Cellular Mechanisms of NETosis.

Neutrophils are critical to innate immunity, including host defense against bacterial and fungal infections. They achieve their host defense role by phagocytosing pathogens, secreting their granules full of cytotoxic enzymes, or expelling neutrophil extracellular traps (NETs) during the process of NETosis. NETs are weblike DNA structures decorated with histones and antimicrobial proteins released by activated neutrophils. Initially described as a means for neutrophils to neutralize pathogens, NET release also occurs in sterile inflammation, promotes thrombosis, and can mediate tissue damage. To effectively manipulate this double-edged sword to fight a particular disease, researchers must work toward understanding the mechanisms driving NETosis. Such understanding would allow the generation of new drugs to promote or prevent NETosis as needed. While knowledge regarding the (patho)physiological roles of NETosis is accumulating, little is known about the cellular and biophysical bases of this process. In this review, we describe and discuss our current knowledge of the molecular, cellular, and biophysical mechanisms mediating NET release as well as open questions in the field.

FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis.

Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.

Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration.

Cell migration toward areas of higher extracellular matrix (ECM) rigidity via a process called "durotaxis" is thought to contribute to development, immune response, and cancer metastasis. To understand how cells sample ECM rigidity to guide durotaxis, we characterized cell-generated forces on the nanoscale within single mature integrin-based focal adhesions (FAs). We found that individual FAs act autonomously, exhibiting either stable or dynamically fluctuating ("tugging") traction. We show that a FAK/phosphopaxillin/vinculin pathway is essential for high FA traction and to enable tugging FA traction over a broad range of ECM rigidities. We show that tugging FA traction is dispensable for FA maturation, chemotaxis, and haptotaxis but is critical to direct cell migration toward rigid ECM. We conclude that individual FAs dynamically sample rigidity by applying fluctuating pulling forces to the ECM to act as sensors to guide durotaxis, and that FAK/phosphopaxillin/vinculin signaling defines the rigidity range over which this dynamic sensing process operates.

NLRP3 is essential for neutrophil polarization and chemotaxis in response to leukotriene B4 gradient.

Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1ß and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3-/-) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3-/- neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.

Mechanical integration of actin and adhesion dynamics in cell migration.

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates.

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture.

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.

Single-shot super-resolution total internal reflection fluorescence microscopy.

We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

Filopodia and focal adhesions: An integrated system driving branching morphogenesis in neuronal pathfinding and angiogenesis.

Single cell branching during development in vertebrates is typified by neuronal branching to form neurites and vascular branches formed by sprouting angiogenesis. Neurons and endothelial tip cells possess subcellular protrusions that share many common features from the morphological to the molecular level. Both systems utilize filopodia as their cellular protrusion organelles and depend on specific integrin-mediated adhesions to the local extracellular matrix for guidance in their pathfinding. We discuss the similar molecular machineries involved in these two types of cell branch formation and use their analogy to propose a new mechanism for angiogenic filopodia function, namely as adhesion assembly sites. In support of this model we provide primary data of angiogenesis in zebrafish in vivo showing that the actin assembly factor VASP participates in both filopodia formation and adhesion assembly at the base of the filopodia, enabling forward progress of the tip cell. The use of filopodia and their associated adhesions provide a common mechanism for neuronal and endothelial pathfinding during development in response to extracellular matrix cues.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

ARAP2 inhibits Akt independently of its effects on focal adhesions.

BACKGROUND INFORMATION: ARAP2, an Arf GTPase-activating protein (Arf GAP) that binds to adaptor protein with PH domain, PTB domain and leucine zipper motifs 1 (APPL1), regulates focal adhesions (FAs). APPL1 affects FA dynamics by regulating Akt. Here, we tested the hypothesis that ARAP2 affects FAs in part by regulating Akt through APPL1. RESULTS: We found that ARAP2 controlled FA dynamics dependent on its enzymatic Arf GAP activity. In some cells, ARAP2 also regulated phosphoAkt (pAkt) levels. However, ARAP2 control of FAs did not require Akt and conversely, the effects on pAkt were independent of FAs. Reducing ARAP2 expression reduced the size and number of FAs in U118, HeLa and MDA-MB-231 cells. Decreasing ARAP2 expression increased pAkt in U118 cells and HeLa cells and overexpressing ARAP2 decreased pAkt in U118 cells; in contrast, ARAP2 had no effect on pAkt in MDA-MB-231 cells. An Akt inhibitor did not block the effect of reduced ARAP2 on FAs in U118. Furthermore, the effect of ARAP2 on Akt did not require Arf GAP activity, which is necessary for effects on FAs and integrin traffic. Altering FAs by other means did not induce the same changes in pAkt as those seen by reducing ARAP2 in U118 cells. In addition, we discovered that ARAP2 and APPL1 had co-ordinated effects on pAkt in U118 cells. Reduced APPL1 expression, as for ARAP2, increased pAkt in U118 and the effect of reduced APPL1 expression was reversed by overexpressing ARAP2. Conversely, the effect of reduced ARAP2 expression was reversed by overexpressing APPL1. ARAP2 is an Arf GAP that has previously been reported to affect FAs by regulating Arf6 and integrin trafficking and to bind to the adaptor proteins APPL1. Here, we report that ARAP2 suppresses pAkt levels in cells co-ordinately with APPL1 and independently of GAP activity and its effect on the dynamic behaviour of FAs. CONCLUSIONS: We conclude that ARAP2 affects Akt signalling in some cells by a mechanism independent of FAs or membrane traffic. SIGNIFICANCE: Our results highlight an Arf GAP-independent function of ARAP2 in regulating Akt activity and distinguish the effect of ARAP2 on Akt from that on FAs and integrin trafficking, which requires regulation of Arf6.

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly.

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF-FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.

Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing.

Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing.

YAP Nuclear Localization in the Absence of Cell-Cell Contact Is Mediated by a Filamentous Actin-dependent, Myosin II- and Phospho-YAP-independent Pathway during Extracellular Matrix Mechanosensing.

Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser(112), however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P)(112)-YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.

Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy.

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T ∼ 20.25 nN/µm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.

Nanoscale architecture of integrin-based cell adhesions.

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.