RESUMEN
Macroendocytosis comprising phagocytosis and macropinocytosis is an actin-driven process regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, nonprotrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction in F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.
Asunto(s)
Fagosomas , Dictyostelium , Forminas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Transducción de Señal , Fagosomas/metabolismo , Actinas/metabolismoRESUMEN
Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.
Asunto(s)
Virus de la Influenza A/inmunología , Macrófagos/inmunología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Succinatos/farmacología , Células A549 , Animales , Carboxiliasas/deficiencia , Carboxiliasas/inmunología , Citocinas/genética , Citocinas/inmunología , Humanos , Macrófagos/virología , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Células THP-1RESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1010219.].
RESUMEN
The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.
Asunto(s)
Citoesqueleto de Actina/genética , Proteínas Portadoras/genética , Proteínas Contráctiles/genética , Melanoma Experimental/genética , Citoesqueleto de Actina/química , Actinas/genética , Animales , Sistemas CRISPR-Cas , Movimiento Celular/genética , Polaridad Celular/genética , Proteínas Contráctiles/química , Dictyostelium/genética , Modelos Animales de Enfermedad , Forminas , Humanos , Melanoma Experimental/patología , Ratones , Microscopía Electrónica , Contracción Muscular/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/genética , Dictyostelium/genética , Hidrólisis , Proteínas de Microfilamentos/genética , Microscopía Fluorescente/métodos , Mutación , Fosfoproteínas/genética , Profilinas/genética , Profilinas/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Endocytic processes are facilitated by both curvature-generating BAR-domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N-BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau-induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect in vivo, where we observed that downregulation of Bin1 in a Drosophila model of tauopathy significantly reduces the appearance of tau-induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau-induced pathobiological changes of the actin cytoskeleton.
Asunto(s)
Actinas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Tauopatías/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteínas tau/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Clonación Molecular , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas tau/metabolismoRESUMEN
Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.
Asunto(s)
Actinas/metabolismo , Dictyostelium/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas ras/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Dictyostelium/metabolismo , Proteínas de Microfilamentos/genética , Mutación , Fagocitosis , Pinocitosis , Proto-Oncogenes Mas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de SeñalRESUMEN
Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Macrófagos/metabolismo , Proteínas de Microfilamentos/metabolismo , Mioblastos/metabolismo , Organogénesis/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Drosophila/fisiología , Morfogénesis/fisiología , Desarrollo de Músculos/fisiología , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.
Asunto(s)
Citoesqueleto de Actina/genética , Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosfoproteínas/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/genética , Embrión de Mamíferos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/fisiología , Unión Proteica , Multimerización de Proteína/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
During Drosophila embryogenesis, the first epithelium with defined cortical compartments is established during cellularization. Actin polymerization is required for the separation of lateral and basal domains as well as suppression of tubular extensions in the basal domain. The actin nucleator mediating this function is unknown. We found that the formin Diaphanous (Dia) is required for establishing and maintaining distinct lateral and basal domains during cellularization. In dia mutant embryos lateral marker proteins, such as Discs-large and Armadillo/ß-Catenin spread into the basal compartment. Furthermore, high-resolution and live-imaging analysis of dia mutant embryos revealed an increased number of membrane extensions and endocytic activity at the basal domain, indicating a suppressing function of dia on membrane invaginations. Dia function might be based on an antagonistic interaction with the F-BAR protein Cip4/Toca-1, a known activator of the WASP/WAVE-Arp2/3 pathway. Dia and Cip4 physically and functionally interact and overexpression of Cip4 phenocopies dia loss-of-function. In vitro, Cip4 inhibits mainly actin nucleation by Dia. Thus, our data support a model in which linear actin filaments induced by Dia stabilize cortical compartmentalization by antagonizing membrane turnover induced by WASP/WAVE-Arp2/3.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Proteínas Portadoras/genética , Drosophila , Proteínas de Drosophila/genética , Forminas , Unión Proteica , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Factores Despolimerizantes de la Actina/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/deficiencia , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Antígenos de Histocompatibilidad Menor , Células 3T3 NIH , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Polimerizacion/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Factor de Células Madre/farmacología , Sus scrofaRESUMEN
BACKGROUND: Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. METHODS AND RESULTS: We have developed induced pluripotent stem cell-derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15-/- macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, ß-oxidation, and NAD(P)H-dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II-V was diminished in ISG15-/- cells. Defective mitochondrial respiration was restored by transduction with wild-type ISG15, but only partially by a conjugation-deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl-itaconate, 4-octyl-itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria-related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read-out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti-oxidant genes NFE2L2, HMOX1, and GPX7, and dimethyl-itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon-stimulated genes, ISG15-/- macrophages maintained their reduced susceptibility to influenza virus infection. CONCLUSIONS: These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate-based medications as additional therapeutics for this rare disorder.
Asunto(s)
Células Endoteliales , Interferones , Aminoácidos de Cadena Ramificada/genética , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/metabolismo , Humanos , Interferones/genética , Fenotipo , Succinatos , Transaminasas/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4-0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates.
RESUMEN
Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas/metabolismo , Seudópodos/metabolismo , Animales , Fenómenos Biomecánicos , Sistemas CRISPR-Cas/genética , Movimiento Celular , Fibroblastos/metabolismo , Forminas , Técnicas de Silenciamiento del Gen , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Modelos Biológicos , Células 3T3 NIH , Fenotipo , Polimerizacion , Seudópodos/ultraestructura , Interferencia de ARNRESUMEN
In response to chemotactic signals, motile cells develop a single protruding front to persistently migrate in direction of the chemotactic gradient. The highly dynamic reorganization of the actin cytoskeleton is an essential part during this process and requires the precise interplay of various actin filament assembly factors and actin-binding proteins (ABPs). Although many ABPs have been implicated in cell migration, as yet only a few of them have been well characterized concerning their specific functions during actin network assembly and disassembly. In this chapter, we describe a versatile method that allows the direct visualization of the assembly of single actin filaments and higher structures in real time by in vitro total internal reflection fluorescence microscopy (TIRF-M) using purified and fluorescently labeled actin and ABPs.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Imagen Molecular , Actinas/química , Animales , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Músculo Esquelético/metabolismo , Unión Proteica , Multimerización de Proteína , Conejos , Coloración y EtiquetadoRESUMEN
Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Dictyostelium/fisiología , Locomoción , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Femenino , Proteínas de Microfilamentos/metabolismo , Miosina Tipo II/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
The cyclic pyrimidine nucleotide cCMP has been suggested to serve as second messenger. However, phosphodiesterases studied so far do not hydrolyze cCMP. Therefore, we searched for alternative cCMP inactivation mechanisms. cCMP is a substrate for multidrug resistance protein 5, indicating that export from the cytosol into the extracellular space is an important inactivation mechanism for cCMP.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Nucleótidos Cíclicos/metabolismo , Transporte Biológico , Células HEK293 , HumanosRESUMEN
Diaphanous-related formins (DRFs) act as downstream effectors of Rho family GTPases and drive the formation and elongation of linear actin filaments in various cellular processes. Here we analyzed the DRF dDia1 from Dictyostelium cells. The biochemical characterization of recombinant dDia1-FH1FH2 by bulk polymerization assays and single filament TIRF microscopy revealed that dDia1 is a rather weak nucleator. Addition of any of the three Dictyostelium profilin isoforms, however, markedly accelerated formin-mediated actin filament barbed end elongation in TIRF assays. Interestingly, filament elongation was significantly faster in presence of DdPFN I (profilin I) when compared to the other two isoforms, suggesting selectivity of dDia1 for DdPFN I. Additionally, we frequently observed dissociation of the formin from growing barbed ends. These findings are consistent with dilution-induced depolymerization assays in presence of dDia1-FH1FH2 showing that dDia1 is a weak capper in comparison with heterodimeric capping protein. To study the physiological role of this formin, we created cell lines lacking dDia1 or overexpressing GFP-tagged dDia1. Of note, constitutively active dDia1 accumulated homogenously in the entire pseudopod suggesting that it controls microfilament architecture to regulate cell migration. Comparison of wild type and dDia1-null cells in random cell migration and chemotaxis toward a cAMP gradient revealed no major differences. By contrast, phototaxis of dDia1-deficient cells during the multicellular stage was markedly impaired. While wild type slugs moved with high directionality toward the light source, the trails of dDia1-null slugs displayed a characteristic V-shaped profile and deviated in angles between 50° and 60° from the path of the incident light. Possibly in conjunction with this defect, dDia1-null cells also formed substantially smaller fruiting bodies. These findings demonstrate dDia1 to be critically involved in collective cell migration during terminal differentiation.
Asunto(s)
Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Citoesqueleto de Actina/metabolismo , Movimiento Celular , Dictyostelium/citología , Endocitosis , Luz , Profilinas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of ß-galactosidase in prespore cells. Together, these findings demonstrate ForC to be critically involved in signalling of the cytoskeleton during various stages of development.