RESUMO
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Queratinócitos/ultraestrutura , Pseudópodes/química , Pseudópodes/ultraestrutura , Animais , Membrana Celular/química , Queratinócitos/química , Microscopia Eletrônica , Peixe-ZebraRESUMO
The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved in vivo through a dynamic process of de novo branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through in vivo time-lapse imaging of the subcellular localization of actin during the process of branching of Drosophila larva sensory neurons, combined with genetic analysis and electron tomography, we have identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation in vivo These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Dendritos/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Animais , Drosophila , Drosophila melanogaster , Células Receptoras Sensoriais/metabolismoRESUMO
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Dictyostelium/genética , Dictyostelium/metabolismo , Embrião não Mamífero , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Peixe-Zebra/genéticaRESUMO
Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Baculoviridae/ultraestrutura , Modelos Estatísticos , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Baculoviridae/química , Baculoviridae/fisiologia , Ensaio Cometa , Tomografia com Microscopia Eletrônica , Expressão Gênica , Genes Reporter , Carpa Dourada , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Melanoma Experimental , Células Sf9 , Spodoptera , Proteína Vermelha FluorescenteRESUMO
Fluorescence nanosectioning within a submicron region above an interface is desirable for many disciplines in the life sciences. A drawback, however, to most current approaches is the a priori need to physically scan a sculptured point spread function in the axial dimension, which can be undesirable for optically sensitive or highly dynamic samples. Here we demonstrate a fluorescence imaging approach that can overcome the need for scanning by exploiting the position-dependent emission spectrum of fluorophores above a simple biocompatible nanostructure. To achieve this we have designed a thin metal-dielectric-coated substrate, where the spectral modification to the total measured fluorescence can be used to estimate the axial fluorophore distribution within distances of 10-150 nm above the substrate with an accuracy of up to 5-10 nm. The modeling and feasibility of the approach are verified and successfully applied to elucidate nanoscale adhesion protein and filopodia dynamics in migrating cells. It is likely that the general principle can find broader applications in, for example, single-molecule studies, biosensing, and studying fast dynamic processes.
Assuntos
Movimento Celular/fisiologia , Metais/química , Microtomia/métodos , Nanoestruturas , Transferência Ressonante de Energia de Fluorescência , Microscopia/métodos , Modelos TeóricosRESUMO
Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 µm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin-Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.
Assuntos
Actinas/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Espaço Intracelular/metabolismo , Melanoma Experimental , Camundongos , Modelos Biológicos , Células NIH 3T3 , Pseudópodes/ultraestrutura , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.
Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Multimerização Proteica , Pseudópodes/metabolismo , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Camundongos , Microscopia de Fluorescência , Faloidina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem com Lapso de TempoRESUMO
Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our "sequential photopatterning" system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science.
Assuntos
Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Movimento Celular/fisiologia , Corantes Fluorescentes/química , Neovascularização Fisiológica/fisiologia , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Química Click , Reagentes de Ligações Cruzadas/química , Corantes Fluorescentes/efeitos da radiação , Humanos , Proteínas Imobilizadas/química , Ligantes , Camundongos , Células NIH 3T3 , Peptídeos/química , Estudo de Prova de Conceito , Propriedades de Superfície , Peixe-ZebraRESUMO
Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
Assuntos
Actinas/metabolismo , Adesão Celular , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Fibroblastos , Adesões Focais , Técnicas de Inativação de Genes , Integrinas/metabolismo , Melanoma Experimental , Camundongos , Células NIH 3T3 , Polimerização , Pseudópodes/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Polyomavirus large T antigen transactivates a variety of genes whose products are involved in S phase induction. These genes are regulated by the E2F family of transcription factors, which are under the control of the pocket protein retinoblastoma protein and its relatives p130 and p107. The viral protein causes a dissociation of E2F-pocket protein complexes that results in transactivation of the genes. This reaction requires the N-terminal binding site for pocket proteins and the J domain that binds chaperones. We found earlier that a mutation of the zinc finger located within the C-terminal domain, a region assumed to function mainly in the replication of viral DNA, also interferes with transactivation. Here we show that binding of the histone acetyltransferase coactivator complex CBP/p300-PCAF to the C terminus correlates with the ability of large T antigen to transactivate genes. This interaction results in promoter-specific acetylation of histones. Inactive mutant proteins with changes within the C-terminal domain were nevertheless able to dissociate the E2F pocket protein complexes, indicating that this dissociation is a necessary but insufficient step in the T antigen-induced transactivation of genes. It has to be accompanied by a second step involving the T antigen-mediated recruitment of a histone acetyltransferase complex.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Modelos Biológicos , Fatores de Transcrição/genética , Ativação Transcricional , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/genética , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , DNA/análise , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Genes Reporter , Immunoblotting , Luciferases/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Testes de Precipitina , Estrutura Terciária de Proteína , Ratos , Células Swiss 3T3 , Fatores de Transcrição/metabolismoRESUMO
In cancers with a highly altered genome, distinct genetic alterations drive subsets rather than the majority of individual tumours. Here we use a sequential search across human tumour samples for transcript outlier data points with associated gene copy number variations that correlate with patient's survival to identify genes with pro-invasive functionality. Employing loss and gain of function approaches in vitro and in vivo, we show that one such gene, MTSS1, promotes the ability of melanocytic cells to metastasize and engages actin dynamics via Rho-GTPases and cofilin in this process. Indeed, high MTSS1 expression defines a subgroup of primary melanomas with unfavourable prognosis. These data underscore the biological, clinical and potential therapeutic implications of molecular subsets within genetically complex cancers.
Assuntos
Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos Nus , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genéticaRESUMO
Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Células Epidérmicas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Peixes , Camundongos , Microinjeções , Miosina Tipo II/metabolismo , Células NIH 3T3 , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.
Assuntos
Citoesqueleto de Actina/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Animais , Recuperação de Fluorescência Após Fotodegradação , Camundongos , Microscopia , PolimerizaçãoRESUMO
Eukaryotic cells can initiate movement using the forces exerted by polymerizing actin filaments to extend lamellipodial and filopodial protrusions. In the current model, actin filaments in lamellipodia are organized in a branched, dendritic network. We applied electron tomography to vitreously frozen 'live' cells, fixed cells and cytoskeletons, embedded in vitreous ice or in deep-negative stain. In lamellipodia from four cell types, including rapidly migrating fish keratocytes, we found that actin filaments are almost exclusively unbranched. The vast majority of apparent filament junctions proved to be overlapping filaments, rather than branched end-to-side junctions. Analysis of the tomograms revealed that actin filaments terminate at the membrane interface within a zone several hundred nanometres wide at the lamellipodium front, and yielded the first direct measurements of filament densities. Actin filament pairs were also identified as lamellipodium components and bundle precursors. These data provide a new structural basis for understanding actin-driven protrusion during cell migration.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Pseudópodes/ultraestrutura , Actinas/fisiologia , Animais , Movimento Celular , Células Cultivadas , Microscopia Crioeletrônica , Citoesqueleto , Peixes , Humanos , Queratinócitos/citologiaRESUMO
Filopodia are rodlike extensions generally attributed with a guidance role in cell migration. We now show in fish fibroblasts that filopodia play a major role in generating contractile bundles in the lamella region behind the migrating front. Filopodia that developed adhesion to the substrate via paxillin containing focal complexes contributed their proximal part to stress fiber assembly, and filopodia that folded laterally contributed to the construction of contractile bundles parallel to the cell edge. Correlated light and electron microscopy of cells labeled for actin and fascin confirmed integration of filopodia bundles into the lamella network. Inhibition of myosin II did not subdue the waving and folding motions of filopodia or their entry into the lamella, but filopodia were not then integrated into contractile arrays. Comparable results were obtained with B16 melanoma cells. These and other findings support the idea that filaments generated in filopodia and lamellipodia for protrusion are recycled for seeding actomyosin arrays for use in retraction.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Pseudópodes/ultraestrutura , Animais , Proteínas de Transporte/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Forma Celular/fisiologia , Corrente Citoplasmática/fisiologia , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Carpa Dourada , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Miosina Tipo II/metabolismo , Paxilina/metabolismo , Transporte Proteico/fisiologia , Pseudópodes/metabolismo , Estresse MecânicoRESUMO
Here we describe the establishment and prediction utilities for a novel nine-amino-acid transactivation domain, 9aa TAD, that is common to the transactivation domains of a large number of yeast and animal transcription factors. We show that the 9aa TAD motif is required for the function of the transactivation domain of Gal4 and the related transcription factors Oaf1 and Pip2. The 9aa TAD possesses an autonomous transactivation activity in yeast and mammalian cells. Using sequence alignment and experimental data we derived a pattern that can be used for 9aa TAD prediction. The pattern allows the identification of 9aa TAD in other Gal4 family members or unrelated yeast, animal, and viral transcription factors. Thus, the 9aa TAD represents the smallest known denominator for a broad range of transcription factors. The wide occurrence of the 9aa TAD suggests that this domain mediates conserved interactions with general transcriptional cofactors. A computational search for the 9aa TAD is available online from National EMBnet-Node Austria at http://www.at.embnet.org/toolbox/9aatad/.