The mechanosensor Piezo1 rings the alarm on epithelial intruders.

Recognition of invasive pathogens by the epithelium that is constantly exposed to microbial products remains incompletely understood. In a recent study, Tadala et al. demonstrated that the entry process of intracellular bacteria is itself a mechanical signal that is detected by the stretch-activated channel Piezo1, which triggers innate immune signaling.

The GAS6-AXL signaling pathway triggers actin remodeling that drives membrane ruffling, macropinocytosis, and cancer-cell invasion.

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.

The roles of Cdc42 and Rac1 in the formation of plasma membrane protrusions in cancer epithelial HeLa cells.

The inducible model of clones generated from the cervical cancer epithelial HeLa cell line has shown the role of DOCK10 as a guanine-nucleotide exchange factor for Rho GTPases Cdc42 and Rac1 and as an inducer of filopodia and plasma membrane (PM) ruffles. In this model, constitutively active (CA) mutants of Cdc42 and Rac1 promote filopodia and ruffles, respectively, as in other models. DOCK9 also induces filopodia and ruffles, although ruffling activity is less prominent. By exploiting this model further, the aim of this work is to provide a more complete understanding of the role of Cdc42 and Rac1, and their interactions with DOCK10 and DOCK9, in regulation of PM protrusions. New clones have been generated from HeLa, including single clones expressing one form of wild-type (WT) or dominant negative (DN) Cdc42 or Rac1, and double clones co-expressing one of them together with either DOCK10 or DOCK9. Expression of WT- and DN-Cdc42 induced filopodia. WT-Cdc42 and, especially, DN-Cdc42 also gave rise to veil protrusions, which were neutralized by DOCK10. Moreover, DN-Cdc42 stimulated the emergence of ruffles, further increased by DOCK10, and WT-Cdc42 also augmented ruffles in presence of DOCK9 and DOCK10. WT-Rac1 greatly increased PM blebbing, as did DN-Rac1 more moderately. In both cases, blebs were enhanced by DOCK10. DN-Rac1 and CA-Rac1 moderately raised filopodia, and DOCK10 and DOCK9 had opposed effects on filopodia (up and down, respectively) in presence of WT-Rac1. As conclusions, we highlight that Cdc42 promotes filopodia regardless of its conformational state, and Rac1 induces blebs in conformations other than CA, especially WT-Rac1, in the inducible HeLa clones. The model could be useful to learn more about the mechanisms underlying PM protrusions.

Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics.

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility.

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

Rare contacts between synapses and microglial processes containing high levels of Iba1 and actin--a postembedding immunogold study in the healthy rat brain.

Although microglia is recognised as the cell-mediating innate immunity in the brain, emerging evidence suggests a role of microglia in synaptic communication and modulation. The ability of microglia to move in the neuropil and contact synapses is crucial for such a function. However, the frequency of microglial contact with synapses is not known. Microglia motility is regulated by actin polymerisation and its interaction with ionising calcium-binding adaptor protein 1 (Iba1). In order to move and make contact with synapses, delicate microglial processes should contain high levels of actin and Iba1. To study this we refined an electron microscopic postembedding immunogold method enabling us to identify and quantitatively study different microglial constituents in intact brain tissue. We show that Iba1 and actin were colocalised at high densities in delicate processes in the rat frontal cortex, and that these delicate processes of microglia contact synaptic elements. About 3.5% of the synapses received direct contact from microglia. There was a marked inverse correlation between the densities of Iba1/actin gold particles and the area of the microglial processes, suggesting that the most delicate processes possess the machinery to provide movement in the neuropil. The low frequency of microglia interaction with synaptic elements suggests that microglia have a limited role in overall regulation of synaptic activity.

Malaria parasites harness Rho GTPase signaling and host cell membrane ruffling for productive invasion of hepatocytes.

Plasmodium sporozoites are the motile forms of the malaria parasites that infect hepatocytes. The initial invasion of hepatocytes is thought to be actively driven by sporozoites, but host cell processes might also play a role. Sporozoite invasion triggers a host plasma membrane invagination that forms a vacuole around the intracellular parasite, which is critical for subsequent intracellular parasite replication. Using fast live confocal microscopy, we observed that the initial interactions between sporozoites and hepatocytes induce plasma membrane ruffles and filopodia extensions. Importantly, we find that these host cell processes facilitate invasion and that Rho GTPase signaling, which regulates membrane ruffling and filopodia extension, is critical for productive infection. Interestingly, sporozoite cell traversal stimulates these processes, suggesting that it increases hepatocyte susceptibility to productive infection. Our study identifies host cell signaling events involved in plasma membrane dynamics as a critical host component of successful malaria parasite infection of hepatocytes.

Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1.

Microorganisms are generally sensed by receptors recognizing microbial molecules, which evoke changes in cellular activities and gene expression. Bacterial pathogens induce secretion of the danger signal ATP as an early alert response of intestinal epithelial cells, initiating overt inflammation. However, what triggers ATP secretion during infection is unclear. Here we show that the inherently mechanosensitive plasma membrane channel PIEZO1 acts as a sensor for bacterial entry. PIEZO1 is mechanically activated by invasion-induced membrane ruffles upstream of Ca2+ influx and ATP secretion. Mimicking mechanical stimuli of pathogen uptake with sterile beads equally elicits ATP secretion. Chemical or genetic PIEZO1 inactivation inhibits mechanically induced ATP secretion. Moreover, chemical or mechanical PIEZO1 activation evokes gene expression in immune and barrier pathways. Thus, mechanosensation of invasion-induced plasma membrane distortion initiates immune signaling upon infection, independently of detection of microbial molecules. Hence, PIEZO1-dependent detection of infection is driven by physical signals instead of chemical ligands.

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils.

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell-substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

Variable Distribution of DOCK-D Proteins between Cytosol and Nucleoplasm in Cell Lines, Effect of Interleukin-4 on DOCK10 in B-Cell Lymphoid Neoplasms, and Validation of a New DOCK10 Antiserum for Immunofluorescence Studies.

Dedicator-of-cytokinesis (DOCK), a family of guanine-nucleotide exchange factors (GEFs), comprises four subfamilies, named from A to D. DOCK-D comprises DOCK9, DOCK10, and DOCK11. The GEF activity involves translocation from the cytoplasm to the plasma membrane (PM), as assessed by the transfection of tagged proteins. However, the cellular localization of endogenous DOCK proteins is poorly understood. In this paper, to gain a better understanding of the role of the DOCK-D proteins, we studied their distribution between cytosol and nucleoplasm in 11 cell lines. DOCK-D proteins were distributed with variable cytosolic or nuclear predominance, although the latter was common for DOCK9 and DOCK11. These results suggest that the DOCK-D proteins may perform new nuclear functions, which remain to be discovered. Furthermore, we found that DOCK10 levels are increased by interleukin-4 (IL-4) in B-cell lymphoid neoplasms other than chronic lymphocytic leukemia (CLL) such as mantle cell lymphoma and diffuse large B-cell lymphoma. We also found evidence for an induction of the cytosolic levels of DOCK10 by IL-4 in CLL. Finally, we obtained a valid DOCK10 antiserum for immunofluorescence (IF) microscopy that, as an antibody against the hemagglutinin (HA) tag, marked PM ruffles and filopodia in HeLa cells with inducible expression of HA-DOCK10.

Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections.

Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. Anat Rec, 302:2224-2232, 2019. © 2019 American Association for Anatomy.

Quantitation of Macropinocytosis in Cancer Cells.

Macropinocytosis has emerged as an important nutrient supply pathway that sustains cell growth of cancer cells within the nutrient-poor tumor microenvironment. By internalizing extracellular fluid through this bulk endocytic pathway, albumin is supplied to the cancer cells, which, after degradation, serves as an amino acid source to meet the high nutrient demands of these highly proliferating cells. Here, we describe a streamlined protocol for visualization and quantitation of macropinosomes in adherent cancer cells grown in vitro. The determination of the "macropinocytic index" provides a tool for measuring the extent to which this internalization pathway is utilized within the cancer cells and allows for comparison between different cell lines and treatments. The protocol provided herein has been optimized for reproducibility and is readily adaptable to multiple conditions and settings.

Cyclophilin A Controls Salmonella Internalization Levels and is Present at E. coli Actin-Rich Pedestals.

Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. Anat Rec, 301:2086-2094, 2018. © 2018 Wiley Periodicals, Inc.

Dock10, a Cdc42 and Rac1 GEF, induces loss of elongation, filopodia, and ruffles in cervical cancer epithelial HeLa cells.

Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells.

Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy.

The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response.