1. Cell Biology
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Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis

  1. Daan Vorselen
  2. Sarah R Barger
  3. Yifan Wang
  4. Wei Cai
  5. Julie A Theriot  Is a corresponding author
  6. Nils C Gauthier
  7. Mira Krendel  Is a corresponding author
  1. Department of Biology and Howard Hughes Medical Institute, University of Washington, United States
  2. Department of Cell and Developmental Biology, State University of New York Upstate Medical University, United States
  3. Department of Mechanical Engineering, Stanford University, United States
  4. IFOM, FIRC Institute of Molecular Oncology, Italy
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Cite this article as: eLife 2021;10:e68627 doi: 10.7554/eLife.68627

Abstract

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f (‘teeth’) that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

Editor's evaluation

This study provides interesting new insights into the roles of mechanical forces generated by actin polymerization and myosin-II contractility in Fc receptor-mediated phagocytosis. The authors identify F-actin 'teeth', which are important for microparticle constriction throughout the phagocytosis process. Moreover, they elucidate the specific roles of Arp2/3 nucleated actin networks, and myosin-II-based contractile structures in phagocytosis.

https://doi.org/10.7554/eLife.68627.sa0

Introduction

Phagocytic uptake of microbial pathogens, apoptotic cells, and debris are essential processes for human health (Boada-Romero et al., 2020; Lim et al., 2017). Given the variety of phagocytic targets, ranging widely in shape, size, and mechanical stiffness, this process requires remarkable plasticity. Phagocytosis is initiated when phagocytic receptors recognize distinct molecular patterns coating the target (Freeman and Grinstein, 2014; Uribe-Querol and Rosales, 2020). Selective engagement and specific exclusion of phagocyte receptors (Freeman et al., 2018; Freeman et al., 2016) enables downstream signaling to initiate the formation of membrane protrusions, which are guided around the target through sequential ligand engagement in a zipper-like fashion to form the phagocytic cup (Griffin and Silverstein, 1974; Jaumouillé and Waterman, 2020; Swanson and Hoppe, 2004). Mechanically, progression of the phagocytic cup is powered by F-actin polymerization that pushes the plasma membrane forward and culminates in the eventual closure of the cup and the formation of a membrane-enclosed phagosome (Vorselen et al., 2020a).

Previously, F-actin within the phagocytic cup was generally considered to be homogenous and the membrane extensions around the target were frequently likened to lamellipodia at the leading edge of a migrating cell (Blanchoin et al., 2014; Davidson and Wood, 2020; Jaumouillé and Waterman, 2020; Small et al., 2002). Yet recent studies have identified dynamic adhesions or podosome-like structures within the phagocytic cup that appear to be sites of actin polymerization (Barger et al., 2019; Ostrowski et al., 2019). This suggests a fundamentally different mechanism for cup shaping based on assembly of individual actin-based protrusions versus a uniform actin meshwork. Podosomes are specialized F-actin adhesive structures that are prominent in myeloid cells and capable of generating forces and degrading extracellular matrix (Linder, 2007; van den Dries et al., 2019a). Interestingly, podosomes are mechanosensitive (Labernadie et al., 2014; van den Dries et al., 2019b), generating greater forces in response to stiffer substrates, and thus podosome-like structures may contribute to the mechanosensitivity observed in phagocytosis, whereby phagocytes engulf stiffer targets more readily than softer ones (Beningo and Wang, 2002; Jaumouillé et al., 2019; Sosale et al., 2015; Vorselen et al., 2020b; Vorselen et al., 2020a). The ‘core’ structure of podosomes is primarily composed of branched F-actin nucleated by the Arp2/3 complex. The Arp2/3 complex itself was initially considered to be critical for all modes of phagocytosis (May et al., 2000), although subsequent work using Arpc2-/- macrophages has revealed that in Fc receptor (FcR)-mediated phagocytosis, a dendritic actin network is only critical for the ingestion of larger targets (>6 μm) (Rotty et al., 2017). This inhibition of phagocytosis upon loss of the Arp2/3 complex activity may relate to the cell’s ability to form IgG-FcR microclusters at the earliest stages of FcR signaling (Jo et al., 2021).

The mechanism of phagocytic cup closure and the involvement of specific myosin motor proteins therein has also remained elusive (Barger et al., 2020; Jaumouillé and Waterman, 2020). The distinct localization of myosin-I at the rim of the phagocytic cup has led to a speculated role in cup closure (Barger et al., 2019; Swanson et al., 1999; Ikeda et al., 2017). Myosin-II has also been hypothesized to orchestrate a ‘purse-string’ contractility based on observations of phagocytes deforming red blood cells during internalization (Araki et al., 2003; Barger et al., 2020; Swanson et al., 1999 ). However, the effect of myosin-II inhibition on phagocytic uptake efficiency is highly variable, with some studies showing no effect (Jaumouillé et al., 2019; Rotty et al., 2017) and others reporting a 40–80% reduction (Olazabal et al., 2002; Sosale et al., 2015; Tsai and Discher, 2008; Yamauchi et al., 2012). Myosin-II inhibition also has limited effect on traction forces tangential to the target surface in 2D spreading assays known as frustrated phagocytosis (Kovari et al., 2016). Together these studies show that the actin dynamics within the membrane extensions of the phagocytic cup must be finely tuned for successful engulfment, but the role of specific myosin motor proteins and any associated contractility in cup closure remains to be elucidated.

It is clear that phagocytosis is driven by mechanical forces, but examining these forces has been challenging due to the limitations of experimental approaches. Changes in cellular tension during phagocytosis measured by micropipette aspiration have been well studied (Heinrich, 2015; Herant et al., 2005), but this technique fails to capture cell-target forces and the spatial variation within the phagocytic cup. Recently, traction force microscopy (TFM) combined with frustrated phagocytosis assays has been used to measure cell-target forces (Barger et al., 2019; Jaumouillé et al., 2019; Kovari et al., 2016; Rougerie and Cox, 2020), however, this assay fails to capture the biologically relevant geometry of the phagocytic cup, which likely affects cytoskeletal dynamics and force generation. Moreover, in its most common application, TFM only measures forces tangential to the target surface, neglecting forces normal to the target surface, which may be critical. With the recent introduction of particle-based force sensing methods (Mohagheghian et al., 2018; Träber et al., 2019), particularly microparticle traction force microscopy (MP-TFM) (Vorselen et al., 2020b), both normal and shear force components can now be studied throughout phagocytosis.

Here, we utilize live-cell imaging combining MP-TFM and lattice light-sheet microscopy (LLSM) to reveal how mechanical forces generated by actin polymerization and myosin contractility drive phagocytic engulfment mediated by FcR. We show that phagocytes assemble F-actin ‘teeth’ that mediate target constriction throughout phagocytosis. Analysis of forces shows a unique signature, in which target constriction, or squeezing, is balanced by pulling forces at the base of the phagocytic cup at early stages and target compression throughout the cup at later stages. Together, normal forces far exceed shear forces at the cell-target interface, pointing to a mechanism fundamentally distinct from lamellipodial spreading in cell motility. We find that target constriction is mediated by Arp2/3-mediated actin polymerization throughout phagocytosis. Moreover, based on both force analysis and precise quantitative measurement of cup progression, we establish a clear role for myosin-II purse string contractility, specifically in phagocytic cup closure. Finally, we present how this force signature might be critical for target selection and ingestion in more complex physiological settings.

Results

LLSM reveals sequence of target deformations induced during live phagocytic engulfment

Given the fast, 3D, and light-sensitive nature of phagocytosis, we used LLSM for high-speed volumetric imaging with minimal phototoxicity to investigate cytoskeletal dynamics and phagocytic forces. To monitor internalization in real time, RAW264.7 macrophages were transfected with mEmerald-Lifeact for labeling of filamentous actin and were fed deformable acrylamide-co-acrylic acid-microparticles (DAAMPs) (Figure 1a). To investigate FcR-mediated phagocytosis, DAAMPs were functionalized with BSA and anti-BSA IgG, as well as AF647-Cadaverine for visualization (Vorselen et al., 2020b; Figure 1b). RAW macrophages were fed DAAMPs with a diameter of 9 µm and a Young’s modulus of 1.4 or 6.5 kPa, which is in the same range as healthy tissue cells and physiological targets of macrophages like apoptotic cells (Cross et al., 2007; Van der Meeren et al., 2020). RAW macrophages typically formed a chalice-shaped phagocytic cup and completed phagocytosis in a similar timeframe (~3 min) as previously reported for much stiffer (2–3 GPa) polystyrene particles (Horsthemke et al., 2017; Figure 1a, Video 1, Figure 1—figure supplement 1). 3D shape reconstructions of the DAAMPs enabled us to examine target deformations as a direct readout of phagocytic forces in real time (Figure 1b–c). We observed target constriction defined by discrete spots of deformation that appeared around the circumference of the DAAMP at the rim of the phagocytic cup (Figure 1d, Video 2). While these deformations were more apparent using the softer 1.4 kPa DAAMPs, qualitatively similar force patterns were observed using stiffer 6.5 kPa targets (Figure 1—figure supplement 2, Video 3) and previously using 0.3 kPa targets with fixed J774 macrophages (Vorselen et al., 2020b). Interestingly, these indentations traveled parallel to the direction of phagocytic cup elongation, along the length of the DAAM particle until cup closure and were associated with ~400 nm maximum target constriction for 1.4 kPa DAAMPs (Video 2, Figure 1—figure supplement 1). To determine whether this behavior was also present in primary macrophages, we also imaged bone marrow-derived macrophages (BMDMs) transfected with mEmerald-Lifeact and observed a similar mechanical progression, albeit with smaller deformations (Figure 1—figure supplement 3). In addition to local deformations in RAW macrophages, we also observed bulk compressive stresses during phagocytic cup progression (~0.5 kPa) and after complete internalization of the DAAMPs (up to 1.5 kPa), leading to a dramatic reduction in DAAMP diameter (Figure 1—figure supplement 1). The spherical appearance of targets and the gradual monotonic increase in compression after completion of engulfment suggests that this compression may relate to the recently observed shrinkage of internalized macropinosomes by osmotic pressure changes regulated by ion flux (Freeman et al., 2020). However, we sometimes observed the appearance of an F-actin shell around the target, similar to previous reports (Liebl and Griffiths, 2009), suggesting that cytoskeletal forces may also contribute to such compression (Figure 1—figure supplement 1, Video 4).

Figure 1 with 3 supplements see all
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 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.

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

Video 2
3D microparticle shape reconstruction shows phagocytic force-induced deformations in real time.

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.

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

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

Forces normal to the target surface are dominant during phagocytic engulfment and lead to strong target constriction

To more closely investigate the role of the actin cytoskeleton in force generation during FcR-mediated engulfment, we performed MP-TFM measurements on fixed cells during phagocytosis. RAW macrophages were exposed to 9 µm, 1.4 kPa DAAMPs, after which they were fixed and stained for F-actin (Figure 2a). Immunostaining of the exposed particle surface allowed precise determination of the stage of engulfment (Figure 2—figure supplement 1). Confocal z-stacks of phagocytic cups enabled 3D target shape reconstructions with super-resolution accuracy and inference of cellular traction forces (Figure 2b and c). The deformation patterns, although slightly lower in magnitude for the fixed samples (Figure 1—figure supplement 1), were similar to those observed in living cells by the LLSM imaging (Figure 2b and Figure 1—figure supplement 1). Specifically, we noted a ring of inhomogeneous F-actin localized along the rim of the phagocytic cup, where high F-actin intensity strongly correlated with inward target deformation.

Figure 2 with 1 supplement see all
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 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 Figure 2—source data 1 and raw images are available on a FigShare repository (Barger et al., 2021a).

Force analysis (see Materials and methods) revealed compressive stresses up to 400 Pa at these sites (Figure 2c), which is substantially greater than our previous findings of stresses of ~100 Pa using softer targets with Young’s modulus 0.3 kPa (Vorselen et al., 2020b), and suggests that force exertion during phagocytosis may be regulated based on target rigidity. The total compressive forces in the phagocytic rim, which lead to target constriction, increased from ~1 nN in early-stage phagocytosis (fraction engulfed ~22%) to ~10 nN in later stages (fraction engulfed >50%). The shear forces were consistent in magnitude with reported values using TFM during frustrated phagocytosis on planar gels of similar rigidity (Rougerie and Cox, 2020) and were ~7-fold lower than the observed normal forces, independent of the stage of phagocytic engulfment (Figure 2d). This suggests that normal forces dominate the mechanical interaction in phagocytosis, which is in stark contrast with lamellipodial extensions during cell migration where shear forces dominate (Case and Waterman, 2015; Legant et al., 2013).

Target constriction coincides with sites of F-actin accumulation and increases with uptake progression

To identify overall trends in F-actin distribution and location of target deformations within the phagocytic cup, we aligned the 3D images of phagocytic cups and analyzed profiles along the phagocytic axis, defined as the axis from the centroid of the cell-target contact area through the target centroid to the opposing target surface (Figure 2e). This analysis confirmed a clear accumulation of F-actin near the front of the phagocytic cup (~5-fold higher than at the cup base), which precisely colocalized with the site of maximal applied inward normal forces regardless of engulfment stage, as illustrated by quantifying the surface mean curvature (Figure 2f). A similar distribution of F-actin and deformation was observed with stiffer 6.5 kPa targets, albeit with smaller target deformations, and interestingly, less F-actin accumulation in the cup rim (Figure 1—figure supplement 2).

To investigate how phagocytic forces change during phagocytic progression, we arranged cups in order by the fraction of their particle surface engulfed, which allowed us to reconstruct phagocytic engulfment over time from fixed cell images (Figure 2g and Figure 2—figure supplement 1). Since DAAMPs cause little optical distortion, measurable features of the phagocytic cups could be analyzed independent of cup orientation and engulfment stage (Vorselen et al., 2020b). We found no marked accumulation of cups at any specific stage, suggesting no bottleneck or rate-limiting steps, which had been previously reported around 50% engulfment (van Zon et al., 2009). However, we did observe a strong increase in global target deformation, measured as the inverse of target sphericity, with phagocytic progression (Spearman’s ρ = –0.62, p = 5.0 × 10–8) (Figure 2h). This decrease in target sphericity was, at least partially, due to a 4% ± 1% (p = 1.9 × 10–7) average increase in DAAMP elongation along the phagocytic axis (Figure 2h), which is consistent with constriction orthogonal to the phagocytic axis. Direct analysis of target constriction and F-actin peak intensity for each phagocytic cup (Figure 2i, Figure 2—figure supplement 1) revealed an apparent contractile ring in almost all (~96%) cups. The location of this actin contractile ring along the phagocytic axis correlated extremely well with phagocytic progression (ρ = 0.93, p = 3.2 × 10–29) and led to target constriction increasing from ~80 nm in early-stage (fraction engulfed <40%) to ~210 nm in late-stage cups (fraction engulfed >70%). This is a direct effect of increasing normal forces at the cup rim (Figure 2j). Strikingly, in early stages of phagocytosis, net pulling (or outward normal) forces were observed throughout the phagocytic cup and particularly at the base (~3 nN total force, > 100 Pa tensile stresses) (Figure 2j), whereas in late-stage phagocytosis strong net compressive stresses were observed.

Arp2/3-mediated actin polymerization drives force generation throughout phagocytosis, whereas myosin-II powers cup closure

The striking observations of target constriction becoming more pronounced later in engulfment inspired us to consider distinct contributions of actin assembly and myosin-mediated contractility to force generation during phagocytosis. To separate the effects of these two actin-dependent processes, we inhibited Arp2/3-mediated and formin-mediated actin polymerization, as well as myosin-II activity using the small molecule inhibitors CK666, SMIFH2, and Blebbistatin, respectively (Figure 3a, Figure 3—figure supplements 25). Although SMIFH2 was recently reported to exhibit off-target effects, most notably myosin-II inhibition (Nishimura et al., 2021), we observed markedly different behaviors upon SMIFH2 treatment compared to direct myosin-II inhibition using Blebbistatin (Figure 3—figure supplement 5). Target deformation analysis and force calculations revealed that target constriction was diminished upon inhibition of the Arp2/3 complex and myosin-II activity, while formin inhibition had a relatively modest effect (Figure 3c,d, Figure 3—figure supplements 2 and 5). The loss of target constriction coincided with a strong reduction (~40%) in F-actin accumulation at the rim of the cup, as well as a 50% broadening of the typical narrow (~2 μm) F-actin band observed in the DMSO control (Figure 3d,e, Figure 3—figure supplements 2 and 5). Of note, upon myosin-II inhibition alone, the loss of F-actin at the rim of the cup was complemented by a small, but significant (p = 0.04), increase in F-actin density at the base of the cup (Figure 3d,f, Figure 3—figure supplement 5). This observation suggests that myosin-II may be promoting actin disassembly at the base of the phagocytic cup during internalization, similar to the role of myosin-II in disassembling the F-actin network at the cell rear during cell motility (Wilson et al., 2010). To understand the implications of these distinct force generation profiles on phagocytic efficiency, we challenged RAW macrophages to engulf 9 μm 1.4 kPa DAAMPs under drug treatment. Similar to previous reports using large polystyrene particles (Rotty et al., 2017), inhibition of the Arp2/3 complex strongly reduced phagocytosis after 15 min and uptake remained low after longer incubation (Figure 3g, Figure 3—figure supplement 2). Meanwhile, uptake was not significantly affected by Blebbistatin treatment and slightly reduced upon SMIFH2 treatment (Figure 3g, Figure 3—figure supplement 5).

Figure 3 with 5 supplements see all
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 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 Figure 2e) 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 Figure 3—source data 1 and raw images are available on a FigShare repository (Barger et al., 2021a).

We then investigated whether the activity of these molecular players may be associated with specific phagocytic stages. Our analysis revealed a significant change in the observed distribution of cup stages upon myosin-II inhibition, but not Arp2/3 or formin perturbation, compared to DMSO-treated control cells (Figure 3h and Figure 3—figure supplement 5). Specifically, in the Blebbistatin-treated cells, we found an >6-fold enrichment of cups that were beyond 90% engulfment, but not yet closed (p = 1.9 × 10–4). This high prevalence of late-stage phagocytic cups suggests a specific role for myosin-II in cup closure. Throughout phagocytosis, general particle deformations, as measured by the decrease in target sphericity, were strongly reduced upon both CK666 and Blebbistatin treatment (Figure 3i). Inhibition of formins generally reduced target deformations, but also increased the cell-to-cell variability in particle deformation, suggesting that formins may play a role in fine-tuning phagocytic force production (Figure 3—figure supplement 5). Whereas overall target deformations were reduced in all stages of phagocytosis upon Arp2/3 inhibition, myosin-II inhibition only significantly affected phagocytic force generation at later stages, after 50% engulfment (Figure 3i). A similar effect was observed when quantifying target constriction and target elongation specifically (Figure 3j, Figure 3—figure supplement 2). This analysis strongly suggests that there is a specific role for myosin-II in late-stage phagocytosis despite previous data that shows myosin-II is dispensable for phagocytosis (Figure 3g; Jaumouillé et al., 2019; Olazabal et al., 2002; Rotty et al., 2017). The strong effect of Arp2/3 inhibition on phagocytic efficiency and target deformations throughout phagocytosis, and the lack of effect on the distribution of cups-in-progress, suggests that branched actin assembly plays an important role throughout phagocytic progression.

Actin-based protrusive teeth drive target constriction and are mechanosensitive

Based on our observations of discrete spots of inward deformation using MP-TFM (Figures 1a, b, 2a and b), and the significant reduction in target deformation after treatment of cells with the Arp2/3 inhibitor CK666, we hypothesized that these local deformations were the result of actin-based protrusions pushing against the surface of the phagocytic target. Indeed, we frequently observed actin-rich puncta that appeared as oblong or triangular tooth-like projections locally indenting the target surface along the internal rim of the phagocytic cup (Figure 4a) and sometimes deeper within the cup (Figure 4b). Similar actin ‘teeth’ were formed by primary murine BMDMs, bone marrow-derived dendritic cells (BMDCs), and HL-60 human neutrophils when challenged with IgG-functionalized DAAMPs, suggesting that these structures are a common feature of phagocytosis (Figure 4c).

Figure 4 with 2 supplements see all
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. Yellow arrows point to actin-based ‘teeth’. (a) Representative images of teeth at the rim of the phagocytic cup deforming the target. Scale bar, 5 μm. (b) Example of actin-based teeth observed within the phagocytic cup. Scale bar, 5 μm. (c) Primary murine bone marrow-derived macrophage (BMDM), as well as primary bone marrow-derived dendritic cell (BMDC) and HL-60 neutrophil-like cells also form actin teeth in response to DAAMP internalization. Scale bar, 5 μm. Zoom scale bar, 2 μm. (d) CK666 and Blebbistatin treatments reduce formation of actin-based teeth within the phagocytic cup. Teeth size and shape are also modestly affected. Violin plots with individual particles (colored markers), means (black cross), median (dashed line). Stars indicate Wilcoxon rank sum test results comparing with the DMSO control with significance levels: p < 0.05*; p < 0.001***. (e) Manually tracked actin teeth trajectories from DAAMP internalization imaged by lattice light-sheet microscopy (LLSM). Particle surface is shown using Mollweide projection (Figure 2—figure supplement 1). Three time points of a single phagocytic event are shown, with different colors representing unique teeth. Circles indicate current or final position of a tracked tooth. Lines connect the previous positions of tracked teeth. Lower left: great circle distance (GCD) of teeth to the equator for two events. Target 1 is visualized above, time 0 corresponds to the time at which engulfment was completed. Lower right: distribution of tooth speeds with average 0.094 ± 0.08 μm/s (=5.6 μm/min) from three phagocytic events (60 teeth in total from targets 1 and 2 in this panel, and from Figure 4—figure supplement 1). Tooth speeds were averaged over the trajectory of individual teeth. (f) Teeth are mostly located at the rim of the cup. Markers represent individual teeth (n = 716). (g) Constriction magnitude correlates with the number of teeth with Spearman’s rank correlation coefficient (r) = 0.42 (p = 4.4*10–4) for individual DMSO phagocytic events (left) and between drug treatments (right). (h) Teeth number and features change with phagocytic progression, with, from left to right, r = 0.2 (p = 0.11 n.s.), r = 0.17 (p = 1.0 × 10–4), r = 0.16 (p = 2.1 × 10–4). (i) Phagocytic cups with actin teeth appear more frequently when cells are challenged with softer targets. RAW macrophages were challenged with 9 μm DAAMPs of 1.4 or 6.5 kPa, fixed and stained for F-actin. (j) Elasticity theory simulations of the relation between tooth size and depth and overall constriction magnitude. Inset shows teeth, simulated as spherical indenters on a spherical target. Bar graph represents pooled data (n = >150 cups) from three independent experiments. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Pooled data was compared using Fisher’s exact test to compare fractions (p = 1.5 × 10–24***). All error bars indicate s.e.m. unless otherwise indicated. Raw data are available in Figure 4—source data 1.

To investigate the nature and biological function of these actin ‘teeth’ more carefully, we identified them on individual particles based on their protrusive nature and high F-actin intensity (Figure 4—figure supplement 1). According to these criteria, teeth were found in almost all phagocytic cups, with ~10 distinct teeth per cup (Figure 4d). Typically ~1 μm in diameter, and protruding ~200 nm into the 1.4 kPa DAAMP targets, they resembled podosomes in size and protrusive nature (Labernadie et al., 2014). Cells treated with the Arp2/3 inhibitor CK666, and to a lesser extent cells treated with the formin inhibitor SMIFH2, exhibited a reduction in the number of actin teeth per cup (80% and 40% reduction, respectively) compared to control cells treated with DMSO (Figure 4d, Figure 3—figure supplement 5). This result suggests that, like podosomes (van den Dries et al., 2019b), target-deforming phagocytic teeth include both Arp2/3- and formin-nucleated actin filaments. Surprisingly, a strong decrease (~50%) in the number of actin teeth was also observed upon myosin-II inhibition. For all treatments, the reduced number of individual teeth that still formed were remarkably similar to those formed by control cells. Although tooth size and depth were reduced upon CK666 or Blebbistatin treatment, the effect size was small (<15%), suggesting that ‘teeth’ are resilient structures that, once formed, have well-defined properties (Figure 4d).

LLSM allowed us to track the dynamics of actin teeth during phagocytosis (Figure 4e), which revealed clear forward movement over the target surface. A few teeth that we initially detected near the rim stayed in place, remaining where they had assembled during early-stage phagocytosis, suggesting that teeth located deeper within the phagocytic cup (as observed in the fixed cell images) may have originated earlier from the cup rim and been left behind as the cup progressed (Figure 4b, Figure 4—figure supplement 1). More commonly, however, teeth moved forward with a speed of ~5.6 μm/min, similar to the previously reported values for podosome-like structures on very stiff substrates during frustrated phagocytosis (Ostrowski et al., 2019). Strikingly, teeth within the same phagocytic cup appeared to move in a coordinated fashion, with similar speed and direction, and even with observed collective speed changes (Figure 4e, Figure 4—figure supplement 1). This suggests that phagocytic teeth, like podosomes (Meddens et al., 2016; Proag et al., 2015), are mechanically interlinked at the mesoscale.

To test whether the actin teeth were mechanosensitive, we challenged RAW macrophages to ingest 9 μm DAAMPs of 1.4 or 6.5 kPa and fixed and stained cells to examine actin teeth formation. Of note, drug treatments had similar effects on uptake of the stiffer particles as the softer particles (Figure 1—figure supplement 2). Because deformations induced on the stiffer targets were smaller, our systematic identification of teeth based on protrusive activity and actin intensity could not be utilized to faithfully compare tooth number or depth between targets of varying stiffness (Figure 1—figure supplement 2). However, by assessing actin distribution, RAW macrophages assembled actin teeth more frequently when fed softer targets (Figure 4i). This is consistent with our findings that there is less overall F-actin accumulation in the cup rim on stiffer targets (Figure 1—figure supplement 2) and suggests that phagocytic teeth may play a role in the overall mechanosensitivity of phagocytosis (Beningo and Wang, 2002; Jaumouillé et al., 2019; Sosale et al., 2015; Vorselen et al., 2020b; Vorselen et al., 2020a).

Given the ring-like organization of phagocytic teeth in the cup rim, combined with their individual protrusive activity, we questioned whether they were sufficient to explain our observations of target constriction orthogonal to the phagocytic axis, or if a separate contractile mechanism is required. We first distinguished the teeth positioned at the rim of the cup (~70% of teeth), which likely contribute to target constriction, from those deeper in the cup, based on their distance from the cup rim (Figure 4f, Figure 4—figure supplement 1). We then determined whether the properties of teeth near the rim correlated with the overall target constriction. Indeed, the number of teeth per cup and tooth size correlated with overall constriction in DMSO-treated cells and between groups treated with actomyosin activity inhibitors CK666, SMIFH2, and Blebbistatin (Figure 4g, Figure 4—figure supplement 1). We further examined whether changes in the teeth could be related to increasing target constriction with phagocytic cup progression. Teeth numbers increased only slightly with phagocytic progression, which suggests that they are formed quickly early on in phagocytosis and are then typically maintained at constant numbers throughout engulfment (Figure 4h). Teeth size and depth increased significantly but modestly during phagocytic progression (Figure 4h, Figure 4—figure supplement 1). Elasticity theory simulations of teeth-like indentations of a spherical target allowed us to test whether teeth protrusive activity is sufficient to explain the extent of overall target constriction in different stages of phagocytosis (Figure 4—figure supplement 2). Remarkably, this revealed that teeth activity is indeed sufficient to account for total target constriction in early-stage phagocytosis (<50% engulfment), but insufficient to explain the greater degree of target constriction later in the process (Figure 4j). This is consistent with additional myosin-II-based contractile forces in late-stage phagocytosis, as suggested by our observations using Blebbistatin.

Regulators of branched actin assembly (Arp2/3 and WASP) and myosin-I isoforms localize to actin teeth while myosin-II forms contractile rings within the phagocytic cup

Due to the resemblance of the phagocytic actin teeth to podosomes in size, protrusive activity (Figure 4d), and dynamics (Figure 4e), we naturally questioned whether these structures were similar in protein composition as well. Given technical challenges with immunohistochemical staining and MP-TFM (see Materials and methods), we transfected the RAW macrophages with fluorescently tagged proteins to assess localization relative to the actin teeth using 3D reconstructions of the DAAMPs. Consistent with our earlier results showing a decrease in the number of teeth after treating cells with the Arp2/3 inhibitor, we found that the Arp2/3 complex and its activator, WASP, colocalized with the actin teeth (Figure 5a and b). Moreover, cortactin and cofilin, actin-binding proteins that are frequently found in association with densely branched actin networks, also localized to the phagocytic teeth (Figure 5—figure supplement 1). In contrast, myosin-II often appeared distinctly behind the actin teeth in an anti-correlated fashion (Figure 5b). In particular, rings composed of myosin-II filaments could be seen within the phagocytic cup (Figure 5a). In cups that were almost closed, myosin-II coalesced into a ring behind the actin rim (Figure 5—figure supplement 1) and clearly localized to sites of target constriction in cases of extreme target deformation (Figure 5c). Meanwhile, the two long-tailed myosin-I isoforms, myo1e and myo1f, localized specifically to the tips of the actin teeth, consistent with our previous observations (Figure 5a; Barger et al., 2019).

Figure 5 with 1 supplement see all
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) 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 (Barger et al., 2021b).

Given both recent and older observations identifying adhesion adaptor proteins at the phagocytic cup (Beningo and Wang, 2002; Greenberg et al., 1990), we were particularly interested in the localization of paxillin and vinculin. In comparison to the branched actin-binding proteins, both paxillin and vinculin localized behind the phagocytic teeth in a punctate-like pattern (Figure 5—figure supplement 1). Altogether, these studies support a model whereby actin teeth are composed of branched actin filaments guided by myosin-I motor proteins. The localization of myosin-II and paxillin/vinculin behind the teeth suggests that these proteins may play a role in the potential interconnection of the teeth, similar to podosomes on 2D surfaces (Meddens et al., 2016; van den Dries et al., 2019b).

Contractile activity may enable resolution of phagocytic conflicts via partial target eating (‘nibbling’) or forfeit of uptake (‘popping’)

While we have found that target constriction is a signature mechanical feature of FcR-mediated phagocytic progression, it is unclear what functional role target constriction might play during phagocytosis, since actin-driven membrane advancement along the target surface should in principle be sufficient for internalization (Herant et al., 2006; Tollis et al., 2010). In addition to the many successful internalization events we observed using LLSM, we also observed some strikingly different target encounters in which RAW macrophages assembled large amounts of F-actin, only to squeeze futilely at the base of the target without completing engulfment. In these cases, the contractile activity resulted in dramatic deformations and even dumbbell-like appearance of the target (Figure 6a and b, Video 5). In addition to this kind of internalization failure by single cells, we also observed incidents where two macrophages engaged one DAAMP target. Similar to previous observations of red blood cells being squeezed into multilobed shapes when attacked by two macrophages simultaneously (Swanson et al., 1999), these conflicts were also observed using primary BMDMs challenged with DAAMPs (Video 6). Although the polymeric targets used in this study prohibit partial target eating because each particle is effectively one single crosslinked macromolecule that cannot easily be severed by cell-exerted forces, this behavior may be reminiscent of trogocytosis, the process which has been observed during immune cell attack of cancer cells whereby phagocytes ingest small bits of their target (Matlung et al., 2018; Morrissey et al., 2018; Velmurugan et al., 2016). By imaging RAW macrophages transfected with GFP-tagged non-muscle myosin-IIa, we observed highly enriched rings of myosin-II signal at DAAMP deformations during attempts of partial target eating (Figure 6c, Video 7). Target encounters involving extreme deformations of the DAAMP also revealed the existence of a ‘popping’ mechanism that could lead to a sudden release of the target (Video 8) or, conversely, a sudden completion of engulfment (Video 9, Figure 6—figure supplement 1). During such events, targets were first gradually deformed to a dumbbell-like shape, followed by a sudden translocation of the particle, as well as an immediate recovery of its original spherical shape and volume (Figure 6d, Video 7). The rapid timescale of this process suggests that it is likely purely mechanical, representing an elastic recoil of the DAAMP. Importantly, these encounters were rather common, with the attempted partial eating attempts (~14%) and popping (~24%) making up almost 40% off all recorded events (Figure 6e). Furthermore, such events, and specifically popping, were mechanosensitive and occurred much less frequently for stiffer 6.5 kPa targets (~1%, n = 89, p = 1.5 × 10–6), resulting in the overall more frequent failure of phagocytosis for soft particles (Figure 6f).

Figure 6 with 1 supplement see all
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 acrylamide-co-acrylic acid-microparticle (DAAMP). Zoomed images of the area marked by the white box showing only the DAAMP channel (inverse grayscale LUT) are shown below. (b) 3D reconstructions showing both the particle shape and F-actin signal over the particle surface corresponding to the event in (a). Scale bar, 3 μm. (c) Maximum intensity projections (MIP) of LLSM stacks showing myosin-II accumulation (orange arrows) during a partial eating event. RAW macrophages (marked by *) were transfected with EGFP-NMMIIA (non-muscle myosin-IIa) to label myosin-II and challenged with IgG-functionalized 1.4 kPa DAAMP. (d) Top: MIPs of LLSM stacks of RAW macrophage suddenly releasing heavily deformed target. Red dashed line outlines DAAMP. Middle: particle position and outline (left), and kymograph of particle position (right). Bottom: particle displacement, sphericity, and apparent diameter over time of the same event shows the sudden nature of the release. (e) Sudden forfeit by a popping mechanism and attempted partial eating are common for 1.4 kPa targets, with ~24% and ~ 14% occurrence of all phagocytic events, respectively. (f) Percentage of failed phagocytic events is dependent on particle rigidity. Data from two to three independent experiments was pooled (n = 89, 91 phagocytic events) and compared using Fisher’s exact test (p = 7.4 × 10–4). (g) Schematic representation of the multiple ways in which target constriction may enable resolving macrophage conflicts in which two cells attempt a single target. All scale bars are 5 μm, unless otherwise indicated. All error bars indicate s.d. estimated by treating phagocytosis as a Bernoulli process. Raw data are available in Figure 6—source data 1.

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

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 (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.

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

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 (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.

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 (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.

Discussion

By combining LLSM and MP-TFM, we report here a detailed analysis of the mechanical progression of phagocytosis and the contributions of several key molecular players. We have discovered that FcR-mediated phagocytosis occurs through a unique mechanism in which normal forces dominate over shear forces in the cell-target interaction. This is in contrast to the current view that the phagocytic cup is equivalent to the leading edge of a migrating cell, where shear forces typically predominate at the cell-substrate interface (Case and Waterman, 2015; Legant et al., 2013). In addition, the fast forward movement of actin teeth, which underlie target constriction, concomitant with phagocytic cup progression across the target is in stark contrast to lamellipodial focal adhesion complexes, which are fixed relative to the substratum (Case and Waterman, 2015). The strong target constriction recently observed in complement-mediated phagocytosis by peritoneal macrophages in vitro (Walbaum et al., 2021) and in phosphatidylserine-mediated phagocytosis by epithelial cells in zebrafish embryos (Hoijman et al., 2021) suggests that strong normal forces and target constriction are likely a general feature of phagocytosis.

We show that these normal forces are primarily generated by protrusive phagocytic actin ‘teeth’ and myosin-II contractility, which make distinct contributions to target constriction. While actin-based puncta have been previously observed during phagocytosis of stiffer polystyrene beads (Barger et al., 2019; Ostrowski et al., 2019), their presence in multiple phagocytic cell types (Figure 4c) and during complement-mediated phagocytosis (Ostrowski et al., 2019) suggests a common role in internalization. Structures with podosome-like features, albeit resistant to CK666 treatment, have also been reported on closed phagosomes containing IgG or serum-opsonized polystyrene targets (Tertrais et al., 2021). We find that phagocytic actin teeth are podosome-like in protein composition, consisting of mostly branched F-actin and actin regulatory proteins. A previous study showed that the Arp2/3 complex was not required for FcR-mediated ingestion of smaller phagocytic targets, while the uptake of particles comparable in size to those in our study (~10 μm) was Arp2/3-dependent (Rotty et al., 2017). This is consistent with our observations that inhibition of the Arp2/3 complex dramatically reduces actin tooth number and size, and ultimately target constriction and uptake efficiency.

Unexpectedly, myosin-II motor inhibition also reduced tooth number and size, albeit to a lesser extent than Arp2/3 inhibition, and also altered tooth spatial distribution (Figure 4—figure supplement 1). Inhibition of myosin-II in studies on podosomes also caused a reduction in podosome stiffness and protrusive activity (Labernadie et al., 2014; Labernadie et al., 2010). In 2D podosomes, myosin-II filaments interconnect radial actin fibers of individual podosomes to create a coherent network (Labernadie et al., 2014; Meddens et al., 2016). The localization of myosin-II behind and in between teeth, as well as the coordinated movement of teeth at the mesoscale, suggests that actin teeth in phagocytosis may be comparably interconnected by actomyosin structures, bearing similarity to the organization of podosome rosettes (Linder, 2007). We find that individual teeth grow larger and stronger with phagocytic progression, correlating with increasing myosin-II constriction observed in late-stage phagocytosis (Figure 7a). These observations may relate to the force feedback mechanism observed in Arp2/3-mediated branched actin networks in vitro showing that mechanical resistance makes self-assembling actin networks stronger (Bieling et al., 2016; Li et al., 2021). Thus, increased myosin-II contraction in late-stage phagocytosis may promote stronger actin teeth. This rapid structural reinforcement by myosin-II has not yet been described for actin cores of podosomes on 2D, and may be unique to phagocytosis.

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 through myosin-II filaments located behind the actin teeth. (b) Ring-like target constriction by protrusive actin teeth and myosin-II activity (red arrows) can be decomposed in forces orthogonal to the phagocytic axis, which are balanced within the ring (yellow arrows) and along the phagocytic axis, which result in a net force exerted by the ring (purple arrows). The local target geometry at the protruding rim of the cup determines the direction of the net force. Before 50% engulfment, this force points outward and is balanced by pulling forces throughout the base of the phagocytic cup (push and lock). Inset, the pulling forces from the cell (dashed arrow) on the target and paired forces from the target on the cell (solid arrow) likely put the receptor-ligand interactions under tension. After 50% engulfment, the net ring force points inward, and is balanced by compressive forces (cyan arrows) throughout the phagocytic cup.

Further potential differences between actin teeth and 2D podosomes should also be noted. Although actin teeth are podosome-like in protein composition, the typical ring-like organization of adhesion proteins surrounding the F-actin core of 2D podosomes is not apparent during phagocytosis. Instead, we, and others (Ostrowski et al., 2019), observe vinculin, for example, to appear more punctate behind actin teeth during phagocytosis (Figure 5—figure supplement 1). This could be due to the difficulty in visualizing the supramolecular architecture in the 3D context of phagocytosis involving curved targets, yet this could also point to differences in the molecular arrangement between actin teeth and 2D podosomes. In the future, higher resolution imaging will be necessary to better compare the architecture of actin teeth and podosomes, including the potential presence of a podosome ‘cap’ in actin teeth (Linder and Cervero, 2020), and may potentially expose changes in mechanosensitive protein localization in response to phagocytic targets of different rigidities. In support of differences between the molecular architecture of actin teeth and podosomes, the nature of force exertion at these structures also likely differs. In 2D podosomes, the protrusive force of the actin ‘core’ is counterbalanced by pulling forces from the surrounding adhesion complexes (Bouissou et al., 2017). This force exertion profile was, at least partly, inferred from the observation of lower global deformation than expected by the protrusive activity of podosome cores alone. In contrast, we observe higher large-scale deformation than expected by the protrusive activity of teeth alone (Figure 4j) and the deformations on the DAAMPs are not indicative of strong local tensile forces by adhesion complexes. Finally, we show that actin teeth are mechanosensitive, forming more frequently when cells are challenged with softer targets, contrary to the reported behavior of podosomes in 2D (Labernadie et al., 2014; van den Dries et al., 2019a; van den Dries et al., 2019b; Figure 4j).

We further show that myosin-II plays an important role in phagocytic cup closure – a stage in phagocytosis that has been notoriously difficult to study experimentally (Marie-Anaïs et al., 2016; Figure 7a). Generally, myosin-II is not deemed important for cup closure or phagocytic progression (Jaumouillé and Waterman, 2020; Swanson et al., 1999), yet we observe myosin-II enrichment at the rim of phagocytic cups (Figure 5c). Moreover, Blebbistatin treatment specifically affects late-stage contractile force generation (Figure 3h) causing cup closure to become a bottleneck step as evidenced by the accumulation of late-stage phagocytic cups (Figure 3g). Because these forces are exerted normal to the target surface, the contribution of myosin-II activity to phagocytic progression has likely gone unnoticed in previous studies using the most common implementation of TFM on a planar substrate, which only measures forces that are tangential to the surface (shear) (Barger et al., 2019; Jaumouillé et al., 2019; Kovari et al., 2016). Seemingly contradictory to its apparent role in force generation at the later stages of cup closure, we do not find that myosin-II inhibition affects uptake efficiency measured as the ratio of fully closed to incompletely closed cups. One potential explanation is that when phagocytosis is stalled beyond 90% engulfment upon myosin-II inhibition, the incomplete closure of the cup may be clearly observed in high-resolution 3D imaging of individual cells, but such late-stage cups may be mistaken for fully internalized particles in the lower resolution 2D microscopy used for evaluating phagocytic efficiency in a large number of cells. Alternatively, a model in which myosin-II has opposing effects on phagocytic progression during early (beneficial) and late-stage (adverse) phagocytosis could also be consistent with our observations. It is also possible that while myosin II-mediated constriction is important for the architecture or normal progression of the phagocytic cup or for cup closure in more challenging environmental contexts, cup closure can still proceed in the absence of functional myosin-II through alternative pathways such as actin polymerization-driven extension of the cup rim. Similarly, in Dictyostelium cytokinesis, myosin-II is required for cortical tension generation in the cleavage furrow but cytokinesis can still be successfully completed on adhesive surfaces via alternative mechanisms (Zang et al., 1997; Zhang and Robinson, 2005). Ultimately, our findings underscore the complexity of the myosin-II contributions to phagocytosis and highlight the power of new biophysical tools to identify its precise functioning.

We show that FcR-mediated phagocytosis is hallmarked by a unique force balance, where before 50% engulfment, target constriction results in an outward force balanced by pulling forces throughout the base of the phagocytic cup (Figure 7b). Given the reduced presence of cytoskeletal components at the base of the cup (Figure 2—figure supplement 1; Freeman and Grinstein, 2014; Jaumouillé and Waterman, 2020), these pulling forces are most likely not due to actin polymerization forces or actomyosin contractility, but instead a result of the target being held in place through receptor-ligand bonds throughout the base of the cup. Hence, the forces acting on receptor-ligand bonds within the cup are likely dependent on the target physical properties and the local geometry at the rim of the cup (Figure 7b). Since the lifetime of such bonds is tension-dependent (Nishi et al., 2017; Zhu et al., 2019), this push-and-lock mechanism may enable sensing of physical target properties through a proofreading mechanism and thereby aid macrophages in target selection. Indeed, we observe that forfeit of uptake via popping, in which the outward-directed forces likely overcome the strength of the receptor-ligand interactions, depends on the local target geometry and target rigidity. In addition, for non-spherical targets (e.g. ellipsoidal), this force balance would result in a net torque aligning the target’s long axis along the phagocytic axis, which has been observed previously and is reported to lead to enhanced uptake efficiency of ellipsoidal targets (Champion and Mitragotri, 2006; Schuerle et al., 2017; Sosale et al., 2015).

We observed that actin teeth formed more frequently when cells were challenged with softer targets (Figure 4i), yet we also associated softer targets with more instances of failed internalization (Figure 6f). This calls into question the effectiveness of actin teeth, and, more generally, target constriction, in driving phagocytic internalization. Aside from a role in cup closure, target constriction could be important for creating a tight apposition between the cell and target, which is essential for receptor engagement (Bakalar et al., 2018). Surprisingly though, we noticed that overly strong target constriction leads to failure of attempted phagocytosis, expressed either as partial eating or a popping mechanism leading in forfeit of uptake. Although these mechanisms may lead to reduced uptake efficiency in isolated phagocyte-target interaction, we suspect they may be critical in more complex phagocytic encounters that occur in vivo, for example, when multiple macrophages approach a single target (Figure 6g), or attempt to engulf adherent and hard-to-reach targets (Colucci-Guyon et al., 2011; Davidson and Wood, 2020; Vorselen et al., 2020a). Altogether, our findings show that actin polymerization-dependent protrusive forces and myosin-II-dependent contractile forces contribute to target deformation and phagocytic internalization, and likely both participate in the mechanosensation required for phagocytic plasticity.

Code availability

The MATLAB code for analysing confocal images and deriving particle shape is publicly available on https://gitlab.com/dvorselen/DAAMparticle_Shape_Analysis; Vorselen, 2021. The Python code used for analysing tractions is provided on https://gitlab.com/micronano_public/ShElastic; Wang and Cai, 2021.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)RAW264.7ATCCTIB-71RRID:CVCL_0493Macrophage-like cell line
Cell line (Homo sapiens)HL-60ATCCCCL-240RRID:CVCL_0002Differentiated into neutrophil-like cells
Biological sample (mouse)Primary bone marrow cellsBarger et al., 2019
AntibodyGoat polyclonal anti-rabbit-Alexa Fluor-405InvitrogenA31556RRID:AB_221605(1:400)
AntibodyRabbit polyclonal anti-mouse BSAMP Bio0865111RRID: AB_2335061(3 mg/mL)
Recombinant DNA reagentEGFP-myo1e (plasmid)Krendel et al., 2007Plasmid construct to transfect and examine myo1e localization
Recombinant DNA reagentEGFP-myo1f (plasmid)Barger et al., 2019Plasmid construct to transfect and examine myo1f localization
Recombinant DNA reagentmEmerald-Lifeact (plasmid)Addgene (a gift from Michael Davidson)#54148Plasmid construct to transfect and monitor F-actin dynamics
Recombinant DNA reagentEGFP-RLC (plasmid)Plasmid construct to transfect and examine myosin-II localization
Recombinant DNA reagentpUB-Halo-WASP (plasmid)This paperPlasmid construct to transfect and examine WASP localization
Recombinant DNA reagentCMV-GFP-NMHCII-A (plasmid)Addgene#11347Plasmid construct to transfect and examine myosin-II localization
Recombinant DNA reagentEGFP-ARP3 (plasmid)This paperPlasmid construct to transfect and examine Arp2/3 complex localization
Recombinant DNA reagentmEmerald-cortactin (plasmid)This paperPlasmid construct to transfect and examine cortactin localization
Recombinant DNA reagentCofilin-EGFP(plasmid)Addgene#50859Plasmid construct to transfect and examine cofilin localization
Recombinant DNA reagentmScarlet-i-paxillin(plasmid)This paperPlasmid construct to transfect and examine paxillin localization
Recombinant DNA reagentpUB-mEmerald-vinculin(plasmid)This paperPlasmid construct to transfect and examine vinculin localization
Peptide, recombinant proteinBSASigmaA305920 mg/mL
Peptide, recombinant proteinAlexa Fluor-488 conjugated phalloidinLife TechnologiesA123791:300
Peptide, recombinant proteinAlexa Fluor-568 conjugated phalloidinLife TechnologiesA123801:300
Peptide, recombinant proteinAlexa Fluor-488 CadaverineThermo Fisher ScientificA306790.2 mM final concentration
Peptide, recombinant proteinAlexa Fluor-647 CadaverineThermo Fisher ScientificA306760.2 mM final concentration
Commercial assay or kitNeon Transfection System 100 µL KitThermo Fisher ScientificMPK10096
Chemical compound, drugCK666EMD MilliporeSML0006150 μM
Chemical compound, drugSMIFH2EMD MilliporeS482610 μM
Chemical compound, drugBlebbistatinEMD MilliporeB056015 μM
Chemical compound, drugDAAM-particlesVorselen et al., 2020bCan be obtained by contacting Julie A Theriot
Software, algorithmFiji (ImageJ)NIHRRID:SCR_002285
Software, algorithmImarisBitplaneRRID:SCR_007370
Software, algorithmMATLABMathworksRRID:SCR_001622
Software, algorithmPythonPython Software foundationRRID:SCR_008394
Software, algorithmCustom MATLAB codeOtherhttps://gitlab.com/dvorselen/DAAMparticle_Shape_Analysis
Software, algorithmCustom Python codeOtherhttps://gitlab.com/micronano_public/ShElastic
OtherVECTASHIELD Antifade Mounting MediumVector Laboratories H-1000

Cell lines

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The RAW 264.7 and HL-60 cell lines were obtained directly from the ATCC and used within passages 3–15. Cell identity was confirmed prior to each experiment using examination of cell morphology by phase-contrast morphology, and cells were regularly tested for mycoplasma contamination using DAPI staining. The cells used in this study do not appear on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee.

Cell culture

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RAW 264.7 (ATCC; male murine cells) were cultured in DMEM, high glucose, containing 10% FBS and 1% antibiotic-antimycotic (Gibco) (cDMEM) at 37°C with 5% CO2. HL-60 cells (ATCC; CCL-240) were cultured in RPMI plus L-glutamine supplemented with 20% FBS and 1% antibiotic-antimycotic (cRPMI). HL-60 cells were differentiated into neutrophil-like cells in culture media containing 1.5% DMSO and used at day 5–6 post-differentiation and plated on 20 μg/mL fibronectin. For the collection of primary murine bone marrow progenitor cells, femurs and tibias of C57BL/6 mice were removed and flushed with cDMEM. Red blood cells were lysed using ACK buffer (0.15 M NH4Cl) and bone marrow progenitor cells were recovered by centrifugation (250× g, 5 min, 4°C), washed once with sterile PBS and plated on tissue culture dishes in cDMEM at 37°C with 5% CO2. For differentiation into BMDMs, non-adherent cells were moved to bacteriological Petri dishes the next day and differentiated over 1 week in cDMEM containing 20 ng/mL recombinant murine M-CSF (Biolegend, 576404). Generation of murine BMDCs has been previously described (Gosavi et al., 2018). In brief, bone marrow progenitor cells were collected in cRPMI and replated in cRPMI containing 20 ng/mL recombinant murine GM-CSF (Peprotech, 315–03) (DC media). On day 3, DC media was supplemented. On days 6 and 8, half of the culture supernatant and nonadherent cells were spun down and resuspended in cRMPI containing 5 ng/mL GM-CSF. DC maturation was assessed on day 10 by flow cytometry using PE-Cd11c (Biolegend, 117307) and FITC-MHC-II (Biolegend, 107605) staining. Cells were used on day 12. All procedures utilizing mice were performed according to animal protocols approved by the IACUC of SUNY Upstate Medical University and in compliance with all applicable ethical regulations.

Chemicals and drugs

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Blebbistatin, CK666, SMIFH2, and fibronectin were purchased from EMD Millipore. Alexa Fluor-488 and Alexa Fluor-568 conjugated phalloidin were purchased from Life Technologies. Janelia Fluor 549 (JF549) HaloTag Ligand was a generous gift from Luke Lavis.

Constructs and transfection

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Human myo1e and myo1f constructs tagged with EGFP have been previously described (Barger et al., 2019). mEmerald-Lifeact was a gift from Michael Davidson (Addgene #54148). Cofilin-EGFP was a gift from James Bamburg (Addgene #50859). Chicken regulatory light chain (RLC) tagged with EGFP was a gift from Klaus Hahn. WASP tagged with myc was a gift from Dianne Cox, and was subcloned into a pUB-Halo-C1 vector. CMV-GFP-NMHCII-A was a gift from Robert Adelstein (Addgene #11347). ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively. Chicken paxillin was a gift from Chris Turner, which was subcloned into mScarlet-i-C1. Chicken vinculin was a gift from Kenneth Yamada (Addgene #50513) and subcloned into pUB-mEmerald-C1. Immunohistochemical staining to determine localization of select podosome-related proteins in relation to the actin teeth did not produce good results, which may be due to the adhesive and porous nature of the IgG-functionalized DAAMPs. As an alternative, we transfected the RAW macrophages with fluorescently tagged proteins. All transfections were accomplished by electroporation (Neon) using the manufacturer’s instructions.

Microparticle synthesis

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DAAMPs were synthesized as previously described (Vorselen et al., 2020b). First, acrylamide mixtures containing 100 mg/mL acrylic components, 150 mM NaOH, 0.3% (v/v) tetramethylethylenediamine, 150 mM MOPS (prepared from MOPS sodium salt, pH 7.4) were prepared. Mass fraction of acrylic acid was 10% and crosslinker mass fraction was 0.65% or 2.3%, for 1.4 and 6.5 kPa particles, respectively. Prior to extrusion, the gel mixture was degassed for 15 min and then kept under nitrogen atmosphere until the extrusion process was complete. Tubular hydrophobic Shirasu porous glass (SPG) were sonicated under vacuum in n-heptane, mounted on an internal pressure micro kit extruder (SPG Technology Co) and immersed into the oil phase (~125 mL) consisting of hexanes (99%) and 3% (v/v) Span 80 (Fluka, 85548 or Sigma-Aldrich, S6760); 10 mL of gel mixture was extruded through SPG membranes under nitrogen pressure of ∼7 kPa, 15 kPa, for membranes with pore size 1.9 and 1.4 μm, respectively; 9 μm, 1.4 kPa particles were synthesized using 1.4 μm pore size membranes, whereas 9 μm, 6.5 kPa particles and 11 μm, 1.4 kPa particles were made using 1.9 μm pore size membranes. The oil phase was continuously stirred at 300 rpm and kept under nitrogen atmosphere. After completion of extrusion, the emulsion temperature was increased to 60°C and polymerization was induced by addition of ~225 mg 2,2’-azobisisobutyronitrile (1.5 mg/mL final concentration). The polymerization reaction was continued for 3 hr at 60°C and then at 40°C overnight. Polymerized particles were subsequently washed (5× in hexanes, 1× in ethanol), dried under nitrogen flow for ∼30 min, and resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4) and stored at 4°C.

Microparticle functionalization

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DAAM-particles were functionalized as previously described (Vorselen et al., 2020b). In brief, DAAMPs were diluted to 5% (v/v) concentration and washed twice in activation buffer (100 mM MES, 200 mM NaCl, pH 6.0). They were then incubated for 15 min in activation buffer supplemented with 40 mg/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 20 mg/mL N-hydroxysuccinimide (NHS), and 0.1% (v/v) Tween 20, while rotating. Afterward they were spun down (16,000× g, 2 min) and washed 4× in PBS, pH 8 (adjusted with NaOH) with 0.1% Tween 20. Immediately after the final wash, the particles were resuspended in PBS, pH 8 with 20 mg/mL BSA (Sigma, A3059) and incubated, rocking for 1 hr. Then cadaverine conjugate was added: either Alexa Fluor-488 Cadaverine (Thermo Fisher Scientific, A-30679) or Alexa Fluor-647 Cadaverine (Thermo Fisher Scientific, A-30676) to a final concentration of 0.2 mM. After 30 min, unreacted NHS groups were blocked with 100 mM TRIS and 100 mM ethanolamine (pH 9). DAAMPs were then spun down (16,000× g, 2 min) and washed 4× in PBS, pH 7.4 with 0.1% Tween 20. BSA-functionalized DAAMPs were resuspended in PBS, pH 7.4 without Tween. Finally, DAAMPs were washed 3× in sterile PBS and opsonized with 3 mg/mL rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 hr at room temperature. DAAMPs were then washed 3× (16,000× g, 2 min) with PBS and resuspended in sterile PBS.

Phagocytosis assays

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DAAMPs were added to a total volume of 400 μL of serum-free DMEM, briefly sonicated in a bath sonicator, and applied to phagocytes in a 12-well plate. To synchronize phagocytosis and initiate DAAMP-phagocyte contact, the plate was spun at 300 × g for 3 min at 4°C. Cells were incubated at 37°C to initiate phagocytosis for a period of 3–5 min. Media was then removed and cells were fixed with 4% PFA/PBS for 15 min. Any unbound DAAMPs were then washed away with 3× washes of PBS and samples were stained with goat anti-rabbit-Alexa Fluor-405 antibodies (Invitrogen, A31556, 1:400) for 30 min to visualize exposed DAAM area. Cells were then washed with PBS (3× for 5 min each) and permeabilized with 0.1% Triton X-100/PBS for 3 min, then stained with Alexa Fluor-568 or -488 conjugated phalloidin (1:300). Coverslips were then mounted using VECTASHIELD Antifade Mounting Medium (Vector Laboratories, H-1000) and sealed with nail polish. For both live and fixed cell assays, cells were randomly selected for imaging, and all cells were considered, including those having already internalized a particle and those interacting with multiple particles. For drug treatments, cells were exposed to the indicated drug concentration for 30 min prior to the assay and DAAM particles were resuspended and exposed to cells in the same drugged media. For phagocytic efficiency assays, cells were incubated 15, 30, or 60 min before fixation. 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. Cells were imaged at 20× magnification and the phagocytic index, defined here as the number of internal particles divided by the number of all cell-associated particles, was determined.

Microscopy

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Confocal images were taken using a PerkinElmer UltraView VoX Spinning Disc Confocal system mounted on a Nikon Eclipse Ti-E microscope equipped with a Hamamatsu C9100-50 EMCCD camera, a 100× (1.4 NA) PlanApo objective, and controlled by Volocity software. Images for protein localization were taken using a Leica TCS SP8 laser scanning confocal microscope with an HC Pl APO 63×/1.4 NA oil CS2 objective at Upstate/Leica Center of Excellence for Advanced Light Microscopy. LLSM images were prone to image artifacts (Figure 1—figure supplement 1), and therefore only confocal image data was used for accurate force analysis.

The LLSM (Chen et al., 2014) utilized was developed by E Betzig and operated/maintained in the Advanced Imaging Center at the Howard Hughes Medical Institute Janelia Research Campus (Ashburn, VA); 488, 560, or 642 nm diode lasers (MPB Communications) were operated between 40 and 60 mW initial power, with 20–50% acousto-optic tunable filter transmittance. The microscope was equipped with a Special Optics 0.65 NA/3.75 mm water dipping lens, excitation objective, and a Nikon CFI Apo LWD 25 × 1.1 NA water dipping collection objective, which used a 500 mm focal length tube lens. Live cells were imaged in a 37°C-heated, water-coupled bath in FluoroBrite medium (Thermo Scientific) with 0–5% FBS and Pen/Strep. Opsonized DAAMPs were added directly to the media bath prior to acquisition. Images were acquired with a Hamamatsu Orca Flash 4.0 V2 sCMOS cameras in custom-written LabView Software. Post-image deskewing and deconvolution was performed using HHMI Janelia custom software and 10 iterations of the Richardson-Lucy algorithm.

Microparticle 3D shape reconstruction

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Image analysis was performed with custom software in MATLAB, similar to as described previously (Vorselen et al., 2020b). Briefly, images were thresholded to estimate the volume and centroid of individual microparticles. Cubic interpolation was then used to calculate the intensity values along lines originating from the particle centroid and crossing the particle edge. Edge coordinates were then directly localized with super-resolution accuracy by fitting a Gaussian to the discrete derivative of these line profiles. This is significantly faster than using pre-processing of the image stacks with the 3D Sobel operator as used previously (Vorselen et al., 2020b). Particle properties, such as sphericity, relative elongation and surface curvature, as well as traction forces, are all sensitive to high-frequency noise, so before further calculations edge coordinates were smoothed. To this end, great circle distances (d) between edge coordinates with indices i and j were first calculated along a perfect sphere: d=arccos(sinθisinθj+cosθicosθjcos(φiφj))R, where R is the equivalent radius of a sphere to the particle. Smoothing was then performed by averaging the radial component of the edge coordinates within the given window size (1 μm2), which is similar to a 2D moving average, but adapted for a spherical surface. A triangulation between edge coordinates was then generated, and the particle surface area S and volume V calculated. Sphericity was calculated as Ψ = (6π1/3V2/3S–1). For surface curvature calculations, first principal curvatures (k1 and k2) of the triangulated mesh were determined as described previously (Ben Shabat and Fischer, 2015; Rusinkiewicz, 2004). The mean curvature was calculated H = (k1 + k2)/2.

Force analysis

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Force calculations were performed using the fast spherical harmonics method within custom Python package ShElastic as described in detail previously (Vorselen et al., 2020b; Wang et al., 2019). Briefly, to derive both normal and shear forces, we solve the inverse problem of inferring the traction forces T in an iterative process. We start with a trial displacement field u, and during optimization the observed particle shape is always matched exactly, while the following cost function is minimized:

(1) f(u)=Eel+αR2(T;Ωt)+βEpen(T)

where RT;Ωt are the cellular forces exerted outside of the cell-target contact area, which is obtained from fluorescent actin and immunostaining (Figure 2—figure supplement 1), Eel is the elastic energy included in this minimization algorithm to penalize unphysical solutions were higher forces producing the same shape, and βEpen is an anti-aliasing term. The weighing parameter α for residual traction outside of the cell-target contact area and β for anti-aliasing were both 1. Similar to most TFM methods, polyacrylamide gels were assumed to be linearly elastic in the small strain regime (ε ≲ 0.1) in which force measurements were made. Tractions were calculated with spherical harmonic coefficients up to lmax = 20, and evaluated on a 21 × 41 grid.

Fluorescent mapping on particle surface and determination of fraction engulfed

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Mapping of fluorescent proteins, phalloidin, and immunostaining to the particle surface was done by determining of the fluorescent intensity along radial lines originating from the particle centroid and passing through each edge coordinate (Figure 1c). Linear interpolation was used to determine the intensity along each line, and the maximum value within a 1 μm distance of the edge coordinate was projected onto the surface. The calculation of the fraction engulfed, alignment of particles using the centroid of the contact area, and obtaining of a stress-free boundary for force calculations was done as described previously (Vorselen et al., 2020b), with the exception that here both the phalloidin stain and the immunostaining of the free particle surface were used to determine the mask (Figure 2—figure supplement 1). For LLSM data, where no staining of the free particle surface was present, alignment was done manually.

Indentation simulations

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Indentation simulations of teeth on a spherical target particle were based on the Hertz contact model (Johnson, 1985). Parameters of the model were estimated from experimental data: the undeformed radius of the target particle was set at Rtarget=3.7μm; the teeth are considered as rigid spherical indenters with radius Rteeth0.5μm; and 10 teeth were simulated for each target, which were equally distributed around the equator of the target sphere (Figure 4—figure supplement 2). The force F and the contact area radius a produced by indentation to absolute depth d for each individual indenter were then evaluated:

F=43ER12d32,a=3FR4E,3

where the effective Young’s modulus E and effective radius R are

1E=1-νtooth2Etooth+1-νtarget2Etarget=34Etarget,1R=1Rteeth+1Rtarget

given that the target particle is near incompressible (νtarget=0.5) and the teeth are rigid compared to the target (Etooth>> Etarget). Considering non-friction contact, the force distribution on the target sphere in the contact area of each tooth can be written as:

pr=p01-ra2,p0=3F2πa2,r0,a,

where r is the radius to the initial contact point, and p0 is the maximum pressure on the contact plane. Given the resulting traction force map Tθ,φ as the boundary condition on the target sphere surface, we solved the elasticity problem, and obtained the displacement map uθ,φ using our ShElastic package (Figure 4—figure supplement 2; Wang et al., 2019). The θ,φ map on the spherical surface has the size of 61 × 121, which is defined by Gauss-Legendre quadrature (Wieczorek and Meschede, 2018). Simulations were carried out for a range of tooth radii Rteeth and absolute depth d to obtain the effective tooth depth and the average constriction along the equator (Figure 4—figure supplement 2), which are directly comparable with experimentally obtained data.

Data availability

All quantitative data generated or analysed during this study are included in the manuscript and supporting files. Source Data files contain all numerical data to generate the figures. All confocal image data is available on FigShare repositories (https://doi.org/10.6084/m9.figshare.16666864 and https://doi.org/10.6084/m9.figshare.16677373).

References

  1. Book
    1. Johnson KL
    (1985) Contact Mechanics
    Cambridge, UK: Cambridge University Press.
    https://doi.org/10.1017/CBO9781139171731
    1. Swanson JA
    2. Johnson MT
    3. Beningo K
    4. Post P
    5. Mooseker M
    6. Araki N
    (1999)
    A contractile activity that closes phagosomes in macrophages
    Journal of Cell Science 112 (Pt 3):307–316.

Decision letter

  1. Pekka Lappalainen
    Reviewing Editor; University of Helsinki, Finland
  2. Suzanne R Pfeffer
    Senior Editor; Stanford University School of Medicine, United States
  3. Pekka Lappalainen
    Reviewer; University of Helsinki, Finland
  4. Renaud Poincloux
    Reviewer; Institute de Pharmacologie et de Biologie Structurale (IPBS), France

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Pekka Lappalainen as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Renaud Poincloux (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. The data obtained by using pharmacological inhibitors (CK666, SMIFH2 and blebbistatin) are not particularly convincing. The authors should at minimum confirm that the compounds efficiently inhibited Arp2/3 and myosin II with the concentrations used here, as well as carry out phagocytosis tests to see the effects of these compounds for engulfment of particles. Ideally, the phagocytosis experiments would be done by using both soft and rigid particles to gain insight into possible different contributions of the 'teeth' and actomyosin ring in phagocytosis of particles of various stiffness. Finally, the SMIFH2 data should be removed (or moved to supplements), because recent studies demonstrated that this inhibitor is not specific for formins.

2. Because the authors almost exclusively use a single macrophage cell line (RAW cells), some key experiments should be repeated with macrophages derived from blood monocytes.

3. The authors should better discuss their results relative to published literature.

Reviewer #1 (Recommendations for the authors):

1. The authors state that the actin-based protrusive 'teeth' are mechanosensitive. However, they apparently only examined the numbers of phagocytic cups with 'teeth' when macrophages were challenged with DAAMPs of 1.4 kPa or 6.5 kPa (Figure 4i). To strengthen this conclusion, a much more thorough comparison of phagocytosis of soft vs. stiff (1.4 kPa vs. 6.5 kPa) microparticles would be informative. The authors should at minimum examine the intensities of F-actin, WASP and Arp2/3 in teeth on soft vs. stiff microparticles, and compare the compressive stresses (assuming that this is feasible also when using 6.5 kPa microparticles).

2. Many conclusions of the study are based on the use of Arp2/3, myosin II, and formin inhibitors. However, from the data presented, it remains unclear how efficient the inhibitor treatments were (i.e. did the inhibitors completely disrupt Arp2/3 and myosin II localizations in the treated cells). Moreover, the authors should be aware that SMIFH2 also inhibits myosins (Nishimura et al., 2021), and this should be taken into account when interpreting the results obtained by using this inhibitor.

3. From the data presented in the manuscript, the roles of Arp2/3 and myosin II for Fc receptor -mediated phagocytosis remain somewhat elusive. Thus, it would be informative to study the effects of Arp2/3 and myosin II inhibitions on the kinetics and efficiency of phagocytosis. Now the authors apparently only analyzed the effects of these inhibitors on the distribution of cup stages.

Reviewer #2 (Recommendations for the authors):

1. The first two figures describing the system and presenting the initial findings are outstanding.

2. Although the mechanical role of cargo has been studied a bit by earlier work, the carefulness of the preparation of the particles and their characterization is excellent.

3. I was a little disappointed that they elected to almost exclusively use a single macrophage cell line (RAW cells), when experiments with cells closer to primary macrophages are quite doable and would have been more convincing and relevant to physiological phagocytosis.

4. If the authors were to investigate the role of myo1e and 1F, I think that would improve the paper and make up for some of the deficiencies of the pharmacological treatments.

5. The meal sharing and pooping stuff is cute, but kind of phenomenological and disconnected from the rest of the paper.

As stated above, clean up the pharmacological stuff in Figure 3 and discuss the results relative to published literature in a more complete and nuanced way, I think this is a great paper.

Reviewer #3 (Recommendations for the authors):

The description of podosome-like structures in phagocytosis should also be discussed in more detail. Podosome-like structures have already been observed at phagocytic cups (Allen and Aderem A, 1996; Labrousse A. et al., 2011; Ostrowski et al., 2019). Recently they have also been observed on closed phagosomes containing IgG-coated beads but not on phagosomes containing naked beads (Tertrais M et al., 2021). Additionally, actin flashes have been described on of mature phagosomes in RAW 264.7 macrophages ingesting IgG-coated particles (Liebl and Griffiths G, 2009). In contrast, Poirier et al., 2020, proposed that actin flashes form at phagosomes only when phagocytosis is mediated by complement receptors but not by Fc receptors (Poirier et al., 2020). The function of Arp2/3 for the formation of actin flashes and podosome like structures has also been started to be documented in these articles. As the difference in phenotype might arise from differences in cell models (primary macrophages vs macrophage cell lines), I would suggest testing whether human macrophages derived from blood monocytes also form force-generating teeth on both phagocytic cups and closed phagosomes and examine whether they are also Arp2/3 dependent.

The introduction and Discussion sections could also be strengthened by introducing and commenting on the work of Sergio Grinstein (Freeman et al., Cell 2016 and 2018, in addition to Ostrowski et al., Dev Cell 2019, already cited).

Line 52. "Podosomes are… capable of generating traction forces". The work from Labernadie et al., Nat. Commun. 2014, Proag et al., ACS Nano 2015 and Bouissou et al., ACS Nano 2017 argue that podosomes are capable of generating both traction and protrusion forces, and not only traction forces.

Lines 70-71. "TFM only measures forces tangential to the target surface". There are many studies using TFM that also consider the normal forces applied to the surface.

Line 99. "1.4 or 6.5 kPa". Why was this range of stiffness chosen? Is it comparable to the stiffness of more physiological particles (bacteria, apoptotic bodies)?

Line 103. "Interestingly…" This result is presented as a new result but similar observations were made in Vorselen et al., Nat. Commun. 2020. I suggest the authors to clarify the difference with their previous observations.

Line 109. "…400 nm maximum target constriction". This is a large constriction, given the size of the particle. How was this value evaluated? And for such a deformation, is the method of evaluating the normal and shear stresses from the deformation of the particle still applicable? What is the constriction limit above which it is no longer applicable?

Lines 111-112. "0.5 kPa" and "1.5kPa". I suggest mentioning that these are values of shear stresses, to avoid confusion with the Young's moduli of the particles.

Lines 129. "The deformation patterns… were similar to those observed in living cells". I am surprised that chemical fixation and the difference in imaging do not alter the assessment of the forces. I suggest to specify how the resolution (LLSM vs confocal) impacts the force evaluation (near the equatorial plane and at the upper apex), and present side by side the distribution of the deformations observed in live and fixed samples.

Lines 136 and 138. "Compressive forces" and "shear forces". The deduction of forces from the particle deformation involves a model and assumptions. In this case, it is not known precisely where the forces are applied, on which surface, in which direction… I therefore suggest the authors to detail and justify the assumptions that have been chosen for this assessment.

Lines 222-225. Are the forces of similar amplitude in these different cells?

Lines 231-236. The partial reduction of formation of "teeth" with CK666, SMIFH2 and Blebbistatin contrasts with the literature describing a complete inhibition of podosomes with CK666 and very slight effects on podosome formation by SMIFH2 and Blebbistatin. I suggest the author to discuss this interesting discrepancy. Still concerning these treatments, did the authors verify their effect on podosomes? And has their effect on the rate of phagocytosis quantified?

Lines 272-275. "Elasticity theory simulations…" Bouissou et al., argued that there is a local balance of forces at the podosome level (central protrusion and peripheral traction), and not only a protrusion force. Given the similarities of teeth to podosomes, it would be interesting to also consider a similar model of force application.

Lines 299-300. Formins are reputed difficult to localize. Were the authors able to observe expected localizations for these formins, e.g. at filopodia? This lack of localization may be inconsistent with the effect of SMIFH2. Could the authors comment?

Lines 302-304. The localizations of paxillin and vinculin are difficult to interpret and seem different from the vinculin localization observed by Ostrowsky et al., One difference may be the model (mouse macrophages vs human macrophages). Could the authors comment on this and/or try to localize these proteins in the phagocytic cup of human macrophages?

Lines 531-547. Were all cells considered or only those with a single phagocytosed particle?

Line 580. Please specify how much smoothing is done.

Lines 584-585. Why are the coordinates smoothed again? Is it for aesthetic reasons? And what is the impact on the evaluation of forces? Could the authors explain?

Figure 4. The authors claim that actin based teeth are interconnected, but it seems to me that this is not yet proven.

Figure 7. Given the similarities with podosomes, could local traction forces also be considered in this model?

Supp. Figure 1j. Could the authors determine an objective criterion to qualify an image quality appropriate or insufficient?

Supp.Figure 2-5. Would it be possible to average the three types of maps (deformation/actin/normalized stain), after classification, to perhaps allow easy and visual comparison of the effects of the treatments?

Supp. Figure 7-8 "physical properties" is imprecise. What do you mean exactly? F-actin intensity? Size? Please clarify.

https://doi.org/10.7554/eLife.68627.sa1

Author response

Essential revisions:

1. The data obtained by using pharmacological inhibitors (CK666, SMIFH2 and blebbistatin) are not particularly convincing. The authors should at minimum confirm that the compounds efficiently inhibited Arp2/3 and myosin II with the concentrations used here, as well as carry out phagocytosis tests to see the effects of these compounds for engulfment of particles.

We carried out new experiments to investigate the effect of CK666, blebbistatin and SMIFH2 on phagocytic efficiency, which we now present in a new figure panel (figure 3g). The new experiments supplement our findings regarding the precise effects of these inhibitors on the cytoskeletal reorganization and force production during phagocytosis with the additional functional information.

As detailed in our response below, we believe that the extremely strong effects on force production during phagocytosis following the use of CK666 and blebbistatin treatment, described in the previous version of the manuscript, provide clear quantitative evidence of the efficacy of these treatments. We observe almost complete disruption of target deformation upon Arp2/3 treatment, and complete disruption of target constriction upon blebbistatin treatment (Figure 3 b-d,h,i). Moreover, we see additional effects of these drug treatments on actin localization (Figure 3 d-f, Figure 4 d). Overall, we are not concerned that these compounds are failing to inhibit their known targets, because they clearly produce strong, specific, and distinct effects on particle deformations.

Ideally, the phagocytosis experiments would be done by using both soft and rigid particles to gain insight into possible different contributions of the 'teeth' and actomyosin ring in phagocytosis of particles of various stiffness.

As detailed in our response below, we have now performed additional experiments to more thoroughly compare phagocytosis of targets with different rigidity. We have now fully devoted figure 1 – supplement 2 to more detailed analysis of uptake of stiffer targets, and refer to our findings and this figure in various places in the text. There, we directly compared actin intensity and constriction magnitude, and discuss the forces involved in phagocytosis of soft vs. stiff beads (1.4 kPa vs. 6.5 kPa) beads. We further included the effect of drug-treatments on uptake efficiency for both softer and stiffer particles (Figure 3g and figure 1 – supplement 2f).

Finally, the SMIFH2 data should be removed (or moved to supplements), because recent studies demonstrated that this inhibitor is not specific for formins.

Although the very clear differences between direct myosin-II inhibition by blebbistatin and SMIFH2 treatment strongly suggest that our observations using the SMIFH2 treatment are not the result of off-target myosin-II inhibition by SMIFH2, we have followed the reviewer’s suggestion and moved all SMIFH2 inhibition data to the supplemental figures (Figure 3 – supplement 5), and we now mention and cite the recent study that reports off-target effects of SMIFH2.

2. Because the authors almost exclusively use a single macrophage cell line (RAW cells), some key experiments should be repeated with macrophages derived from blood monocytes.

We have performed additional experiments and analyzed additional data to include more details on uptake by primary bone marrow derived macrophages (BMDMs). We have chosen BMDM over human blood monocytes because we do not have the appropriate protocols to conduct experiments on human-derived macrophages, and we have previous experience working with primary murine macrophages derived from bone marrow.

Compared to RAW macrophages, transfecting and imaging the primary cells was non-trivial. Moreover, we observed that force exertion by primary BMDMs is lower than force exertion by RAW macrophages, as evidenced by significantly smaller target deformations. To observe meaningful deformations and derive cellular forces, we had to challenge BMDMs with targets of lower rigidity (0.3 kPa), which are taken up less efficiently (as we and others have reported, phagocytosis is a mechanosensitive process that is less efficient for lower rigidity targets). Nevertheless, we now show that uptake mediated by primary BMDMs largely follows the same mechanical progression, and that they also form teeth on such softer targets (Figure 1 – supplement 3).

In addition to these quantitative differences between RAW and primary macrophages, we also observe some qualitative differences that will require substantial additional work to elucidate (for example BMDMs appear to exert compressive forces at the base of the phagocytic cup, and target constriction doesn’t always appear right at the rim of the cup but can lag significantly behind the cup rim). We now have included a supplementary figure panel to show the typical progression of primary-macrophage mediated phagocytosis (Figure 1 – supplement 3), and briefly mention the similarities and differences in the main text:

“To determine whether this behavior was also present in primary macrophages, we also imaged bone marrow-derived macrophages transfected with mEmerald-Lifeact and observed a similar mechanical progression, albeit with smaller deformations (Figure 1 – supplement 3).”

We believe a more in-depth analysis of primary cells would be best presented in a subsequent manuscript given the already substantial amount of data and new analysis in the current submission.

3. The authors should better discuss their results relative to published literature.

As detailed in our response to specific reviewer comments, we have expanded our introduction and Discussion sections to better discuss our results in light of other published literature, including a more thorough discussion of the similarities and differences between actin teeth and podosomes as well as the role of the Arp2/3 complex and myosin-II in phagocytosis. All changes compared to the original version have been emphasized in blue in the related manuscript file submitted along with the revised manuscript.

Reviewer #1 (Recommendations for the authors):

1. The authors state that the actin-based protrusive 'teeth' are mechanosensitive. However, they apparently only examined the numbers of phagocytic cups with 'teeth' when macrophages were challenged with DAAMPs of 1.4 kPa or 6.5 kPa (Figure 4i). To strengthen this conclusion, a much more thorough comparison of phagocytosis of soft vs. stiff (1.4 kPa vs. 6.5 kPa) microparticles would be informative. The authors should at minimum examine the intensities of F-actin, WASP and Arp2/3 in teeth on soft vs. stiff microparticles, and compare the compressive stresses (assuming that this is feasible also when using 6.5 kPa microparticles).

We thank the Reviewer for this helpful suggestion. We have now performed additional experiments to more thoroughly compare phagocytosis of targets varying in rigidity. We have included new figure panels that directly compare actin intensity and constriction magnitude, and discuss the forces involved in phagocytosis of soft vs. stiff beads (1.4 kPa vs. 6.5 kPa) beads. We have now fully devoted Figure 1 – supplement 2 to a more detailed analysis of uptake of stiffer targets, and refer to our findings and this figure in various places in the text. Overall, stiff (6.5 kPa) target uptake shows a qualitatively very similar signature as soft target (1.4 kPa) uptake. The target deformations are (expectedly) smaller for stiffer targets, but we also, interestingly, find less actin accumulation on stiffer targets.

We have also attempted a more systematic analysis of teeth on targets of varying rigidity. This, however, turns out to be a challenging task. We currently automatically detect teeth based on local protrusive activity (as measured by target curvature) and actin intensity (Figure 4 – supplement 1a). However, picking a comparable threshold for identification of teeth on particles of varying rigidities is non-trivial. A key difficulty here is that the stiffer targets deform much less (as now quantified in Figure 1 – supplement 2). This raises the possibility that teeth induce deformations that are below our detection limit, which would not only bias our estimate of tooth numbers, but also the indentation and force generation by the remaining detected teeth. We tested various thresholding strategies for detecting teeth on stiff targets, but this led to different overall conclusions about teeth numbers and protrusive activity. These difficulties in systematic identification and analysis of teeth also make it extremely hard for us to compare WASP and Arp2/3 intensity at teeth as requested by the reviewer. The significant cup-to-cup variation, and difference in transfection efficiency and hence stain intensity between cells, makes such measurements critically dependent on a systematic comparison between a larger number of cups and teeth. We believe that these are hurdles that we may be able to overcome but will require significant further work and therefore in our opinion best reserved for a future study. We have rewritten our paragraph on teeth mechanosensitivity to clarify this point (see pg. 10, line 299).

2. Many conclusions of the study are based on the use of Arp2/3, myosin II, and formin inhibitors. However, from the data presented, it remains unclear how efficient the inhibitor treatments were (i.e. did the inhibitors completely disrupt Arp2/3 and myosin II localizations in the treated cells). Moreover, the authors should be aware that SMIFH2 also inhibits myosins (Nishimura et al., 2021), and this should be taken into account when interpreting the results obtained by using this inhibitor.

As mentioned later in this document, we have decided to move all SMIFH2 data to the SI and note these recent findings regarding SMIFH2 on pg. 8 (line 217). Both CK666 and Blebbistatin inhibit the activity of the Arp2/3 complex and myosin II respectively; this mode of action is not expected to ”completely disrupt” target protein localization. It is therefore more common to use inhibition of formation of certain structures or force exertion as an indicator of treatment efficacy. For example, the paper in which CK-666 was initially introduced (Nolen et al., 2009) used inhibition of actin structures (podosomes specifically) as a readout.

In our study, we see extremely clear effects on force production after use of CK666 and blebbistatin treatment, which gives a very quantitative readout of the efficacy of these treatments. We observe almost complete disruption of all target deformations upon Arp2/3 treatment, and complete disruption of target constriction upon blebbistatin treatment (Figure 3 b-d,h,i). Moreover, we see additional effects of these drug treatments on actin localization (Figure 3 d-f, Figure 4 d). Altogether, we believe that these data provide very strong evidence of the efficacy of these drugs.

Furthermore, we have now examined the F-actin staining at the ventral portion of the RAW cells in our phagocytosis experiments, and similar to Nolen et al., (2009) we observe complete disassembly of podosome actin structures (marked by red squares) and a dramatic change in cell morphology (increase in spiky filopodia) upon CK666 treatment, as shown in the confocal image slices (Author response image 1).

Author response image 1

3. From the data presented in the manuscript, the roles of Arp2/3 and myosin II for Fc receptor -mediated phagocytosis remain somewhat elusive. Thus, it would be informative to study the effects of Arp2/3 and myosin II inhibitions on the kinetics and efficiency of phagocytosis. Now the authors apparently only analyzed the effects of these inhibitors on the distribution of cup stages.

We would like to point out that in addition to the effect of CK666 and Blebbistatin on the distribution of cup stages, in our initial submission we also studied the effect of these inhibitors on (1) actin localization and spatial organization in the phagocytic cup (Figure 3d-f, 4d, Figure 3 – supplement 1b, Figure 4 – supplement 1e), (2) global deformations of phagocytic targets (Figure 4d,i,j Figure 3 – supplement 1b,e), (3) cellular forces generated during phagocytosis (Figure 3a,b), and (4) how all these depend on precise stage of phagocytic progression (Figure 3i,j, Figure 3 – supplement 1c,e, Figure 4 – supplement 1g).

For this revised submission, we have now also included data on the efficiency of phagocytic uptake using the DAAM particles under drug treatments at various timepoints. This data is presented in a new figure panel (3g), Figure 3 – supplement 1d (kinetics), and 2f (stiff particles). We see a very strong inhibition of phagocytic uptake after 15 minutes by the Arp2/3 inhibitor, and no pronounced effect after myosin II inhibition. These observations hold true at later timepoints, and are also very similar when using stiffer 6.5 kPa targets. We now also discuss these results in light of the previous literature in our Discussion section.

Reviewer #2 (Recommendations for the authors):

1. The first two figures describing the system and presenting the initial findings are outstanding.

2. Although the mechanical role of cargo has been studied a bit by earlier work, the carefulness of the preparation of the particles and their characterization is excellent.

We thank the reviewer for these kind words about our work.

3. I was a little disappointed that they elected to almost exclusively use a single macrophage cell line (RAW cells), when experiments with cells closer to primary macrophages are quite doable and would have been more convincing and relevant to physiological phagocytosis.

We appreciate this criticism and have now included more information on the phagocytic behavior of murine primary bone-marrow derived macrophages (BMDM) (see new Figure 1 – supplement 3). Compared to RAW macrophages, transfecting and imaging the primary cells was non-trivial. Moreover, as evidenced by significantly smaller target deformations, force exertion by primary BMDMs is lower than force exertion by RAW cells. This results in an additional complication, which is that to observe meaningful deformations and derive cellular forces, we had to use targets of lower rigidity (0.3 kPa), which are taken up less efficiently (since as we and others have reported, phagocytosis is a mechanosensitive process that is less efficient for softer targets). Nevertheless, we now show that uptake by primary BMDMs follows a qualitatively similar mechanical progression, including formation of protrusive actin-rich teeth and overall target constriction.

4. If the authors were to investigate the role of myo1e and 1F, I think that would improve the paper and make up for some of the deficiencies of the pharmacological treatments.

Although we agree with the Reviewer that this would be very interesting, we believe that the manuscript, as is, provides sufficient new insights into the biophysics of phagocytosis, as well as the roles of specific actin structures and regulators on phagocytic progression. We worry that adding results on myosin 1e/f would be too ambitious, and feel like this would be more appropriate for a follow-up study.

5. The meal sharing and pooping stuff is cute, but kind of phenomenological and disconnected from the rest of the paper.

We agree with the reviewer that these observations are necessarily less quantitative than most of the analysis in this paper. Since these observations aren’t particularly rare (up to 30% of soft particle uptake), we believe there is value in reporting this behavior. Moreover, the relation between phagocytic force exertion and uptake efficiency is currently poorly understood, and as the reviewer pointed out, the forces generated during phagocytosis by Arp2/3-branched actin polymerization and myosin-II motor proteins could be more important depending on the context in which phagocytosis is taking place. These observations of popping and meal-sharing are an indication of how force exertion may differently affect isolated cell-target interactions than more complex cell-target encounters.

As stated above, clean up the pharmacological stuff in Figure 3 and discuss the results relative to published literature in a more complete and nuanced way, I think this is a great paper.

Reviewer #3 (Recommendations for the authors):

The description of podosome-like structures in phagocytosis should also be discussed in more detail. Podosome-like structures have already been observed at phagocytic cups (Allen and Aderem A, 1996; Labrousse A. et al., 2011; Ostrowski et al., 2019). Recently they have also been observed on closed phagosomes containing IgG-coated beads but not on phagosomes containing naked beads (Tertrais M et al., 2021). Additionally, actin flashes have been described on of mature phagosomes in RAW 264.7 macrophages ingesting IgG-coated particles (Liebl and Griffiths G, 2009). In contrast, Poirier et al., 2020, proposed that actin flashes form at phagosomes only when phagocytosis is mediated by complement receptors but not by Fc receptors (Poirier et al., 2020). The function of Arp2/3 for the formation of actin flashes and podosome like structures has also been started to be documented in these articles. As the difference in phenotype might arise from differences in cell models (primary macrophages vs macrophage cell lines), I would suggest testing whether human macrophages derived from blood monocytes also form force-generating teeth on both phagocytic cups and closed phagosomes and examine whether they are also Arp2/3 dependent.

We thank the Reviewer for this interesting comment. The frequency or characteristics of actin teeth may well differ among phagocyte cell types, however we would like to point out that we already tested and observed their presence in human neutrophil-like cells and murine primary bone marrow derived macrophages as well as primary dendritic cells during phagocytosis. To address the reviewer’s comments, we have added more data on force generation, evaluated as target deformations, induced by actin teeth in primary murine bone marrow derived macrophages (BMDMs) during live cell lattice light sheet imaging (new figure 1 – supplement 3). Although there are quantitative differences between BMDM and RAW cell force exertion (BMDMs generally induce less target deformation than RAW cells), the qualitative signature, including overall target constriction and observation of distinct actin-rich puncta associated with local protrusive activity, appears very similar. The potential difference between human and murine primary phagocyte behavior is an interesting question, especially given the known differences among these cell types with respect to podosome formation. Because we do not have the approved protocols to conduct experiments with human blood-derived monocytes, potential comparisons will have to be addressed in a future study.

We now have also expanded our discussion of previous observations of podosome-like structures in phagocytosis, including the work by Tertrais et al., (2021). The idea to look at actin structures on closed phagosomes is very interesting. As the reviewer points out, actin flashes have been observed around phagocytic targets coated with different ligands, including bacteria like Listeria. These actin flashes may also be Arp2/3-mediated, and may, as the work by Tetrais et al., reports, contain podosome like structures. In addition, we have previously seen distinct deformations of targets after completion of engulfment (Vorselen et al., 2020). These deformations far exceeded the deformations caused by phagocytic teeth and originated from the site of cup closure. Together, to our knowledge there are still many unknowns around the regulation and dynamics of actin structures on closed phagosomes, and how this depends on target properties and engulfment mechanisms. Albeit very interesting, going into the actin dynamics associated with relocation and maturation of phagosomes and the potential role of teeth or podosome-like structures in that process, would, in our opinion, warrant an entire study by itself.

The introduction and Discussion sections could also be strengthened by introducing and commenting on the work of Sergio Grinstein (Freeman et al., Cell 2016 and 2018, in addition to Ostrowski et al., Dev Cell 2019, already cited).

We have now rewritten the introduction and Discussion sections to more fully comment on this past literature, including the suggested works. Thank you for this suggestion.

Line 52. "Podosomes are… capable of generating traction forces". The work from Labernadie et al., Nat. Commun. 2014, Proag et al., ACS Nano 2015 and Bouissou et al., ACS Nano 2017 argue that podosomes are capable of generating both traction and protrusion forces, and not only traction forces.

We have removed the word traction from this sentence. We now also discuss the similarities and potential differences between actin tooth and podosome force exertion in our Discussion section.

Lines 70-71. "TFM only measures forces tangential to the target surface". There are many studies using TFM that also consider the normal forces applied to the surface.

We thank the reviewer for pointing out this mistake. We fully agree and are aware of applications of TFM that do measure normal forces. We have adapted this sentence to reflect this: “Moreover, in its most common application, TFM only measures forces tangential to the target surface, neglecting forces normal to the target surface, which may be critical.”

Line 99. "1.4 or 6.5 kPa". Why was this range of stiffness chosen? Is it comparable to the stiffness of more physiological particles (bacteria, apoptotic bodies)?

The stiffness range chosen is similar to healthy tissue cells as well as apoptotic cells. We added the following to clarify this:

“RAW macrophages were fed DAAMPs with a diameter of 9 µm and a Young’s modulus of 1.4 or 6.5 kPa, which is in the same range as tissue cells and physiological targets of macrophages like apoptotic cells34,35. Raw macrophages typically formed a chalice-shaped phagocytic cup and completed phagocytosis in similar timeframe (~3 minutes) as previously reported for much stiffer (2-3 GPa) polystyrene particles36 (Figure 1a, Video 1, Figure 1 – supplement 1e).”

In addition, there are also practical reasons to use relatively soft particles because deformations on much stiffer particles are not well observed and hence forces cannot be inferred.

Line 103. "Interestingly…" This result is presented as a new result but similar observations were made in Vorselen et al., Nat. Commun. 2020. I suggest the authors to clarify the difference with their previous observations.

Our previous work was mostly focused on establishing the microparticle traction force technology. Indeed, a similar observation was made as those described here, but the previous measurements were of a single phagocytic cup on a fixed cell. While we believe that this current description as a common feature of phagocytosis is still novel, we have moved the word “Interestingly” to the more novel discovery of the teeth movement by live-cell imaging. We have also included the following note in this section: “While these deformations were more apparent using the softer 1.4 kPa DAAMPs, the same force pattern was observed using stiffer 6.5 kPa targets (Figure 1 – supplement 2a-b, Video 3) and previously using 0.3 kPa targets using J774 macrophages.”

Line 109. "…400 nm maximum target constriction". This is a large constriction, given the size of the particle. How was this value evaluated? And for such a deformation, is the method of evaluating the normal and shear stresses from the deformation of the particle still applicable? What is the constriction limit above which it is no longer applicable?

We thank the reviewer for this comment and are aware of the limitations of our methodology, which assumes linear elastic particle behavior, for very large deformations, where this assumption is likely to fail.

Firstly, we added a clarification to figure 1 – supplement 1c, which contains the data on maximum target constriction, as to how this value was determined:

“. For each video, the single timepoint in which target constriction was maximal was identified, and the exact constriction value was determined as described in Figure 3 – supplement 1a.”

A typical rule of thumb is that linear elasticity can be assumed for strains of up to 0.1. In our case, a maximum constriction of 400 nm, would amount to a strain of 0.09: still within this range. In addition, this is the constriction in a single frame with maximal constriction selected from a video, and the vast majority of images captured in fixed cell data (which were exclusively used for force calculations) will hence have considerably lower deformation. The constriction values observed in fixed cell data are presented in Figure 2i, and typically ~200 nm (strains < 0.05), which is well within the limits were particle behavior can be reasonably assumed to be linearly elastic. We added a clarification to our methods section regarding this assumption:

“Similar to most TFM methods, polyacrylamide gels were assumed to be linearly elastic in the small strain regime (ε ≲ 0.1) in which force measurements were made.”

Lines 111-112. "0.5 kPa" and "1.5kPa". I suggest mentioning that these are values of shear stresses, to avoid confusion with the Young's moduli of the particles.

The stresses mentioned on line 111 and 112 (now line 142 and 143) are not shear stresses but bulk compressive stresses. Our methodology allows tracking the overall change of particle volume and combined with measuring the bulk modulus of the particles (which was done previously, Vorselen et al., 2020) allows determining bulk compressive stresses. To clarify this point, we changed the wording of this sentence: “In addition, we observed bulk compressive stresses during phagocytic cup progression (~ 0.5 kPa) and after complete internalization of the DAAMPs (up to 1.5 kPa), leading to a dramatic reduction in DAAMP diameter (Figure 1 – supplement 1e-f).”

Lines 129. "The deformation patterns… were similar to those observed in living cells". I am surprised that chemical fixation and the difference in imaging do not alter the assessment of the forces. I suggest to specify how the resolution (LLSM vs confocal) impacts the force evaluation (near the equatorial plane and at the upper apex), and present side by side the distribution of the deformations observed in live and fixed samples.

We thank the reviewer for this comment. We would like to note that the difference in particle reconstruction, and later force evaluation, is not only dependent on resolution, and instead relies on many factors, including signal-to-noise ratio (as detailed in Vorselen et al., 2020) and potential imaging artifacts, as observed in the majority of the lattice light sheet data presented here. Specifically imaging artifacts, which can result in large apparent target deformations outside the cell-target contact area, can make it impossible to converge to a solution when solving the elasticity theory problem, which is the reason we did not include any force calculations based on the LLSM data.

As illustrated in figure 2 – supplement 1b, there is also significant particle-to-particle variation, so a comparison between the live cell data and fixed cell data needs to be based on a systematic analysis. This is why we choose, next to qualitative comparison of the deformation patterns, to quantitatively compare target constriction at ~50% engulfment in both live and fixed cells (Figure 1 – supplement 1D). This is a relatively straight-forward deformation to measure quantitatively, and revealed 250 nm average target constriction in the live-cell data and a ~40% lower 180 nm average constriction in the fixed-cell data. We have updated the main text, and the supplemental figure to make this comparison clearer:

“The deformation patterns, although slightly lower in magnitude for the fixed samples (Figure 1 – supplement 1), were similar to those observed in living cells by the LLSM imaging (Figure 2b, Figure 1 – supplement 1).”

“d, Average target constriction at 50% engulfment. Average is 0.25 ± 0.04 μm (s.e.m., n = 23 cups), which is ~40% higher than observed with fixed cell data at similar stage at 0.18 ± 0.02 μm (s.e.m., n = 19) (main text figure 2i). This indicates partial relaxation of the particle to a more spherical shape during fixation, and suggests that the fixed cell force measurements are a slight underestimate of the real phagocytic forces.”

We also added in the caption of figure 1 – supplement 1h:

“Shape reconstruction in MP-TFM is critically dependent on particle edge localization detection, which shows clear irregularities (yellow arrows) because of the striping artifact. Such artifacts result in large apparent particle deformations inside and outside the cell-target contact area, and can make it impossible to converge on a solution when solving the elasticity theory problem to infer tractions from target deformations. Because of this, we only used the confocal image data for force analysis.”

Lines 136 and 138. "Compressive forces" and "shear forces". The deduction of forces from the particle deformation involves a model and assumptions. In this case, it is not known precisely where the forces are applied, on which surface, in which direction… I therefore suggest the authors to detail and justify the assumptions that have been chosen for this assessment.

To make our methodology and assumptions clearer, we have added a section in the methods (Force analysis) to give more detail on the methodology and assumptions that go into it. We also added a note on previous line 136, now line 164, (“see methods”). We do not make any explicit assumptions about the direction and location where the cell applies forces, other than that we restrict them to the cell-target contact area, which is determined from F-actin and exposed particle immunostaining (Figure 2 – supplement 1).

“Force calculations were performed using the spherical harmonics method within custom Python package ShElastic as described in detail previously21,66. Briefly, to derive forces, we solve the inverse problem of inferring the tractions in an iterative process to closely match the observed particle shapes and by limiting cellular force exertion to the cell-target contact area as obtained from fluorescent phalloidin and exposed particle immunostaining. In addition, the elastic energy is included in this minimization algorithm to penalize unphysical solutions where higher forces produce the same shape. Similar to most TFM methods, polyacrylamide gels were assumed to be linearly elastic in the small strain regime (ε ≲ 0.1) in which force measurements were made. Tractions were calculated with spherical harmonic coefficients up to lmax = 20, and evaluated on a 21 × 41 grid. Chosen values for the weighing parameter a for residual traction outside of the cell-target contact area and b for anti-aliasing were both 1.”

For a more in-depth discussion of the methodology, we also refer to our previous work (Vorselen et al., 2020), in which the methodology is described in much more detail and its performance evaluated using test cases.

Lines 222-225. Are the forces of similar amplitude in these different cells?

The force analysis suggested by the reviewer would be very interesting. However, such an analysis would require larger data sets and careful analysis. As observed in the RAW cell data, we see significant cell-to-cell variation, and dependence on other factors, such as cup stage, of force exertion. Moreover, we currently do not know if there are differences in the mechanical progression of phagocytosis depending on the cell type, beyond tooth involvement. Given that such differences are likely to occur, and given that it is very challenging to decouple force exertion by various cytoskeletal structures (teeth and the contractile ring, for example), it is non-trivial to make direct comparisons between force exertion by teeth between these cell lines. We believe that a comparison of force exertion between different cell types requires an in-depth biophysical comparison between phagocytosis by these cell types, and is beyond the scope of the current manuscript, but would be extremely interesting for future studies.

Lines 231-236. The partial reduction of formation of "teeth" with CK666, SMIFH2 and Blebbistatin contrasts with the literature describing a complete inhibition of podosomes with CK666 and very slight effects on podosome formation by SMIFH2 and Blebbistatin. I suggest the author to discuss this interesting discrepancy. Still concerning these treatments, did the authors verify their effect on podosomes? And has their effect on the rate of phagocytosis quantified?

We have moved all SMIFH2 data to supplemental figures, given the recent report of off-target effects of SMIFH2 treatment, so in our response to this comment we focus on the effect of CK666 and Blebbistatin treatment. Firstly, we note that the reduction of tooth numbers in our experiments is larger for CK666 treatment than Blebbistatin treatment, with the number of teeth is close to 0 for CK666 treatment. Of note, the concentration of CK-666 used in this study inhibits all podosome-like actin structures on the ventral side of RAW macrophages (see earlier Figure).

Differences in findings between our tooth quantification and previous podosome quantification are likely at least partially based on the method of identification of teeth/podosomes. We use a combined metric based on both protrusive activity and F-actin intensity. This is necessary, because at the location of the teeth (the front of the phagocytic cup) actin itself is already strongly enriched. Given these different ways of identification of actin structures, but also potential other differences between these experiments, we are hesitant to make direct quantitative comparisons between these studies.

Nevertheless, we now address the effect of Blebbistatin treatment on podosomes and teeth in our Discussion section. Given that we use protrusive activity in tooth identification, we believe our results are consistent with previous reports on podosome activity, which we now note in the manuscript:

“Unexpectedly, myosin-II motor inhibition also reduces tooth number and size, albeit to a lesser extent than Arp2/3 inhibition, and also alters tooth spatial distribution (Figure 4 – supplement 1). Similarly, inhibition of myosin-II in studies on podosomes cause a reduction in podosome stiffness and protrusive activity18,58.”

In addition, actin teeth and podosomes may have significant differences, which we now also discuss more thoroughly in our Discussion sections paragraph starting at line 441, (page 15). Finally, we have performed additional experiments to investigate the effect of these inhibitors on phagocytic efficiency, and we present this data in new figure panels 3g and Figure 3 – supplement 1d. In these experiments we find that the CK-666 treatment has a much more dramatic effect on the phagocytic uptake than the Blebbistatin or SMIFH2 treatments.

Lines 272-275. "Elasticity theory simulations…" Bouissou et al., argued that there is a local balance of forces at the podosome level (central protrusion and peripheral traction), and not only a protrusion force. Given the similarities of teeth to podosomes, it would be interesting to also consider a similar model of force application.

We thank the Reviewer for bringing up this very interesting point. In the work by Bouissou et al., the argument for the local balance of forces at the podosome level is, at least in part, based on the observation that local bulges around each podosome, instead of a large-scale bulge, is only expected if the protrusive forces are locally balanced around each podosome. We believe that this points to an interesting difference with our observations in phagocytosis. As we show in Figure 4j, we see higher, not less, large-scale deformation than expected by the protrusive activity of teeth alone. This suggests that there may be significant differences in the force balance between podosomes and phagocytic teeth. We now discuss this interesting difference and its implications in the Discussion section (lines 454-459). We also expanded our discussion of the more general similarities and differences between podosomes and phagocytic teeth.

Lines 299-300. Formins are reputed difficult to localize. Were the authors able to observe expected localizations for these formins, e.g. at filopodia? This lack of localization may be inconsistent with the effect of SMIFH2. Could the authors comment?

We agree with the Reviewer on this point of inconsistency. We have no reason to believe that the formin constructs we used in this manuscript were unfit for localization studies. To note, mDia1, but not mDia2, has been observed to localize to the phagocytic cups during complement-mediated phagocytosis (Jaumouille et al., 2019). mDia2 and FHOD1 have been shown not to localize to the actin cores of macrophage podosomes. We believe formins such as INF2 or FMNL1, which have been described in macrophage podosomes, are likely to localize to the actin teeth and thus may play a role in actin nucleation. However, to avoid any confusion on this topic, we have removed the mDia2 and FHOD1 localization figures and moved all SMIFH2 data to the SI (Figure 3 – supplement 5).

Lines 302-304. The localizations of paxillin and vinculin are difficult to interpret and seem different from the vinculin localization observed by Ostrowsky et al., One difference may be the model (mouse macrophages vs human macrophages). Could the authors comment on this and/or try to localize these proteins in the phagocytic cup of human macrophages?

There may be many reasons for differences in localization observed between our experiments and those by Ostrowski et al., including cell type (mouse vs. human), but also target rigidity (1.3 kPa DAAMP vs. > 1 GPa polystyrene bead), as well as antibody choice and imaging modality (confocal vs. STED). Moreover, we note that the majority of the work done in the manuscript by Ostrowski was in frustrated phagocytosis assays, and the ring-like distribution of adhesion proteins around an actin-core typically associated with podosomes was much less clear in their assays with spherical targets, which could be due to either technical differences in imaging, or that the structures that are formed during frustrated phagocytosis assays are not identical to those formed during phagocytosis of a target with more physiological curvature. We believe that the results of their localization experiments on spherical targets are more in line with our results. To address this Reviewer’s concern, we have replaced the confocal image of vinculin recruitment in the SI with one that is hopefully clearer in illustrating this point (Figure 5 – Supplement 1c), and now comment on the comparison of the protein distributions we observe with those reported for podosomes and in phagocytosis previously in the Discussion section (lines 441-452).

Lines 531-547. Were all cells considered or only those with a single phagocytosed particle?

To address this point, we added the following brief clarification in our “Phagocytic assays” section of the methods:

“For both live and fixed cell assays, cells were randomly selected for imaging, and all cells were considered, including those having already internalized a particle and those interacting with multiple particles.”

Line 580. Please specify how much smoothing is done.

Please see our reply on line 584-585 below.

Lines 584-585. Why are the coordinates smoothed again? Is it for aesthetic reasons? And what is the impact on the evaluation of forces? Could the authors explain?

We thank the reviewer for their careful attention to the methods section. We now realize this section was unclear and have adapted the wording to clarify the following: only a single smoothing step of the edge coordinates was performed, and original line 580 and 584-584 were referring to a single procedure, where lines 584-585 were detailing the method of smoothing and amount of smoothing performed. We rewrote this section to make this clearer:

“Particle properties, such as sphericity, relative elongation and surface curvature, as well as traction forces, are all sensitive to high-frequency noise, so before further calculations edge coordinates were smoothed. To this end, great circle distances (d) between edge coordinates with indices i and j were first calculated along a perfect sphere: d= arccos(sinθisinθj+ cosθicosθjcos(φiφj))R, where R is the equivalent radius of a sphere to the particle. Smoothing was then performed by averaging the radial component of the edge coordinates within the given window size (1 µm2), which is similar to a 2D moving average, but adapted for a spherical surface.”

More generally, calculation of sphericity, relative elongation, surface curvature and traction forces are all sensitive to high frequency noise. For the latter two, this is largely due to the fact that the equivalent of a derivative is used in these calculations, where taking a discrete derivative of noisy data is bound to amplifying the noise. In addition, for computational efficiency traction force calculations are done using spherical harmonic coefficients up to lmax = 20 on a 21 x 41 grid. This uses only ~900 points, which is fewer than the ~5000 vertices determined in the original particle shape, and smoothing helps to better reflect the somewhat larger scale deformations of the particle surface.

Smoothing also helps for accurate calculation of relative elongation. This metric (determined by taking the ratio of the length of the phagocytic axis to the average of the two orthogonal axes), is prone to individual outlying vertices. Sphericity is also highly affected by high frequency contributions as those strongly affect the surface area calculation but have minimal effect on the volume.

We believe that an in depth discussion of these factors goes beyond the scope of this paper, but we have added a short clarification (see the section above). In addition, we added the clarification that force calculations were done up using spherical harmonic coefficients up to lmax = 20 on a 21 x 41 grid.

Figure 4. The authors claim that actin based teeth are interconnected, but it seems to me that this is not yet proven.

We agree with the Reviewer that we have not provided definitive evidence to conclude that teeth are interconnected. Therefore, we have now adapted the phrasing in Figure 4, the abstract, and our model to reflect that teeth are “likely interconnected”. However, we believe that our data tracking the teeth, and specifically that teeth within the same phagocytic cup appeared to move in a coordinated fashion, with similar speed and direction, and even with observed collective speed changes, supports a model where the teeth are functionally interconnected.

Figure 7. Given the similarities with podosomes, could local traction forces also be considered in this model?

As alluded to in our response to the reviewer comment regarding line 272-275, we believe that there currently is not enough evidence to include local tensile forces in this model.

Supp. Figure 1j. Could the authors determine an objective criterion to qualify an image quality appropriate or insufficient?

We thank the reviewer for this suggestion. In this case, because of the image artifacts present in the LLSM data were frequently observed, we were not able to use any of the lattice light sheet videos for force analysis. We therefore believe that our current criterium: to only use the confocal imaging data for force analysis, is a clear and justifiable choice. Coming up with an objective criterion is not trivial in the context of these artifacts that seem to be unique to lattice light sheet microscopy and their origin is not well understood. We changed the wording in the figure panel 1j, and its caption, to clarify this. We also removed the upper row of the figure panels to clarify the figure and message (Figure 1 – supplement 1e).

Supp.Figure 2-5. Would it be possible to average the three types of maps (deformation/actin/normalized stain), after classification, to perhaps allow easy and visual comparison of the effects of the treatments?

We appreciate this suggestion and the value of trying to capture the signature of these complex maps in a simplified manner. Since the particles are only aligned along the phagocytic axis, particles have an arbitrary position with respect to rotation around this axis. Therefore, any features orthogonal to the phagocytic axis will be mostly averaged out when averaging the maps. In addition, since each of these cups is in a different stage of engulfment (ranging from <20% to >90%), averaging the maps directly, without some normalization for the fraction engulfment, is likely not very informative. We believe that visualization of the deformation, curvature and fluorescent intensity profiles along the phagocytic axis (averaged in the directions orthogonal to the phagocytic axis) and then normalized to the fraction engulfed, is the most natural way for easy and visual comparison of the treatments, and this is how we choose to present this data in figure 2f and figure 3d in the main text.

Supp. Figure 7-8 "physical properties" is imprecise. What do you mean exactly? F-actin intensity? Size? Please clarify.

To address the reviewers concern we have changed this wording to “teeth number, size and indentation”.

https://doi.org/10.7554/eLife.68627.sa2

Article and author information

Author details

  1. Daan Vorselen

    Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing
    Contributed equally with
    Sarah R Barger
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7800-4023
  2. Sarah R Barger

    Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, United States
    Present address
    Molecular, Cellular, Developmental Biology, Yale University, New Haven, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Daan Vorselen
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6941-7256
  3. Yifan Wang

    Department of Mechanical Engineering, Stanford University, Stanford, United States
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  4. Wei Cai

    Department of Mechanical Engineering, Stanford University, Stanford, United States
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  5. Julie A Theriot

    Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, United States
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    jtheriot@uw.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2334-2535
  6. Nils C Gauthier

    IFOM, FIRC Institute of Molecular Oncology, Milan, Italy
    Contribution
    Conceptualization, Supervision, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  7. Mira Krendel

    Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, United States
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing, Investigation
    For correspondence
    krendelm@upstate.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7008-9069

Funding

American Heart Association (Predoctoral fellowship 18PRE34070066)

  • Sarah R Barger

National Institute of Diabetes and Digestive and Kidney Diseases (R01DK083345)

  • Mira Krendel

Associazione Italiana per la Ricerca sul Cancro (Investigator Grant 20716)

  • Nils C Gauthier

Howard Hughes Medical Institute

  • Julie A Theriot

Cancer Research Institute (CRI Irvington fellowship)

  • Daan Vorselen

National Institute of General Medical Sciences (R01GM138652)

  • Mira Krendel

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

LLSM imaging was performed at the Advanced Imaging Center (AIC) – Howard Hughes Medical Institute (HHMI) Janelia Research Campus. We thank John M Heddleston, Jesse Aaron, and Teng-Leong Chew of the AIC for helpful discussion. The AIC is jointly funded by the Gordon and Betty Moore Foundation and the Howard Hughes Medical Institute. We thank Lorenzo L Labitigan for critical review of the manuscript and Sharon Chase for help with animal experiments.

Ethics

This study was performed in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures utilizing mice were performed according to the animal protocol (IACUC# 364) approved by the IACUC of SUNY Upstate Medical University and in compliance with all applicable ethical regulations.

Senior Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Reviewing Editor

  1. Pekka Lappalainen, University of Helsinki, Finland

Reviewers

  1. Pekka Lappalainen, University of Helsinki, Finland
  2. Renaud Poincloux, Institute de Pharmacologie et de Biologie Structurale (IPBS), France

Publication history

  1. Preprint posted: March 15, 2021 (view preprint)
  2. Received: March 21, 2021
  3. Accepted: October 27, 2021
  4. Accepted Manuscript published: October 28, 2021 (version 1)
  5. Version of Record published: November 11, 2021 (version 2)

Copyright

© 2021, Vorselen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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