Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements

Neutrophils are important effector cells in the host defense against invading micro-organisms. One of the mechanisms they employ to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli, i.e. phorbol 12-myristate 13-acetate, monosodium urate crystals and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either ARPC1B- or MKL1-deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics there is a lack of translocation of NE to the nucleus, which may well explain the impaired NET formation. Collectively, our data illustrate the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

Geometric control of Myosin-II orientation during axis elongation

The actomyosin cytoskeleton is a crucial driver of morphogenesis. Yet how the behavior of large scale cytoskeletal patterns in deforming tissues emerges from the interplay of geometry, genetics, and mechanics remains incompletely understood. Convergent extension flow in D. melanogaster embryos provides the opportunity to establish a quantitative understanding of the dynamics of anisotropic non-muscle myosin II. Cell-scale analysis of protein localization in fixed embryos suggests that there are complex rules governing how the control of myosin anisotropy is regulated by gene expression patterns. However, technical limitations have impeded quantitative and dynamic studies of this process at the whole embryo level, leaving the role of geometry open. Here we combine in toto live imaging with quantitative analysis of molecular dynamics to characterize the distribution of myosin anisotropy and corresponding genetic patterning. We found pair rule gene expression continuously deformed, flowing with the tissue frame. In contrast, myosin anisotropy orientation remained nearly static, aligned with the stationary dorsal-ventral axis of the embryo. We propose myosin recruitment by a geometrically defined static source, potentially related to the embryo-scale epithelial tension, and account for transient deflections by the interplay of cytoskeletal turnover with junction reorientation by flow. With only one parameter, this model quantitatively accounts for the time course of myosin anisotropy orientation in wild-type, twist, and even-skipped embryos as well as embryos with perturbed egg geometry. Geometric patterning of the cytoskeleton suggests a simple physical strategy to ensure a robust flow and formation of shape.

JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer’s disease.

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

Structural basis of the super- and hyper-relaxed states of myosin II

Super-relaxation is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster (relaxed) state, is ubiquitous and thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail forming an interacting-heads motif, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Salt-Mediated Stiffening, Destruction, and Resculpting of Actomyosin Network

Cells dynamically change their viscoelastic properties by restructuring networks of actin filaments in the cytoskeleton, enabling diverse mechanical processes such as mobility and apoptosis. This restructuring is modulated, in part, by actin-binding proteins, such as myosin II, as well as counterions such as Mg2+ and K+. While high concentrations of Mg2+ can induce bundling and crosslinking of actin filaments, high concentrations of K+ destabilize myosin II minifilaments necessary to crosslink actin filaments. Here, we elucidate how the mechanics and structure of actomyosin networks evolve under competing effects of varying Mg2+ and K+ concentrations. Specifically, we couple microfluidics with optical tweezers microrheology to measure the time-varying linear viscoelastic moduli of actin networks crosslinked via myosin II as we cycle between low and high Mg2+ and K+ concentrations. Our complementary confocal imaging experiments correlate the time-varying viscoelastic properties with salt-mediated structural evolution. We find that the elastic modulus displays an intriguing non-monotonic time dependence in high-salt conditions, that correlates with structural changes, and that this process is irreversible, with the network evolving to a new steady-state as Mg2+ and K+ decrease back to their initial concentrations.