Jeremy D. Rotty

Microglia maintain brain homeostasis via iC3b-mediated synaptic pruning. The Arp2/3 complex has been implicated in iC3b-mediated macrophage phagocytosis, but it is unclear whether it is similarly required in microglia in the CNS. We examined the question of CR3-dependent clearance of iC3b in microglia using a combination of in vitro and in situ physical confinement studies. Arp2/3 inhibition decreased iC3b phagocytosis and cell motility in vitro. Furthermore, microglia-like cells remove immobilized iC3b from the substrate in an Arp2/3-dependent fashion, in a process reminiscent of trogocytic synaptic pruning. We also used a novel approach to immobilize an iC3b gradient onto a substrate and demonstrate Arp2/3-dependent haptotactic migration toward increasing iC3b concentrations. While Arp2/3-deficient microglia robustly respond to ATP via chemotaxis within mouse hippocampal slices, they demonstrate a persistent inability to stably interact with iC3b-coated beads. The present study establishes new approaches to systematically interrogate molecular pathways relevant to synaptic pruning, advances the understanding of iC3b phagocytosis as a haptotactic response, and confirms that the Arp2/3-dependent haptotactic response is important for microglia function in the CNS microenvironment.

Physical confinement is not routinely considered as a factor that influences phagocytosis, which is typically investigated using unconfined in vitro assays. BV2 microglia-like cells were used to interrogate the impact of confinement on IgG-mediated phagocytosis side by side with unconfined cells. Confinement acted as a potent phagocytic driver, greatly increasing the fraction of phagocytic cells in the population compared to the unconfined setting. Arp2/3 complex and myosin II contributed to this effect. Remarkably, confinement partially rescued phagocytic uptake upon myosin II disruption. In addition, cells under confinement were partially resistant to the actin-depolymerizing drug cytochalasin D. Unexpectedly, we observed that bead uptake stimulated persistent migration, a process we term ‘phagocytic priming’. Integrin-dependent adhesion was required for phagocytic priming in unconfined and confined settings, but was dispensable for phagocytic uptake. The cytoskeletal requirements for phagocytic priming differed depending on confinement state. Myosin II and Arp2/3 complex were required for phagocytic priming under confinement, but not in unconfined settings. As with phagocytosis, cytoskeleton-dependent priming of motility varies based on physical confinement status. Phagocytic priming may be a crucial innate immune mechanism by which cells respond to wounds or trauma with increased surveillance of the local microenvironment.

Cells sense and respond to their environment in a myriad of ways. In many instances, they must integrate simultaneous cues ranging from the physical properties and composition of the extracellular matrix to guidance cues that stimulate chemotaxis or haptotaxis. How cells make sense of multiple simultaneous cues is an ongoing physiologically relevant area of research. The present study seeks to contribute to the understanding of multi-cue sensing by understanding how the transition to a confined setting with or without an added haptotactic gradient alters macrophage migration. We found that the transition to confinement is itself a directional cue capable of driving persistent migration hours after macrophages enter the confined environment. Next, we found that a haptotactic fibronectin gradient made cells even more directionally persistent under confinement. Finally, Arp2/3 complex deletion rendered macrophages unresponsive to the haptotactic gradient, but they retained directionally persistent migration due to their transition to confinement. These findings may be particularly relevant for cells that move from an adherent 2D environment into a confining 3D environment, like leukocytes and circulating tumor cells that extravasate into peripheral tissue.

Microglia maintain brain homeostasis by performing iC3b-mediated synaptic pruning on excessive dendritic spines during neurodevelopment. Cellular interaction with iC3b is mediated by the Arp2/3 complex in other cell types, but understudied in microglia. Using a combination of in vitro and in situ physical confinement studies, we examined CR3-dependent clearance of iC3b in microglia and the role the Arp2/3 complex plays in enabling this clearance. We demonstrated Arp2/3 complex inhibition decreased phagocytosis and cell motility in vitro . Furthermore, we demonstrate that microglia-like cells are able to remove immobilized iC3b from the substrate in an Arp2/3-dependent fashion, in a process reminiscent of trogocytic synaptic pruning. We also used a novel approach to immobilize an iC3b gradient onto a substrate and demonstrate Arp2/3-dependent haptotactic migration toward increasing iC3b concentrations. Microglia demonstrate a persistent inability to stably interact with iC3b-coated beads in hippocampal slice cultures upon Arp2/3 complex inhibition. As a whole, the present study establishes new approaches to systematically interrogate molecular pathways relevant to synaptic pruning, advances the understanding of iC3b phagocytosis as a haptotactic response, and confirms that the Arp2/3-dependent haptotactic response is relevant for microglia in their normal physiological microenvironment.

Cells sense and respond to their environment in a myriad of ways. In many instances they must integrate simultaneous cues ranging from the physical properties and composition of the extracellular matrix to guidance cues that stimulate chemotaxis or haptotaxis. How cells make sense of multiple simultaneous cues is an ongoing physiologically relevant question. The present study seeks to contribute to the understanding of multi-cue sensing by understanding how the transition to a confined setting with or without an added haptotactic gradient alters macrophage migration. We found that the transition to confinement is itself a directional cue capable of driving persistent migration hours after macrophages enter the confined environment. Next, we found that a haptotactic fibronectin gradient made cells even more directionally persistent under confinement. Finally, Arp2/3 complex deletion rendered macrophages unresponsive to the haptotactic gradient, but they retained directionally persistent migration due to their transition to confinement. These findings may be particularly relevant for cells that move from an adherent 2D environment into a confining 3D environment, like leukocytes and circulating tumor cells that extravasate into peripheral tissue. Summary Statement: Macrophages migrate persistently after they transition from 2D adhesion to an adhesive confined environment. Migration is enhanced further if cells sense a fibronectin gradient. This may have relevance to immune and cancer cell behavior.

Physical confinement is not routinely considered as a factor that influences phagocytosis, which is typically assayed in unconfined settings in vitro . BV-2 microglia-like cells were used to interrogate the impact of confinement on IgG-mediated phagocytosis side by side with unconfined cells. Confinement acted as a potent phagocytic driver, greatly increasing the fraction of phagocytic cells in the population compared to the unconfined setting. Arp2/3 complex and myosin II contributed to this effect. Remarkably, confinement partially rescued phagocytic uptake upon myosin II disruption. In addition, cells under confinement were partially resistant to the actin-depolymerizing drug cytochalasin D. Unexpectedly, we observed that bead uptake stimulated persistent migration, a process we term ‘phagocytic priming’. Integrin-dependent adhesion was required for phagocytic priming in unconfined and confined settings, but was dispensable for phagocytic uptake. The cytoskeletal requirements for phagocytic priming differed depending on confinement state. Myosin II and Arp2/3 complex were required for phagocytic priming under confinement, but not in unconfined settings. As with phagocytosis, cytoskeleton-dependent priming of motility shifts depending on physical confinement status. Phagocytic priming may facilitate innate immune function by driving cells to more efficiently surveil their local microenvironment in response to wounds or trauma.

Dynamic cycles of actin remodeling drive membrane protrusion and retraction events essential for macrophage function. Phosphoregulation of actin-associated proteins plays a key role, but the factors that determine the spatiotemporal balance between kinases and phosphatases is less well understood in this context. Here, we identify the Protein Phosphatase 1 (PP1)-binding protein Phactr4 as a critical regulator of cytoskeletal remodeling. Phactr4 loss disrupts lamellipodial architecture, which results in uncoordinated migration and disrupted iC3b-mediated phagocytosis. Unstable membrane dynamics underlie the Phactr4 knockdown phenotypes. Phactr4 is recruited to the leading edge via interaction with active Arp2/3 complex, and strongly correlates with membrane retraction. Phactr4 loss leads to ezrin hyperphosphorylation, and membrane protrusion defects in these cells are reversed by ezrin inhibition. Our findings position Phactr4 as a critical PP1-dependent coordinator of cytoskeletal remodeling during macrophage migration and phagocytosis. Recent reports have linked Phactr4 to several human disease states, which may be due to its influence on actin dynamics.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). The present work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses to macrophages in 2D environments. Laminin 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared to cells on fibronectin. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. The present study also demonstrates that laminin 111 exerts a suppressive effect toward fibronectin, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the cell’s ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory modes based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in 3D and in vivo contexts warrants further study.