RESUMEN
Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.
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Proteínas de la Membrana , Microscopía Fluorescente , Transporte de Proteínas , Microscopía Fluorescente/métodos , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/análisis , Membrana Celular/metabolismo , Membrana Celular/química , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , AnimalesRESUMEN
Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape.
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Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Actinas , Proteínas Activadoras de GTPasa , Animales , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Actinas/metabolismo , Ratones , Extensiones de la Superficie Celular/metabolismo , Humanos , Línea Celular , Guanosina Trifosfato/metabolismo , HidrólisisRESUMEN
Fundamental limits of cellular deformations, such as hyperextension of a living cell, remain poorly understood. Here, we describe how the single-celled protist Lacrymaria olor, a 40-micrometer cell, is capable of reversibly and repeatably extending its necklike protrusion up to 1200 micrometers in 30 seconds. We discovered a layered cortical cytoskeleton and membrane architecture that enables hyperextensions through the folding and unfolding of cellular-scale origami. Physical models of this curved crease origami display topological singularities, including traveling developable cones and cytoskeletal twisted domain walls, which provide geometric control of hyperextension. Our work unravels how cell geometry encodes behavior in single cells and provides inspiration for geometric control in microrobotics and deployable architectures.
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Forma de la Célula , Extensiones de la Superficie Celular , Cilióforos , Citoesqueleto , Membrana Celular/ultraestructura , Citoesqueleto/ultraestructura , Cilióforos/citología , Cilióforos/fisiología , Extensiones de la Superficie Celular/ultraestructura , Microtúbulos/ultraestructuraRESUMEN
The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells.
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Propiedades de Superficie , Porosidad , Humanos , Extensiones de la Superficie Celular/ultraestructura , Extensiones de la Superficie Celular/metabolismo , Membrana Celular/metabolismo , Adhesión Celular , AnimalesRESUMEN
To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.
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Burkholderia , Sistemas de Secreción Tipo VI , Burkholderia/metabolismo , Burkholderia/fisiología , Sistemas de Secreción Tipo VI/metabolismo , Humanos , Membrana Celular/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas Bacterianas/metabolismo , Actinas/metabolismo , Dinamina II/metabolismo , Autofagia , Galectinas/metabolismo , Interacciones Huésped-Patógeno , Extensiones de la Superficie Celular/metabolismo , Animales , Proteínas Asociadas a Microtúbulos , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by formation of lamellipodia and/or filopodia, while amoeboid migration is based on bleb formation. Changing of migrational conditions can lead to alteration in the character of cell movement. For example, inhibition of the Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility mode. Ability of the cells to switch from one type of motility to another is called migratory plasticity. Cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryo fibroblasts REF52 with normal VIF organization, fibroblasts with vimentin knockout (REF-/-), and fibroblasts with mutation inhibiting assembly of the full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase in the number of cells with blebs. The fibroblasts with short fragments of vimentin demonstrate the significant increase in number of cells forming blebs both spontaneously and in the presence of CK-666. Disruption of the VIF organization did not lead to the significant changes in the microtubules network or the level of myosin light chain phosphorylation, but caused significant reduction in the focal contact system. The most pronounced and statistically significant decrease in both size and number of focal adhesions were observed in the REF117 cells. We believe that regulation of the membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that organization of VIFs determines the type of cell protrusions, which, in turn, determines the character of cell movement. A novel role of VIFs as a regulator of membrane blebbing, essential for manifestation of the migratory plasticity, is shown.
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Adhesiones Focales , Filamentos Intermedios , Ratas , Animales , Filamentos Intermedios/metabolismo , Adhesiones Focales/metabolismo , Vimentina/genética , Vimentina/metabolismo , Vimentina/farmacología , Microtúbulos/metabolismo , Movimiento Celular , Extensiones de la Superficie Celular/metabolismoRESUMEN
The migratory dynamics of cells can be influenced by the complex microenvironment through which they move. It remains unclear how the motility machinery of confined cells responds and adapts to their microenvironment. Here, we propose a biophysical mechanism for a geometry-dependent coupling between cellular protrusions and the nucleus that leads to directed migration. We apply our model to geometry-guided cell migration to obtain insights into the origin of directed migration on asymmetric adhesive micropatterns and the polarization enhancement of cells observed under strong confinement. Remarkably, for cells that can choose between channels of different size, our model predicts an intricate dependence for cellular decision making as a function of the two channel widths, which we confirm experimentally.
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Extensiones de la Superficie Celular , Movimiento CelularRESUMEN
BACKGROUND: The neuronal transdifferentiation of adult bone marrow cells (BMCs) is still considered an artifact based on an alternative explanation of experimental results supporting this phenomenon obtained over decades. However, recent studies have shown that following neural induction, BMCs enter an intermediate cellular state before adopting neural-like morphologies by active neurite extension and that binucleated BMCs can be formed independent of any cell fusion events. These findings provide evidence to reject the idea that BMC neural transdifferentiation is merely an experimental artifact. Therefore, understanding the intermediate states that cells pass through during transdifferentiation is crucial given their potential application in regenerative medicine and disease modelling. METHODS: In this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can adopt during neural-like differentiation using live-cell nuclear fluorescence labelling, time-lapse microscopy, and confocal microscopy analysis. RESULTS: Here, we showed that after neural induction, hBM-MSCs enter an intermediate cellular state in which the nuclei are able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning occur because human cell nuclei somehow sense their environment. In addition, we showed the process of direct interactions between cell nuclei, which opens the possibility of a new level of intercellular interaction. CONCLUSIONS: The present study advances the understanding of the intermediate stage through which hBM-MSCs pass during neural transdifferentiation, which may be crucial to understanding the mechanisms of these cell conversion processes and eventually harness them for use in regenerative medicine. Importantly, our study provides for the first time evidence that the nuclei of hBM-MSC-derived intermediate cells somehow sense their environment, generating cellular protrusions to contact other cells. In summary, human mesenchymal stromal cells could not only help to increase our understanding of the mechanisms underlying cellular plasticity but also facilitate the exact significance of nuclear positioning in cellular function and in tissue physiology.
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Células Madre Adultas , Neuronas , Adulto , Humanos , Diferenciación Celular/fisiología , Extensiones de la Superficie Celular , Núcleo Celular , Células de la Médula Ósea , Células CultivadasRESUMEN
Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.
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Actinas , Forminas , Neuropéptidos , Citoesqueleto de Actina/química , Actinas/química , Forminas/química , Neuropéptidos/química , Extensiones de la Superficie Celular/química , Neuronas/metabolismo , Microscopía ElectrónicaRESUMEN
LncRNAs are emerging as important regulators of gene expression by controlling transcription in the nucleus and by modulating mRNA translation in the cytoplasm. In this study, we reveal a novel function of lncRNA SNHG15 in mediating breast cancer cell invasion through regulating the local translation of CDH2 mRNA. We show that SNHG15 preferentially localizes at the cellular protrusions or cell leading edge and that this localization is directed by IMP1, a multifunctional protein involved in many aspects of RNA regulation. We demonstrate that SNHG15 also forms a complex with nucleolin, allowing nucleolin to be co-transported with SNHG15 to the cell protrusions, where the accumulated nucleolin is able to bind to CDH2 mRNA. Interaction with nucleolin stabilizes local CDH2 mRNA and regulates its translation, thus promoting cell invasive potential. Our findings reveal an underlying mechanism by which lncRNA could serve as a carrier to transport a protein regulator into a specific cell compartment to enhance target mRNA expression.
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MicroARNs , ARN Largo no Codificante , Línea Celular Tumoral , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proliferación Celular/genética , Extensiones de la Superficie Celular/metabolismo , MicroARNs/genética , Regulación Neoplásica de la Expresión Génica , NucleolinaRESUMEN
Protrusions at the leading-edge of a cell play an important role in sensing the extracellular cues during cellular spreading and motility. Recent studies provided indications that these protrusions wrap (coil) around the extracellular fibers. However, the physics of this coiling process, and the mechanisms that drive it, are not well understood. We present a combined theoretical and experimental study of the coiling of cellular protrusions on fibers of different geometry. Our theoretical model describes membrane protrusions that are produced by curved membrane proteins that recruit the protrusive forces of actin polymerization, and identifies the role of bending and adhesion energies in orienting the leading-edges of the protrusions along the azimuthal (coiling) direction. Our model predicts that the cell's leading-edge coils on fibers with circular cross-section (above some critical radius), but the coiling ceases for flattened fibers of highly elliptical cross-section. These predictions are verified by 3D visualization and quantitation of coiling on suspended fibers using Dual-View light-sheet microscopy (diSPIM). Overall, we provide a theoretical framework, supported by experiments, which explains the physical origin of the coiling phenomenon.
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Extensiones de la Superficie Celular , Señales (Psicología) , Endocitosis , Proteínas de la Membrana , Modelos TeóricosRESUMEN
To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate that, similar to rounded cells in 2D culture, rounded cells in 3D collagen contain large amounts of CSE and use it to cover developing protrusions. Upon retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. We present high-resolution imaging of F-actin and microtubules (MTs) for different cell lines in a 3D environment and demonstrate the correlated changes between CSE and protrusion dynamics. To coordinate CSE storage and release with protrusion formation and motility, we expect cells to have specific mechanisms for regulating CSE, and we hypothesize that MTs play a substantial role in this mechanism by reducing cell surface dynamics and stabilizing CSE. We also suggest that different effects of MT depolymerization on cell motility, such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by this role of MTs in CSE regulation.
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Actinas , Colágeno , Actinas/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Microtúbulos/metabolismo , Seudópodos/metabolismo , Movimiento Celular/fisiología , Extensiones de la Superficie CelularRESUMEN
Adherent cells migrate on layered tissue interfaces to drive morphogenesis, wound healing, and tumor invasion. Although stiffer surfaces are known to enhance cell migration, it remains unclear whether cells sense basal stiff environments buried under softer, fibrous matrix. Using layered collagen-polyacrylamide gel systems, we unveil a migration phenotype driven by cell-matrix polarity. Here, cancer (but not normal) cells with stiff base matrix generate stable protrusions, faster migration, and greater collagen deformation because of "depth mechanosensing" through the top collagen layer. Cancer cell protrusions with front-rear polarity produce polarized collagen stiffening and deformations. Disruption of either extracellular or intracellular polarity via collagen crosslinking, laser ablation, or Arp2/3 inhibition independently abrogates depth-mechanosensitive migration of cancer cells. Our experimental findings, validated by lattice-based energy minimization modeling, present a cell migration mechanism whereby polarized cellular protrusions and contractility are reciprocated by mechanical extracellular polarity, culminating in a cell-type-dependent ability to mechanosense through matrix layers.
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Extensiones de la Superficie Celular , Colágeno , Colágeno/metabolismo , Movimiento Celular/fisiología , Morfogénesis , Extensiones de la Superficie Celular/metabolismo , Matriz Extracelular/metabolismoRESUMEN
Imaging two or more fluorescent biosensors in the same living cell can reveal the spatiotemporal coordination of protein activities. However, using multiple Förster resonance energy transfer (FRET) biosensors together is challenging due to toxicity and the need for orthogonal fluorophores. Here we generate a biosensor component that binds selectively to the activated conformation of three different proteins. This enabled multiplexed FRET with fewer fluorophores, and reduced toxicity. We generated this MultiBinder (MB) reagent for the GTPases RhoA, Rac1, and Cdc42 by combining portions of the downstream effector proteins Pak1 and Rhotekin. Using FRET between mCherry on the MB and YPet or mAmetrine on two target proteins, the activities of any pair of GTPases could be distinguished. The MB was used to image Rac1 and RhoA together with a third, dye-based biosensor for Cdc42. Quantifying effects of biosensor combinations on the frequency, duration, and velocity of cell protrusions and retractions demonstrated reduced toxicity. Multiplexed imaging revealed signaling hierarchies between the three proteins at the cell edge where they regulate motility.
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Técnicas Biosensibles , Proteína de Unión al GTP cdc42 , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Transducción de Señal , Transferencia Resonante de Energía de Fluorescencia/métodos , Extensiones de la Superficie Celular , Colorantes , Técnicas Biosensibles/métodos , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.
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Anoicis , Carcinogénesis , Extensiones de la Superficie Celular , Supervivencia Celular , Melanoma , Transducción de Señal , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Septinas/metabolismo , Extensiones de la Superficie Celular/química , Extensiones de la Superficie Celular/metabolismo , Carcinogénesis/genética , Adhesión Celular , Quinasas MAP Reguladas por Señal Extracelular , Fibroblastos , Mutación , Forma de la Célula , Imagenología Tridimensional , Quinasas de Proteína Quinasa Activadas por MitógenosRESUMEN
Oxidative damage has been implicated in the pathogenesis of numerous disorders by affecting the normal functions of several tissues. Further, oxidative stress acts within cells to influence cell morphology and the behavior of cell migration. The movement and migration of cells are crucial during the development of organisms as they transition from embryo to adult, and for the homeostasis of adult tissues. Epicatechin (EC) is a natural flavonoid derived mostly from tea, chocolate, and red wine. We investigated the protective impact of EC on D-galactose(D-gal)/rotenone-injured NIH3T3 cells and found alterations in cell dynamics throughout the procedure. The results reveal that D-gal/rotenone stimulation can cause the cell area to expand and the number of cellular protrusions to increase. EC intervention can considerably minimize the oxidative damage of rotenone on NIH3T3 cells (p < 0.05) but showed little influence on cell damage induced by D-gal. Furthermore, the corrective ability of EC as an antioxidant is reflected in a dose-dependent effect on cell movement, including variations in movement speed and distance. Overall, from the perspective of cell morphology and cell motility, EC has a good protective impact on cells harmed by rotenone induced oxidative damage, as well as corrective properties as an antioxidant to balance intracellular oxidative stress, which allowing for a more comprehensive evaluation of antioxidant performance of EC.
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Antioxidantes , Catequina , Animales , Ratones , Antioxidantes/farmacología , Catequina/farmacología , Células 3T3 NIH , Estrés Oxidativo/efectos de los fármacos , Rotenona/farmacología , Galactosa/farmacología , Forma de la Célula/efectos de los fármacos , Extensiones de la Superficie Celular/efectos de los fármacos , Sustancias Protectoras/farmacologíaRESUMEN
Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.
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Extensiones de la Superficie Celular , Proteínas de Drosophila , Animales , Movimiento Celular/fisiología , Extensiones de la Superficie Celular/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores ErbB , Receptores de Quimiocina , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BAR (Bin, Amphiphysin, and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse BAR) domain containing tools derived from the fusion of the Arabidopsis thaliana cryptochrome 2 photoreceptor and I-BAR protein domains ("CRY-BARs") with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane-binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and phosphatidylinositol 4,5-bisphosphate-binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.
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Citoesqueleto de Actina , Proteínas de Arabidopsis , Membrana Celular , Optogenética , Citoesqueleto de Actina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Dominios Proteicos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Extensiones de la Superficie Celular/química , Optogenética/métodos , Humanos , Células HEK293RESUMEN
The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading ("Brownian zipper hypothesis") as well as protrusion-dominated spreading ("protrusive zipper hypothesis"). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.
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Extensiones de la Superficie Celular , Citoesqueleto , Adhesión Celular , Movimiento Celular , Humanos , FagocitosisRESUMEN
Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.