Much remains to be learned about the molecular mechanisms underlying a class of human disorders called actinopathies. These genetic disorders are characterized by loss-of-function mutations in actin-associated proteins that affect immune cells, leading to human immunopathology. However, much remains to be learned about how cytoskeletal dysregulation promotes immunological dysfunction. The current study reveals that the macrophage actin cytoskeleton responds to LPS/IFNγ stimulation in a biphasic manner that involves cellular contraction followed by cellular spreading. Myosin II inhibition by blebbistatin blocks the initial contraction phase and lowers iNOS protein levels and nitric oxide secretion. Conversely, conditional deletion of Arp2/3 complex in macrophages attenuates spreading and increases nitric oxide secretion. However, iNOS transcription is not altered by loss of myosin II or Arp2/3 function, suggesting post-transcriptional regulation of iNOS by the cytoskeleton. Consistent with this idea, proteasome inhibition reverses the effects of blebbistatin and rescues iNOS protein levels. Arp2/3-deficient macrophages demonstrate two additional phenotypes: defective MHCII surface localization, and depressed secretion of the T cell chemokine CCL22. These data suggest that interplay between myosin II and Arp2/3 influences macrophage activity, and potentially impacts adaptive-innate immune coordination. Disrupting this balance could have detrimental impacts, particularly in the context of Arp2/3-associated actinopathies.

Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase–mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc– and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2 ^fl/fl ) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreER ^T2 ; Arpc2 ^fl/fl ). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting cofactors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell–extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.

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.

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.

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.