Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis
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Curated by eLife
Evaluation Summary:
This study is of great interest to cell biologists studying phagocytosis. The work describes a new method for studying phagocytosis; the engulfment of large cargos such as pathogens by the immune system. With this new method, they describe a mechanical force at the rim of the phagocytic cup that function like teeth. This work will advance the field in a new direction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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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.
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Author Response:
Reviewer #2 (Public Review):
In this manuscript, the authors use lattice light sheet microscopy and custom made soft micro-particles to examine the forces generated during phagocytosis and assess the molecular functions and localization of various components of the system. The imaging is truly fantastic and the discovery of phagocytic 'teeth' that exert force normal to the bead surface is a real advance to the field.
However, the functional studies using pharmacological inhibitors are more problematic. Specifically, the authors use pharmacological agents to test the roles of NMII (Blebb), the Arp2/3 complex (CK666) and supposedly formins (SMIFH2). The formin inhibitor is particularly problematic since it has known off target effects such as NMII (Sellers et al) and has never really be validated in terms of …
Author Response:
Reviewer #2 (Public Review):
In this manuscript, the authors use lattice light sheet microscopy and custom made soft micro-particles to examine the forces generated during phagocytosis and assess the molecular functions and localization of various components of the system. The imaging is truly fantastic and the discovery of phagocytic 'teeth' that exert force normal to the bead surface is a real advance to the field.
However, the functional studies using pharmacological inhibitors are more problematic. Specifically, the authors use pharmacological agents to test the roles of NMII (Blebb), the Arp2/3 complex (CK666) and supposedly formins (SMIFH2). The formin inhibitor is particularly problematic since it has known off target effects such as NMII (Sellers et al) and has never really be validated in terms of specificity or potency. I realize this drug has been used a lot in the literature, but so was BDM before it was finally discredited. It doesn't really give much of an effect and, combined with the fact the two formins checked are not in the cup, this data should just be removed from the paper.
We thank the Reviewer for pointing out the issue with SMIFH2. To address the reviewer’s comment, we have moved all data regarding SMIFH2 treatment to the SI (Figure 3 – supplement 5),and made a note in the manuscript on its potential off-target effects, including citation of the work by Nishimura et al. (pg. 8, line 217). While we are familiar with the work by Nishimura et al, we would also like to point out that we see very different effects of SMIFH2 treatment as compared to the direct myosin-II inhibition by blebbistatin, with the drug concentrations that we are using (see Figure 3).
As for the Arp2/3 inhibition, the data showing that this complex is important for generating force at the 'teeth' is convincing, however, the only real straightforward test of whether the complex is required for phagocytosis (Fig. 3g) shows that this drug has no effect on the fraction of engulfed particles. Doesn't this mean that the forces generated by branched actin are irrelevant for the kinetics of phagocytosis? That would be consistent with the published literature showing that genetic deletion of the Arp2/3 complex has only a partial inhibitory effect on FcR phagocytosis of rigid particles (a point the authors avoid discussing). Perhaps the forces generated by Arp2/3-branched actin are important under more challenging conditions such as where sheer flow was affecting the cells/particles, but this part of the paper is problematic.
We thank the Reviewer for pointing out the potentially complex role of the role of Arp2/3 in phagocytosis. It is indeed true that there may not be a one-to-one correlation between phagocytic teeth (and force exertion) and uptake efficiency, and this could further depend on the context in which phagocytosis is taking place. We note that for our experiment in Figure 3g (now figure 3h) we solely measure cups that are undergoing phagocytosis, and hence conclusions about the uptake efficiency and fraction engulfed particles cannot be drawn from this data. Instead, this data implies that there is no specific stage in phagocytosis that is affected more by Arp2/3-inhibition than other stages, and this analysis would for example not show any significant differences if phagocytic cup formation is approximately uniformly slowed during pseudopod progression. We have added a clarification in the text to make this point clearer:
“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 this complex has an important role throughout phagocytic progression.”
We have now performed additional experiments that show that Arp2/3 inhibition leads to a strong reduction in uptake in our phagocytosis assays. We present this data in a new figure panel (Fig. 3g). We observe a similar effect of Arp2/3 inhibition on uptake of 4 times stiffer beads (Figure 1 – supplement 2f). This is consistent with previous work with rigid particles comparable in size (~ 10 m) to the particles in our study (Rotty et al., 2017), where a significant reduction in phagocytic uptake efficiency upon Arp2/3 inhibition was also reported. We now also discuss the role of the Arp2/3 complex in more depth in our discussion section, including the results of our new uptake efficiency experiments and previous results in published literature (pg. 14, starting at line 419).
We agree that the relation of force exertion to phagocytic efficiency may depend on the context of the phagocyte and target. They could be relevant in the case of external flow, and we discuss in our manuscript how we believe that these forces may also be more critical during partial target eating of targets that are hard to engulf fully (too large, or hard-to-reach) as well as when multiple phagocytes approach a single target.
The blebbistatin data are interesting, but also somewhat contradictory with the literature showing this drug does not affect the uptake of rigid particles. It would be helpful if the authors could compare soft and much more rigid particles with this treatment to test this idea. The localization of myosin at the end stage of phagocytosis is very nice.
To address this concern, we have compared uptake of 1.4 kPa and 6.5 kPa particles, and potential differential effects of inhibitor treatments depending on target rigidity (Fig. 3g, Figure 1 – supplement 2f). We opted for making this comparison over a comparison between much stiffer (e.g. polystyrene) targets, as it will be non-trivial to ensure that these targets are identical in other chemical properties (ligand density, surface charge, etc.) and therefore that any potential difference can be solely attributed to target rigidity. Importantly, we have previously shown that there is a strong mechano-dependent difference in uptake efficiency for the 1.4 and 6.5 kPa beads (Vorselen et al, 2020). In these experiments, we see no clear detrimental effect of myosin-II inhibition on uptake efficiency independent of target rigidity similar to some past literature. We believe that this may be because the cup stages in which we saw high enrichment were at very late stages of engulfment (> 90% engulfment). That such cups aren’t fully closed was clear when performing high-resolution 3D imaging, but may be missed in lower resolution 2D imaging that we, and others, used for evaluating phagocytic efficiency, and hence these cups may be hard to distinguish from fully internalized particles. We now discuss these results and this possible explanation for the apparent discrepancy starting on page 16 line 476.
Reviewer #3 (Public Review):
Having revealed the role of class 1 myosins myosin 1e and myosin 1f during phagocytosis and having recently developed an innovative method to reveal the forces generated in this process, Daan Vorselen and colleagues studied the molecular mechanisms involved in force production during macrophage phagocytosis. In particular, they documented the involvement in force generation at the phagosome of Arp2/3-based actin protrusions, called « teeth », which assemble into a ring-like superstructure, and of myosin-II, which presumably plays a role in phagosome closure. Finally, they document phagocytosis failures in the form of popping mechanisms. The imaging and mechanical analyses of this article are particularly impressive, and these data allow the authors to propose a new model for the generation and balance of forces during phagocytosis.
This precise work thus paves the way to understanding the generation and transmission of forces during phagocytosis. However, some information could be added to strengthen the impact of the manuscript. In particular, current knowledge about the role of the Arp2/3 complex during phagocytosis could be detailed in the introduction, and what this article adds to the literature on this subject could be discussed better.
We thank the Reviewer for their kind words about our work. We now discuss the role of the Arp2/3 complex in more depth in our discussion section, including the results of our new uptake efficiency experiments upon inhibitor treatment, as well as previous results in published literature.
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Evaluation Summary:
This study is of great interest to cell biologists studying phagocytosis. The work describes a new method for studying phagocytosis; the engulfment of large cargos such as pathogens by the immune system. With this new method, they describe a mechanical force at the rim of the phagocytic cup that function like teeth. This work will advance the field in a new direction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This interesting study focused on the roles of mechanical forces generated by actin polymerization and myosin II contractility in Fc receptor -mediated phagocytosis. Through an elegant combination of microparticle traction force microscopy and lattice light-sheet microscopy, the authors identify F-actin 'teeth' that are important for microparticle constriction throughout the phagocytosis process, as well as elucidate the specific roles of Arp2/3 nucleated actin networks, and myosin II -based contractile structures in the process.
The data are of good technical quality and the study provides interesting new insights into Fc receptor -mediated phagocytosis.
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Reviewer #2 (Public Review):
In this manuscript, the authors use lattice light sheet microscopy and custom made soft micro-particles to examine the forces generated during phagocytosis and assess the molecular functions and localization of various components of the system. The imaging is truly fantastic and the discovery of phagocytic 'teeth' that exert force normal to the bead surface is a real advance to the field.
However, the functional studies using pharmacological inhibitors are more problematic. Specifically, the authors use pharmacological agents to test the roles of NMII (Blebb), the Arp2/3 complex (CK666) and supposedly formins (SMIFH2). The formin inhibitor is particularly problematic since it has known off target effects such as NMII (Sellers et al) and has never really be validated in terms of specificity or potency. I …
Reviewer #2 (Public Review):
In this manuscript, the authors use lattice light sheet microscopy and custom made soft micro-particles to examine the forces generated during phagocytosis and assess the molecular functions and localization of various components of the system. The imaging is truly fantastic and the discovery of phagocytic 'teeth' that exert force normal to the bead surface is a real advance to the field.
However, the functional studies using pharmacological inhibitors are more problematic. Specifically, the authors use pharmacological agents to test the roles of NMII (Blebb), the Arp2/3 complex (CK666) and supposedly formins (SMIFH2). The formin inhibitor is particularly problematic since it has known off target effects such as NMII (Sellers et al) and has never really be validated in terms of specificity or potency. I realize this drug has been used a lot in the literature, but so was BDM before it was finally discredited. It doesn't really give much of an effect and, combined with the fact the two formins checked are not in the cup, this data should just be removed from the paper.
As for the Arp2/3 inhibition, the data showing that this complex is important for generating force at the 'teeth' is convincing, however, the only real straightforward test of whether the complex is required for phagocytosis (Fig. 3g) shows that this drug has no effect on the fraction of engulfed particles. Doesn't this mean that the forces generated by branched actin are irrelevant for the kinetics of phagocytosis? That would be consistent with the published literature showing that genetic deletion of the Arp2/3 complex has only a partial inhibitory effect on FcR phagocytosis of rigid particles (a point the authors avoid discussing). Perhaps the forces generated by Arp2/3-branched actin are important under more challenging conditions such as where sheer flow was affecting the cells/particles, but this part of the paper is problematic.
The blebbistatin data are interesting, but also somewhat contradictory with the literature showing this drug does not affect the uptake of rigid particles. It would be helpful if the authors could compare soft and much more rigid particles with this treatment to test this idea. The localization of myosin at the end stage of phagocytosis is very nice.
Altogether, with some revisions, this work is perfect for the broad readership and will have a significant impact on the field.
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Reviewer #3 (Public Review):
Having revealed the role of class 1 myosins myosin 1e and myosin 1f during phagocytosis and having recently developed an innovative method to reveal the forces generated in this process, Daan Vorselen and colleagues studied the molecular mechanisms involved in force production during macrophage phagocytosis. In particular, they documented the involvement in force generation at the phagosome of Arp2/3-based actin protrusions, called « teeth », which assemble into a ring-like superstructure, and of myosin-II, which presumably plays a role in phagosome closure. Finally, they document phagocytosis failures in the form of popping mechanisms. The imaging and mechanical analyses of this article are particularly impressive, and these data allow the authors to propose a new model for the generation and balance of …
Reviewer #3 (Public Review):
Having revealed the role of class 1 myosins myosin 1e and myosin 1f during phagocytosis and having recently developed an innovative method to reveal the forces generated in this process, Daan Vorselen and colleagues studied the molecular mechanisms involved in force production during macrophage phagocytosis. In particular, they documented the involvement in force generation at the phagosome of Arp2/3-based actin protrusions, called « teeth », which assemble into a ring-like superstructure, and of myosin-II, which presumably plays a role in phagosome closure. Finally, they document phagocytosis failures in the form of popping mechanisms. The imaging and mechanical analyses of this article are particularly impressive, and these data allow the authors to propose a new model for the generation and balance of forces during phagocytosis.
This precise work thus paves the way to understanding the generation and transmission of forces during phagocytosis. However, some information could be added to strengthen the impact of the manuscript. In particular, current knowledge about the role of the Arp2/3 complex during phagocytosis could be detailed in the introduction, and what this article adds to the literature on this subject could be discussed better.
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