RESUMEN
Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.
Asunto(s)
Actinas , Proteínas de Microfilamentos , Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Movimiento CelularRESUMEN
Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.
Asunto(s)
División Celular Asimétrica/fisiología , Meiosis/fisiología , Oocitos/citología , Oocitos/fisiología , Partenogénesis/fisiología , Rhabditoidea/citología , Rhabditoidea/embriología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Núcleo Celular/fisiología , Femenino , Microtúbulos/fisiología , Oviparidad/fisiología , Huso Acromático/fisiologíaRESUMEN
Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Forminas/metabolismo , Animales , Ratones , Polimerizacion , Conejos , PorcinosRESUMEN
The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, which links the cytoskeleton to the nuclear interior. We demonstrate that nesprin-2 accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are mobile at time scales similar to the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient for this accumulation. Actin filaments are organized in a barrel structure around the nucleus in the direction of movement. Using two-photon ablation and cytoskeleton-inhibiting drugs, we demonstrate an actomyosin-dependent pulling force on the nucleus from the front of the cell. The elastic recoil upon ablation is dampened when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant toward the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.
Asunto(s)
Núcleo Celular , Proteínas Nucleares , Actinas/genética , Movimiento Celular , Membrana Nuclear , Proteínas Nucleares/genéticaRESUMEN
The cell shape changes that ensure asymmetric cell divisions are crucial for correct development, as asymmetric divisions allow for the formation of different cell types and therefore different tissues. The first division of the Caenorhabditis elegans embryo has emerged as a powerful model for understanding asymmetric cell division. The dynamics of microtubules, polarity proteins, and the actin cytoskeleton are all key for this process. In this review, we highlight studies from the last five years revealing new insights about the role of actin dynamics in the first asymmetric cell division of the early C. elegans embryo. Recent results concerning the roles of actin and actin binding proteins in symmetry breaking, cortical flows, cortical integrity, and cleavage furrow formation are described.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Caenorhabditis elegans/metabolismo , Morfogénesis , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Miosinas/genética , Miosinas/metabolismoRESUMEN
RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20-30 µm inside a living Caenorhabditis elegans worm.
Asunto(s)
Iluminación , Microscopía Fluorescente , Nanotecnología/métodos , Animales , Caenorhabditis elegans , Línea Celular , Supervivencia Celular , Toma de Decisiones , Hipocampo/citología , Humanos , Procesamiento de Imagen Asistido por Computador , Neuronas/citología , Ratas Sprague-Dawley , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Matrix metalloproteinases (MMPs) are associated with decreased patient prognosis but have failed as anti-invasive drug targets despite promoting cancer cell invasion. Through time-lapse imaging, optical highlighting, and combined genetic removal of the five MMPs expressed during anchor cell (AC) invasion in C. elegans, we find that MMPs hasten invasion by degrading basement membrane (BM). Though irregular and delayed, AC invasion persists in MMP- animals via adaptive enrichment of the Arp2/3 complex at the invasive cell membrane, which drives formation of an F-actin-rich protrusion that physically breaches and displaces BM. Using a large-scale RNAi synergistic screen and a genetically encoded ATP FRET sensor, we discover that mitochondria enrich within the protrusion and provide localized ATP that fuels F-actin network growth. Thus, without MMPs, an invasive cell can alter its BM-breaching tactics, suggesting that targeting adaptive mechanisms will be necessary to mitigate BM invasion in human pathologies.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Membrana Basal/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Polimerizacion , Citoesqueleto de Actina/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismoRESUMEN
During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Membrana Basal/metabolismo , Caenorhabditis elegans/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mecanotransducción Celular , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/genética , Animales , Membrana Basal/citología , Fenómenos Biomecánicos , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Movimiento Celular , Células Eucariotas/citología , Células Eucariotas/metabolismo , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Forminas , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Morfogénesis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimerizacion , Proteína Fluorescente RojaRESUMEN
In cells, actin filaments continuously assemble and disassemble while maintaining an apparently constant network structure. This suggests a perfect balance between dynamic processes. Such behavior, operating far out of equilibrium by the hydrolysis of ATP, is called a dynamic steady state. This dynamic steady state confers a high degree of plasticity to cytoskeleton networks that allows them to adapt and optimize their architecture in response to external changes on short time-scales, thus permitting cells to adjust to their environment. In this Review, we summarize what is known about the cellular actin steady state, and what gaps remain in our understanding of this fundamental dynamic process that balances the different forms of actin organization in a cell. We focus on the minimal steps to achieve a steady state, discuss the potential feedback mechanisms at play to balance this steady state and conclude with an outlook on what is needed to fully understand its molecular nature.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Ecosistema , Microtúbulos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , HumanosRESUMEN
Highly regulated cell migration events are crucial during animal tissue formation and the trafficking of cells to sites of infection and injury. Misregulation of cell movement underlies numerous human diseases, including cancer. Although originally studied primarily in two-dimensional in vitro assays, most cell migrations in vivo occur in complex three-dimensional tissue environments that are difficult to recapitulate in cell culture or ex vivo Further, it is now known that cells can mobilize a diverse repertoire of migration modes and subcellular structures to move through and around tissues. This review provides an overview of three distinct cellular movement events in Caenorhabditis elegans-cell invasion through basement membrane, leader cell migration during organ formation, and individual cell migration around tissues-which together illustrate powerful experimental models of diverse modes of movement in vivo We discuss new insights into migration that are emerging from these in vivo studies and important future directions toward understanding the remarkable and assorted ways that cells move in animals.
Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Movimiento Celular , Actinas/metabolismo , Animales , Biomarcadores , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Movimiento Celular/genética , Regulación de la Expresión Génica , Humanos , Mioblastos/citología , Mioblastos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Transducción de SeñalRESUMEN
Despite their fundamental importance in the regulation of cell physiology, the mechanisms that confer cell adaptability to changes in the microenvironment are poorly understood. A recent study in Cell (Mueller et al., 2017) examines the capability of branched actin networks to respond and adapt to mechanical load in vivo.
Asunto(s)
Citoesqueleto de Actina , ActinasRESUMEN
Actin filament dynamics have been studied for decades in pure protein solutions or in cell extracts, but a breakthrough in the field occurred at the turn of the century when it became possible to reconstitute networks of actin filaments, growing in a controlled but physiological manner on surfaces, mimicking the actin assembly that occurs at the plasma membrane during cell protrusion and cell shape changes. The story begins with the bacteria Listeria monocytogenes, the study of which led to the reconstitution of cellular actin polymerization on a variety of supports including plastic beads. These studies made possible the development of liposome-type substrates for filament assembly and micropatterning of actin polymerization nucleation. Based on the accumulated expertise of the last 15 years, many exciting approaches are being developed, including the addition of myosin to biomimetic actin networks to study the interplay between actin structure and contractility. The field is now poised to make artificial cells with a physiological and dynamic actin cytoskeleton, and subsequently to put these cells together to make in vitro tissues. This article is part of a Special Issue entitled: Mechanobiology.
Asunto(s)
Citoesqueleto de Actina/química , Proteínas Bacterianas/química , Listeria monocytogenes/química , Proteínas de la Membrana/química , Citoesqueleto de Actina/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Humanos , Listeria monocytogenes/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.
Asunto(s)
Actinas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Citoesqueleto/química , Citoesqueleto/metabolismo , Animales , HumanosRESUMEN
The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.
Asunto(s)
Citoesqueleto de Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/química , Actinas/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Unión al ADN/química , Familia de Proteínas del Síndrome de Wiskott-Aldrich/química , Animales , Caenorhabditis elegans , Movimiento Celular , Desarrollo Embrionario , Humanos , Profilinas/química , SeudópodosRESUMEN
Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the "molecular clutch" description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of Major Sperm Protein (MSP), which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.
RESUMEN
Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Animales , Movimiento Celular/fisiología , Humanos , Morfogénesis/fisiología , Uniones Estrechas/metabolismoRESUMEN
Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an 'outside geometry'. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin-streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Forma de la Célula/fisiología , Modelos Biológicos , Miosina Tipo II/metabolismo , Polimerizacion , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Fenómenos Biomecánicos/fisiología , Biomimética , Biotina , Humanos , Liposomas/metabolismo , Profilinas/metabolismo , EstreptavidinaRESUMEN
Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.
Asunto(s)
Citoesqueleto de Actina/química , Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Módulo de Elasticidad , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Actinas/química , Animales , Sitios de Unión , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
Cell motility is locally achieved by the interplay between lamellipodia and filopodia at the protrusion sites. The actin cytoskeleton rearranges from a highly branched short filamentous network to well aligned elongated bundles from lamellipodia to filopodia, respectively. This process is governed predominantly by actin binding proteins, VASP and fascin, at the leading edge of migratory cells. Here we use an Arp2/3-complex dependent bead motility assay to study the effect of fascin both on its own and in the presence of VASP. The Young's modulus of phalloidin stabilized comets grown in the presence of fascin increased linearly with concentration above a 0.5 µM threshold. Inversely, the initial velocity of the comets decreased linearly with fascin concentration above the same threshold. Interestingly, VASP and fascin together increased the Young's modulus of the comets compared to those grown in the presence of only one of the two proteins. This effect was most remarkable at low concentration, below 0.5 and 0.15 µM for fascin and VASP, respectively. Our results showed that fascin and VASP work cooperatively to enhance the Young's modulus of the actin network within the comets.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Módulo de Elasticidad , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Bovinos , Movimiento Celular , Humanos , Microscopía de Fuerza Atómica , Faloidina/metabolismo , Seudópodos/metabolismo , ConejosRESUMEN
The comet motility assay, inspired by Listeria locomotion, has been used extensively as an in vitro model to study the structural and motile properties of the actin cytoskeleton. However, there are no quantitative measurements of the mechanical properties of these actin comets. In this work, we use nanoindentation based on atomic force microscopy to measure the elastic modulus of actin comets grown on 1-µm-diameter beads in an Arp2/3 (actin-related proteins 2 and 3)-complex-dependent fashion in the absence and in the presence of VASP (vasodilator-stimulated phosphoprotein). Recruitment of VASP to the bead surface had no effect on the initial velocity or morphology of the comets. Instead, we observed an improved contact of the comets with the beads and an increased elastic modulus of the comets. The VASP-mediated increase in elastic modulus was dependent on both concentration and ionic strength. In conclusion, we propose that VASP plays a mechanical role in Arp2/3-complex-dependent motility by amplifying the elastic modulus of the thus assembled actin network and, consequently, by strengthening its cohesion for persistent protrusion.