAW macrophage expressing mEmerald-Lifeact (gray) ingesting deformable acrylamide- co -acrylic acid-micro (DAAM)-particles (DAAMPs) (9 μm,1.4 kPa) (blue). Contractile activity on the DAAMP leads to sudden ‘popping’ of target toward the cell to complete ingestion. Concentrated F-actin ring appears to lag behind this event. Merged images (left) with single DAAMP channel in gray (right) to highlight target displacement. Time stamp: min:s. Scale bar, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques:

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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 using LLSM. ( a ) Time lapse montage (min:s) of maximum intensity projections in x/y and x/z. Scale bar, 5 μm. ( b,c ) Schematic of the combined LLSM and MP-TFM experimental approach and analysis, respectively. ( d ) Front and side view of reconstructed DAAM-particle internalized in ( a ) showing target deformations and F-actin localization on particle surface. Colorscale represents the deviation of each vertex from a perfect sphere with radius equal to the median radial distance of edge coordinates to the particle centroid. Scale bar, 3 μm. Figure 1—source data 1. Numeric data for and .

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Transfection

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( 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 were stained for F-actin, and particles with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, second to fourth column: single confocal slices through particle centroid. Scale bar, 5 μm. ( b ) 3D shape reconstructions of deformable acrylamide- co -acrylic acid-microparticle (DAAMP) in ( a ) revealing detailed target deformations induced during phagocytosis and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, and cups are aligned with the phagocytic axis (see e ) from left to right. Scale bars, 3 μm. ( c ) Normal and shear stresses inferred from the shape deformations of the targets in ( a,b ). Negative normal forces denote (inward) pushing forces. ( d ) Averages of absolute magnitudes of normal and shear stresses in phagocytic cups (n = 18). Violin plots show individual phagocytic events (blue markers), mean (black cross) and median (dashed line). *Two-sided Wilcoxon rank sum test: p = 2.0 × 10 –4 . ( e ) Schematic representation of phagocytic parametrization. Normalized cup position indicates the position along the phagocytic axis relative to the rim of the cup, with 0 the cup base and 1 the rim of the phagocytic cup. ( f ) Average profiles of target deformation and F-actin intensity along the phagocytic axis, where 0 and 1 are the cup base and rim, respectively. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets (in 30 bins). Only targets beyond 40% engulfment were included (54 out of 68 events in total). ( g ) Cumulative distribution function of the engulfment stage of randomly selected phagocytic events before completion of engulfment (n = 68). Dashed red line indicates a linear fit. ( h ) Target sphericity and elongation dependence on phagocytic stage. Blue squares indicate individual measurements, black lines indicate averages within five bins. Middle graph inset schematic shows how relative elongation was determined. ( i ) Analysis of the radially symmetric component of particle deformation and F-actin fluorescence along the phagocytic axis for all phagocytic events (n = 68). Marker and line styles as in ( h ). ( j ) Analysis of forces in the contractile ring at the cup rim and throughout the remainder of the cup for 18 cups selected for force analysis. All error bars indicate s.e.m. unless indicated otherwise. Raw data are available in and raw images are available on a FigShare repository . Figure 2—source data 1. Numeric data for .

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Staining, Shear, Fluorescence, Marker

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( 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 were treated with DMSO, CK666 (150 μM), and Blebbistatin (15 μM) for 30 min prior to phagocytic challenge. Each target is approximately 60% engulfed. Fixed cells were stained for F-actin, and particles were labeled with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, second to third column: single confocal slices through particle centroid. Scale bar, 5 μm. ( b ) Particle shape reconstructions from ( a ) revealing cell-induced target deformations and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, cups are aligned with the phagocytic axis (see ) from left to right. Scale bars, 3 μm. ( c ) Normal and shear stresses derived from target deformations. Negative normal forces denote (inward) pushing forces. ( d ) Average profiles of target deformation and F-actin intensity along the phagocytic axis. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets before 40% engulfment were excluded. ( e, f ) Violin plots showing individual phagocytic events (colored markers), mean (black cross), and median (dashed line). ( e ) F-actin peak intensity and band width. ( f ) F-actin intensity in the cup (behind the rim), measured right (3 μm) behind the main peak for each particle. ( g ) Phagocytic efficiency upon drug treatment evaluated as the number of internalized particles divided by the total number of cell-associated particles. Uptake was evaluated 15 min after addition of particles and normalized to internalization by DMSO-treated cells. Three independent experiments were performed where 80–200 particles were measured per condition for each experiment. ***p = 0.0007 (t-test result for hypothesis, mean = 1). ( h ) Upper panel, cumulative distribution function of the engulfment stage of randomly selected phagocytic events before completion of engulfment ( n = 68, 63, 73 respectively) from three independent experiments. Two sample Kolmogorov-Smirnov test was used (p = 0.016*). Lower panel, fraction late-stage cups. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Fisher’s exact test was used to compare fractions (p = 1.9 × 10 –4 )***. ( i ) Sphericity and ( j ) constriction magnitude of DAAM-particle changes with phagocytic progression upon drug treatment. Colored markers indicate individual events, black lines indicate averages of five bins. Right column, violin plots of all events. Marker and line styles as in ( e ). All statistical tests were two-sided Wilcoxon rank sum test comparing with the DMSO control (gray) over the same bin with significance levels: p < 0.05*; p < 0.01**; p < 0.001***, unless otherwise indicated. All error bars indicate s.e.m. unless indicated otherwise. Raw data are available in and raw images are available on a FigShare repository . Figure 3—source data 1. Numeric data for and .

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Staining, Labeling, Shear, Derivative Assay, Marker, Control

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( 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 visualization. Cells were treated with 10 μM SMIFH2 for 30 min prior to phagocytic challenge. The target is approximately 60% engulfed. Fixed cells were stained for F-actin, and particles were labeled with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projection (MIP) of confocal z-stacks, second to third column: single confocal slices through particle centroid. Scale bar, 5 μm. ( b ) Particle shape reconstructions from ( a ) revealing cell-induced target deformations and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, cups are aligned with the phagocytic axis (see ) from left to right. Scale bars, 3 μm. ( c ) Normal and shear stresses derived from target deformations. Negative normal forces denote (inward) pushing forces. ( d ) Average profiles of target deformation and F-actin intensity along the phagocytic axis. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets before 40% engulfment were excluded. ( e, f ) Violin plots showing individual phagocytic events (colored markers), mean (black cross), and median (dashed line). ( e ) F-actin peak intensity and band width. ( f ) F-actin intensity in the cup (behind the rim), measured right (3 μm) behind the main peak for each particle. ( g ) Phagocytic efficiency evaluated as the number of internalized particles divided by the total number of cell-associated particles upon SMIFH2 treatment. Three independent experiments were performed, and uptake was evaluated 15 min after addition of particles and normalized to internalization by DMSO-treated cells. t-Test result for hypothesis that the mean = 1 revealed no significant (n.s.) difference to DMSO control cells (p = 0.09). ( h ) Left panel, cumulative distribution function of the engulfment stage of randomly selected phagocytic events before completion of engulfment (n = 68 DMSO control and 55 SMIFH2-treated) from three independent experiments. Two sample Kolmogorov-Smirnov test revealed no significant difference. Right panel, fraction late-stage cups. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Fisher’s exact test was used to compare fractions (n.s.). ( i ) Sphericity and ( j ) constriction magnitude of DAAM particle is not affected by SMIFH2 treatment. Colored markers indicate individual events, black lines indicate averages of five bins. Right columns, violin plots of all events. Marker and line styles as in ( e ). Two-sided Wilcoxon rank sum test comparing with the DMSO control (gray) over the same bins revealed no significant differences. ( k ) SMIFH2 treatment reduces formation of actin-based teeth within the phagocytic cup. Teeth size and shape are not affected. *** indicates Wilcoxon rank sum test results comparing with the DMSO control with p < 0.001. All error bars indicate s.e.m. unless indicated otherwise. Raw data are available in and .

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Staining, Labeling, Shear, Derivative Assay, Control, Marker

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( 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) functionalized with IgG and AF647-Cadaverine to assess localization to actin teeth (yellow arrowheads). Images are maximum intensity projections of confocal z-stacks. White boxes in leftmost panels indicate the site of the zoomed images to the right. Scale bar, 5 μm. Zoom scale bar, 1 μm. ( b ) DAAM-particle reconstructions for examples shown in ( a ) showing target deformations and localization of fluorescent proteins with respect to actin teeth. Scale bar, 3 μm. ( c ) Myosin-II condensing into thick concentric rings (marked by orange arrowheads) during late-stage phagocytosis of a highly deformed target. Images are maximum intensity projections of confocal z-stacks. Scale bar, 5 μm. Zoom scale bar, 1 μm. Raw images are available on a FigShare repository .

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Transfection, Binding Assay

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: 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) functionalized with IgG and AF647-Cadaverine. ( a ) Actin-binding proteins, cortactin, and cofilin localize to phagocytic teeth (yellow arrowheads) during DAAM-particle internalization. ( b ) Myosin-II forming concentric rings (orange arrowhead) during late-stage phagocytosis. ( c ) Adaptor proteins, vinculin and paxillin, localize at the base of phagocytic teeth in a punctate pattern (red arrowheads). DAAMPs are 9 μm, 1 kPa. Images are maximum intensity projections of confocal z-stacks. Scale bar, 5 μm. Zoom scale bar, 1 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Transfection, Binding Assay

Journal: eLife

Article Title: Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

doi: 10.7554/eLife.68627

Figure Lengend Snippet: ( 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 deformable acrylamide- co -acrylic acid-micro (DAAM)-particle, showing strong deformation and a sudden internalization step. Bottom: DAAM-particle-only channel (inverted grayscale). ( b ) Left: particle position and outline with color-coded kymograph of particle position. Right: particle displacement, sphericity, and apparent diameter over time of the same event shows the sudden nature of the internalization. Scale bars, 5 μm.

Article Snippet: Similar staining of the exposed DAAM area (using Alexa Fluor-488-AffiniPure Fab Fragment Goat Anti-Rabbit IgG [Jackson Immunoresearch, 111-547-003, 1:1000]) was performed to differentiate fully internalized from adherent and partly internalized particles.

Techniques: Microscopy