Nonmuscle Myosin II Regulation Directs Its Multiple Roles in Cell Migration and Division.

Nonmuscle myosin II (NMII) is a multimeric protein complex that generates most mechanical force in eukaryotic cells. NMII function is controlled at three main levels. The first level includes events that trigger conformational changes that extend the complex to enable its assembly into filaments. The second level controls the ATPase activity of the complex and its binding to microfilaments in extended NMII filaments. The third level includes events that modulate the stability and contractility of the filaments. They all work in concert to finely control force generation inside cells. NMII is a common endpoint of mechanochemical signaling pathways that control cellular responses to physical and chemical extracellular cues. Specific phosphorylations modulate NMII activation in a context-dependent manner. A few kinases control these phosphorylations in a spatially, temporally, and lineage-restricted fashion, enabling functional adaptability to the cellular microenvironment. Here, we review mechanisms that control NMII activity in the context of cell migration and division.

CD69 controls the uptake of L-tryptophan through LAT1-CD98 and AhR-dependent secretion of IL-22 in psoriasis.

The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9(+) γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues.

Meeting Report - Workshop 'Actin-based mechanosensation and force generation in health and disease'.

International experts in the fields of cellular motility, force generation and mechanosensation met in Baeza, a UNESCO World Heritage city, from the 10th to the 13th of November, 2019. The meeting, part of the 'Current Trends in Biomedicine' series, took place at the 'Sede Antonio Machado', a beautiful 17th century building turned into a conference center of the Universidad Internacional de Andalucía (UNIA), which sponsored the event. The meeting was organized by Alexis Gautreau, Pekka Lappalainen and Miguel Vicente-Manzanares, with the support of the European Molecular Biology Organization (EMBO) and the Spanish-based company IMPETUX. Fifty scientists presented recent results during the talks, poster sessions and thematic discussions. As Baeza itself served as a crossroads of medieval Christian, Moorish and Jewish cultures, the meeting brought together cell biologists, biochemists, biophysicists and engineers from around the world that provided an integrated vision of the role of the actin cytoskeleton, force generation and mechanosensation in diverse physiological processes and pathologies.

Panobinostat Synergistically Enhances the Cytotoxicity of Microtubule Destabilizing Drugs in Ovarian Cancer Cells.

Ovarian cancer (OC) is one of the most common gynecologic neoplasia and has the highest mortality rate, which is mainly due to late-stage diagnosis and chemotherapy resistance. There is an urgent need to explore new and better therapeutic strategies. We have previously described a family of Microtubule Destabilizing Sulfonamides (MDS) that does not trigger multidrug-mediated resistance in OC cell lines. MDS bind to the colchicine site of tubulin, disrupting the microtubule network and causing antiproliferative and cytotoxic effects. In this work, a novel microtubule-destabilizing agent (PILA9) was synthetized and characterized. This compound also inhibited OC cell proliferation and induced G2/M cell cycle arrest and apoptosis. Interestingly, PILA9 was significantly more cytotoxic than MDS. Here, we also analyzed the effect of these microtubule-destabilizing agents (MDA) in combination with Panobinostat, a pan-histone deacetylase inhibitor. We found that Panobinostat synergistically enhanced MDA-cytotoxicity. Mechanistically, we observed that Panobinostat and MDA induced α-tubulin acetylation and that the combination of both agents enhanced this effect, which could be related to the observed synergy. Altogether, our results suggest that MDA/Panobinostat combinations could represent new therapeutic strategies against OC.

Non-muscle myosin II takes centre stage in cell adhesion and migration.

Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

L-selectin expression is regulated by CXCL8-induced reactive oxygen species produced during human neutrophil rolling.

Neutrophils destroy invading microorganisms by phagocytosis by bringing them into contact with bactericidal substances, among which ROS are the most important. However, ROS also function as important physiological regulators of cellular signaling pathways. Here, we addressed the involvement of oxygen derivatives in the regulation of human neutrophil rolling, an essential component of the inflammatory response. Flow experiments using dihydroethidium-preloaded human neutrophils showed that these cells initiate an early production of intracellular ROS during the rolling phase of the adhesion cascade, a phenomenon that required cell rolling, and the interaction of the chemokine receptor CXCR2 with their ligand CXCL8. Flow cytometry experiments demonstrated that L-selectin shedding in neutrophils is triggered by ROS through an autocrine-paracrine mechanism. Preincubation of neutrophils with the NADPH oxidase complex inhibitor diphenyleniodonium chloride significantly increased the number of rolling neutrophils on endothelial cells. Interestingly, the same effect was observed when CXCL8 signaling was interfered using either a blocking monoclonal antibody or an inhibitor of its receptor. These findings indicate that, in response to CXCL8, neutrophils initiate ROS production during the rolling phase of the inflammatory response. This very early ROS production might participate in the modulation of the inflammatory response by inducing L-selectin shedding in neutrophils.

Organizing polarized delivery of exosomes at synapses.

Exosomes are extracellular vesicles that transport different molecules between cells. They are formed and stored inside multivesicular bodies (MVB) until they are released to the extracellular environment. MVB fuse along the plasma membrane, driving non-polarized secretion of exosomes. However, polarized signaling potentially directs MVBs to a specific point in the plasma membrane to mediate a focal delivery of exosomes. MVB polarization occurs across a broad set of cellular situations, e.g. in immune and neuronal synapses, cell migration and in epithelial sheets. In this review, we summarize the current state of the art of polarized MVB docking and the specification of secretory sites at the plasma membrane. The current view is that MVB positioning and subsequent exosome delivery requires a polarizing, cytoskeletal dependent-trafficking mechanism. In this context, we propose scenarios in which biochemical and mechanical signals could drive the polarized delivery of exosomes in highly polarized cells, such as lymphocytes, neurons and epithelia.

Full L1-regularized Traction Force Microscopy over whole cells.

BACKGROUND: Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. RESULTS: Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. CONCLUSIONS: The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.

Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner.

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an alpha-actinin-actin template that elongates centripetally from nascent adhesions. Alpha-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.

Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer.

Mechanotransduction encompasses the role of mechanical forces in controlling cell behavior by activating signal transduction pathways. Most forces at a cellular level are caused by myosin II, which contracts and cross-links actin. Myosin II-dependent forces are transmitted through the actin cytoskeleton to molecular endpoints that promote specific cellular outcomes, e.g., cell proliferation, adhesion, or migration. For example, most adhesive and migratory phenomena are mechanically linked by a molecular clutch comprised of mechanosensitive scaffolds. Myosin II activation and mechanosensitive molecular mechanisms are finely tuned and spatiotemporally integrated to coordinate morphogenetic events during development. Mechanical events dependent on myosin II also participate in tumor cell proliferation, invasion, and metastatic dissemination. Specifically, tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for proliferation and/or dissemination. These observations position myosin II-dependent force generation and mechanotransduction at the crossroads between normal development and cancer.

Role of the cytoskeleton during leukocyte responses.

The cytoskeleton is a cellular network of structural, adaptor and signalling molecules that regulates most cellular functions that are related to the immune response, including migration, extravasation, antien recognition, activation and phagocytosis by different subsets of leukocytes. Recently, a large number of regulatory elements and structural constituents of the leukocyte cytoskeleton have been identified. In this review, we discuss the composition and regulation of the different cytoskeletal elements and their role in immune responses.

Engines of change: Nonmuscle myosin II in mechanobiology.

The emergence of mechanobiology has unveiled complex mechanisms by which cells adjust intracellular force production to their needs. Most communicable intracellular forces are generated by myosin II, an actin-associated molecular motor that transforms adenosine triphosphate (ATP) hydrolysis into contraction in nonmuscle and muscle cells. Myosin II-dependent force generation is tightly regulated, and deregulation is associated with specific pathologies. Here, we focus on the role of myosin II (nonmuscle myosin II, NMII) in force generation and mechanobiology. We outline the regulation and molecular mechanism of force generation by NMII, focusing on the actual outcome of contraction, that is, force application to trigger mechanosensitive events or the building of dissipative structures. We describe how myosin II-generated forces drive two major types of events: modification of the cellular morphology and/or triggering of genetic programs, which enhance the ability of cells to adapt to, or modify, their microenvironment. Finally, we address whether targeting myosin II to impair or potentiate its activity at the motor level is a viable therapeutic strategy, as illustrated by recent examples aimed at modulating cardiac myosin II function in heart disease.

A primer on cancer-associated fibroblast mechanics and immunosuppressive ability.

Cancer-associated fibroblasts (CAFs) are a major point of interest in modern oncology. Their interest resides in their ability to favor tumor growth without carrying genetic mutations. From a translational standpoint, they are potential therapeutic targets, particularly for hard-to-treat solid cancers. CAFs can be defined as non-tumor cells within the tumor microenvironment that have the morphological traits of fibroblasts, are negative for lineage-specific markers (e.g., leukocyte, endothelium), and enhance tumor progression in a multi-pronged manner. Two often-mentioned aspects of CAF biology are their ability to alter the mechanics and architecture of the tumor microenvironment, and also to drive local immunosuppression. These two aspects are the specific focus of this work, which also contains a brief summary of novel therapeutic interventions under study to normalize or eliminate CAFs from the tumor microenvironment.

Host Cell Targets for Unconventional Antivirals against RNA Viruses.

The recent COVID-19 crisis has highlighted the importance of RNA-based viruses. The most prominent members of this group are SARS-CoV-2 (coronavirus), HIV (human immunodeficiency virus), EBOV (Ebola virus), DENV (dengue virus), HCV (hepatitis C virus), ZIKV (Zika virus), CHIKV (chikungunya virus), and influenza A virus. With the exception of retroviruses which produce reverse transcriptase, the majority of RNA viruses encode RNA-dependent RNA polymerases which do not include molecular proofreading tools, underlying the high mutation capacity of these viruses as they multiply in the host cells. Together with their ability to manipulate the immune system of the host in different ways, their high mutation frequency poses a challenge to develop effective and durable vaccination and/or treatments. Consequently, the use of antiviral targeting agents, while an important part of the therapeutic strategy against infection, may lead to the selection of drug-resistant variants. The crucial role of the host cell replicative and processing machinery is essential for the replicative cycle of the viruses and has driven attention to the potential use of drugs directed to the host machinery as therapeutic alternatives to treat viral infections. In this review, we discuss small molecules with antiviral effects that target cellular factors in different steps of the infectious cycle of many RNA viruses. We emphasize the repurposing of FDA-approved drugs with broad-spectrum antiviral activity. Finally, we postulate that the ferruginol analog (18-(phthalimide-2-yl) ferruginol) is a potential host-targeted antiviral.

Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-Like Changes in Human Microvascular Endothelial Cells.

Dengue virus (DENV) is a pathogenic arbovirus that causes human disease. The most severe stage of the disease (severe dengue) is characterized by vascular leakage, hypovolemic shock, and organ failure. Endothelial dysfunction underlies these phenomena, but the causal mechanisms of endothelial dysfunction are poorly characterized. This study investigated the role of c-ABL kinase in DENV-induced endothelial dysfunction. Silencing c-ABL with artificial miRNA or targeting its catalytic activity with imatinib revealed that c-ABL is required for the early steps of DENV infection. DENV-2 infection and conditioned media from DENV-infected cells increased endothelial expression of c-ABL and CRKII phosphorylation, promoted expression of mesenchymal markers, e.g., vimentin and N-cadherin, and decreased the levels of endothelial-specific proteins, e.g., VE-cadherin and ZO-1. These effects were reverted by silencing or inhibiting c-ABL. As part of the acquisition of a mesenchymal phenotype, DENV infection and treatment with conditioned media from DENV-infected cells increased endothelial cell motility in a c-ABL-dependent manner. In conclusion, DENV infection promotes a c-ABL-dependent endothelial phenotypic change that leads to the loss of intercellular junctions and acquisition of motility.

Correction: Escudero-Flórez et al. Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-like Changes in Human Microvascular Endothelial Cells. Viruses 2023, 15, 1437.

Ryan L [...].

Characterization of mutant versions of the R-RAS2/TC21 GTPase found in tumors.

The R-RAS2 GTP hydrolase (GTPase) (also known as TC21) has been traditionally considered quite similar to classical RAS proteins at the regulatory and signaling levels. Recently, a long-tail hotspot mutation targeting the R-RAS2/TC21 Gln72 residue (Q72L) was identified as a potent oncogenic driver. Additional point mutations were also found in other tumors at low frequencies. Despite this, little information is available regarding the transforming role of these mutant versions and their relevance for the tumorigenic properties of already-transformed cancer cells. Here, we report that many of the RRAS2 mutations found in human cancers are highly transforming when expressed in immortalized cell lines. Moreover, the expression of endogenous R-RAS2Q72L is important for maintaining optimal levels of PI3K and ERK activities as well as for the adhesion, invasiveness, proliferation, and mitochondrial respiration of ovarian and breast cancer cell lines. Endogenous R-RAS2Q72L also regulates gene expression programs linked to both cell adhesion and inflammatory/immune-related responses. Endogenous R-RAS2Q72L is also quite relevant for the in vivo tumorigenic activity of these cells. This dependency is observed even though these cancer cell lines bear concurrent gain-of-function mutations in genes encoding RAS signaling elements. Finally, we show that endogenous R-RAS2, unlike the case of classical RAS proteins, specifically localizes in focal adhesions. Collectively, these results indicate that gain-of-function mutations of R-RAS2/TC21 play roles in tumor initiation and maintenance that are not fully redundant with those regulated by classical RAS oncoproteins.

STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells.

Two-color spatio-temporal image cross-correlation spectroscopy (STICCS) is a new, to our knowledge, image analysis method that calculates space-time autocorrelation and cross-correlation functions from fluorescence intensity fluctuations. STICCS generates cellular flow and diffusion maps that reveal interactions and cotransport of two distinct molecular species labeled with different fluorophores. Here we use computer simulations to map the capabilities and limitations of STICCS for measurements in complex heterogeneous environments containing micro- and macrostructures. We then use STICCS to analyze the co-flux of adhesion components in migrating cells imaged using total internal reflection fluorescence microscopy. The data reveal a robust, time-dependent co-fluxing of certain integrins and paxillin in adhesions in protrusions when they pause, and in adhesions that are sliding and disassembling, demonstrating that the molecules in these adhesions move as a complex. In these regions, both α6ß1- or αLß2-integrins, expressed in CHO.B2 cells, co-flux with paxillin; an analogous cotransport was seen for α6ß1-integrin and α-actinin in U2OS. This contrasts with the behavior of the α5ß1-integrin and paxillin, which do not co-flux. Our results clearly show that integrins can move in complexes with adhesion proteins in protrusions that are retracting.