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Membrane-induced interactions play an important role in organizing membrane proteins. Measurements of the interactions between two and three membrane deforming objects have revealed their nonadditive nature. They are thought to lead to complex many-body effects, however, experimental evidence is lacking. We here present an experimental method to measure many-body effects in membrane-mediated interactions using colloidal spheres placed between a deflated giant unilamellar vesicles and a planar substrate. The confined colloidal particles cause a large deformation of the membrane while not being physicochemically attached to it and interact through it. Two particles attract with a maximum force of 0.2 pN. For three particles, compact equilateral triangles were preferred over linear arrangements. We use numerical energy minimization to establish that the attraction stems from a reduction in the membrane-deformation energy caused by the particles. Confining up to 36 particles, we find a preference for hexagonally close packed clusters. However, with increasing number of particles the order of the confined particles decreases, at the same time, diffusivity of the particles increases. Our experiments show that the nonadditive nature of membrane-mediated interactions affects the interactions and arrangements and ultimately leads to spherical aggregates with liquid-like order of potential importance for cellular processes.
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Cluster formation of microscopic swimmers is key to the formation of biofilms and colonies, efficient motion and nutrient uptake, but, in the absence of other interactions, requires high swimmer concentrations to occur. Here we experimentally and numerically show that cluster formation can be dramatically enhanced by an anisotropic swimmer shape. We analyze a class of model microswimmers with a shape that can be continuously tuned from spherical to bent and straight rods. In all cases, clustering can be described by Michaelis-Menten kinetics governed by a single scaling parameter that depends on particle density and shape only. We rationalize these shape-dependent dynamics from the interplay between interlocking probability and cluster stability. The bent rod shape promotes assembly in an interlocking fashion even at vanishingly low particle densities and we identify the most efficient shape to be a semicircle. Our work provides key insights into how shape can be used to rationally design out-of-equilibrium self-organization, key to creating active functional materials and processes that require two-component assembly with high fidelity.
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The membrane curvature of cells and intracellular compartments continuously adapts to enable cells to perform vital functions, from cell division to signal trafficking. Understanding how membrane geometry affects these processes in vivo is challenging because of the biochemical and geometrical complexity as well as the short time and small length scales involved in cellular processes. By contrast, in vitro model membranes with engineered curvature would provide a versatile platform for this investigation and applications to biosensing and biocomputing. Here, we present a strategy that allows fabrication of lipid membranes with designed shape by combining 3D micro-printing and replica-molding lithography with polydimethylsiloxane to create curved micrometer-sized scaffolds with virtually any geometry. The resulting supported lipid membranes are homogeneous and fluid. We demonstrate the versatility of the system by fabricating structures of interesting combinations of mean and Gaussian curvature. We study the lateral phase separation and how local curvature influences the effective diffusion coefficient. Overall, we offer a bio-compatible platform for understanding curvature-dependent cellular processes and developing programmable bio-interfaces for living cells and nanostructures.
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Active colloidal microswimmers serve as archetypical active fluid systems, and as models for biological swimmers. Here, by studying in detail their velocity traces, we find robust power-law intermittency with system-dependent exponential cut off. We model the intermittent motion by an interplay of the field gradient-dependent active force, which depends on a fluid gradient and is reduced when the swimmer moves, and the locally fluctuating hydrodynamic drag, that is set by the wetting properties of the substrate. The model closely describes the velocity distributions of two disparate swimmer systems: AC field activated and catalytic swimmers. The generality is highlighted by the collapse of all data in a single master curve, suggesting the applicability to further systems, both synthetic and biological.
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Floppy microscale spring networks are widely studied in theory and simulations, but no well-controlled experimental system currently exists. Here, we show that square lattices consisting of colloid-supported lipid bilayers functionalized with DNA linkers act as microscale floppy spring networks. We extract their normal modes by inverting the particle displacement correlation matrix, showing the emergence of a spectrum of soft modes with low effective stiffness in addition to stiff modes that derive from linker interactions. Evaluation of the softest mode, a uniform shear mode, reveals that shear stiffness decreases with lattice size. Experiments match well with Brownian particle simulations, and we develop a theoretical description based on mapping interactions onto a linear response model to describe the modes. Our results reveal the importance of entropic steric effects and can be used for developing reconfigurable materials at the colloidal length scale.
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Many cell functions require a concerted effort from multiple membrane proteins, for example, for signaling, cell division, and endocytosis. One contribution to their successful self-organization stems from the membrane deformations that these proteins induce. While the pairwise interaction potential of two membrane-deforming spheres has recently been measured, membrane-deformation-induced interactions have been predicted to be nonadditive, and hence their collective behavior cannot be deduced from this measurement. We here employ a colloidal model system consisting of adhesive spheres and giant unilamellar vesicles to test these predictions by measuring the interaction potential of the simplest case of three membrane-deforming, spherical particles. We quantify their interactions and arrangements and, for the first time, experimentally confirm and quantify the nonadditive nature of membrane-deformation-induced interactions. We furthermore conclude that there exist two favorable configurations on the membrane: (1) a linear and (2) a triangular arrangement of the three spheres. Using Monte Carlo simulations, we corroborate the experimentally observed energy minima and identify a lowering of the membrane deformation as the cause for the observed configurations. The high symmetry of the preferred arrangements for three particles suggests that arrangements of many membrane-deforming objects might follow simple rules.
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Proteínas de Membrana , Método de Monte CarloRESUMO
The impact of space radiation and microgravity on DNA damage responses has been discussed controversially, largely due to the variety of model systems engaged. Here, we performed side-by-side analyses of human hematopoietic stem/progenitor cells (HSPC) and peripheral blood lymphocytes (PBL) cultivated in a 2D clinostat to simulate microgravity before, during and after photon and particle irradiation. We demonstrate that simulated microgravity (SMG) accelerates the early phase of non-homologous end joining (NHEJ)-mediated repair of simple, X-ray-induced DNA double-strand breaks (DSBs) in PBL, while repair kinetics in HSPC remained unaltered. Repair acceleration was lost with increasing LET of ion exposures, which increases the complexity of DSBs, precluding NHEJ and requiring end resection for successful repair. Such cell-type specific effect of SMG on DSB repair was dependent on the NF-кB pathway pre-activated in PBL but not HSPC. Already under unperturbed growth conditions HSPC and PBL suffered from SMG-induced replication stress associated with accumulation of single-stranded DNA and DSBs, respectively. We conclude that in PBL, SMG-induced DSBs promote repair of radiation-induced damage in an adaptive-like response. HSPC feature SMG-induced single-stranded DNA and FANCD2 foci, i.e., markers of persistent replication stress and senescence that may contribute to a premature decline of the immune system in space.
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Reparo do DNA , Sistema Hematopoético , Humanos , DNA de Cadeia Simples , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Dano ao DNARESUMO
Colloidal molecules are ideal model systems for mimicking real molecules and can serve as versatile building blocks for the bottom-up self-assembly of flexible and smart materials. While most colloidal molecules are rigid objects, the development of colloidal joints has made it possible to endow them with conformational flexibility. However, their unrestricted range of motion does not capture the limited movement and bond directionality that is instead typical of real molecules. In this work, we create flexible colloidal molecules with an in situ controllable motion range and bond directionality by assembling spherical particles onto cubes functionalized with complementary surface-mobile DNA. By varying the sphere-to-cube size ratio, we obtain colloidal molecules with different coordination numbers and find that they feature a constrained range of motion above a critical size ratio. Using theory and simulations, we show that the particle shape together with the multivalent bonds creates an effective free-energy landscape for the motion of the sphere on the surface of the cube. We quantify the confinement of the spheres on the surface of the cube and the probability to change facet. We find that temperature can be used as an extra control parameter to switch in situ between full and constrained flexibility. These flexible colloidal molecules with a temperature switching motion range can be used to investigate the effect of directional yet flexible bonds in determining their self-assembly and phase behavior, and may be employed as constructional units in microrobotics and smart materials.
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Endocytosis is a key cellular process involved in the uptake of nutrients, pathogens, or the therapy of diseases. Most studies have focused on spherical objects, whereas biologically relevant shapes can be highly anisotropic. In this letter, we use an experimental model system based on Giant Unilamellar Vesicles (GUVs) and dumbbell-shaped colloidal particles to mimic and investigate the first stage of the passive endocytic process: engulfment of an anisotropic object by the membrane. Our model has specific ligand-receptor interactions realized by mobile receptors on the vesicles and immobile ligands on the particles. Through a series of experiments, theory, and molecular dynamics simulations, we quantify the wrapping process of anisotropic dumbbells by GUVs and identify distinct stages of the wrapping pathway. We find that the strong curvature variation in the neck of the dumbbell as well as membrane tension are crucial in determining both the speed of wrapping and the final states.
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Ring, or cyclic, polymers have unique properties compared to linear polymers, due to their topologically closed structure that has no beginning or end. Experimental measurements on the conformation and diffusion of molecular ring polymers simultaneously are challenging due to their inherently small size. Here, we study an experimental model system for cyclic polymers, that consists of rings of flexibly linked micron-sized colloids with n=4-8 segments. We characterize the conformations of these flexible colloidal rings and find that they are freely jointed up to steric restrictions. We measure their diffusive behavior and compare it to hydrodynamic simulations. Interestingly, flexible colloidal rings have a larger translational and rotational diffusion coefficient compared to colloidal chains. In contrast to chains, their internal deformation mode shows slower fluctuations for nâ²8 and saturates for higher values of n. We show that constraints stemming from the ring structure cause this decrease in flexibility for small n and infer the expected scaling of the flexibility as function of ring size. Our findings could have implications for the behavior of both synthetic and biological ring polymers, as well as for the dynamic modes of floppy colloidal materials.
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HYPOTHESIS: Colloidal molecules with anisotropic shapes and interactions are powerful model systems for deciphering the behavior of real molecules and building units for creating materials with designed properties. While many strategies for their assembly have been developed, they typically yield a broad distribution or are limited to a specific type. We hypothesize that the shape and relative sizes of colloidal particles can be exploited to efficiently direct their assembly into colloidal molecules of desired valence. EXPERIMENTS: We exploit electrostatic self-assembly of negatively charged spheres made from either polystyrene or silica onto positively charged hematite cubes. We thoroughly analyze the role of the shape and size ratio of particles on the cluster size and yield of colloidal molecules. FINDINGS: Using a combination of experiments and simulations, we demonstrate that cubic particle shape is crucial to generate high yields of distinct colloidal molecules over a wide variety of size ratios. We find that electrostatic repulsion between the satellite spheres is important to leverage the templating effect of the cubes, leading the spheres to preferentially assemble on the facets rather than the edges and corners of the cube. The sixfold symmetry of cubes favors the assembly of molecules with six, four, and two satellite spheres at appropriate size ratios and interaction strength. Furthermore, we reveal that our protocol is not affected by the specific choice of the material of the colloidal particles. Finally, we show that the permanent magnetic dipole moment of the hematite cubes can be utilized to separate colloidal molecules from non-assembled satellite particles. Our simple and effective strategy might be extended to other templating particle shapes, thereby greatly expanding the library of colloidal molecules that can be achieved with high yield and purity.
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Coloides , Poliestirenos , Compostos Férricos , Dióxido de SilícioRESUMO
Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.
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Reabsorção Óssea , Osteoclastos , Reabsorção Óssea/metabolismo , Citocinas/metabolismo , Humanos , Osteoclastos/metabolismo , Raios XRESUMO
Cooperative motion in biological microswimmers is crucial for their survival as it facilitates adhesion to surfaces, formation of hierarchical colonies, efficient motion, and enhanced access to nutrients. Here, we confine synthetic, catalytic microswimmers along one-dimensional paths and demonstrate that they too show a variety of cooperative behaviours. We find that their speed increases with the number of swimmers, and that the activity induces a preferred distance between swimmers. Using a minimal model, we ascribe this behavior to an effective activity-induced potential that stems from a competition between chemical and hydrodynamic coupling. These interactions further induce active self-assembly into trains where swimmers move at a well-separated, stable distance with respect to each other, as well as compact chains that can elongate, break-up, become immobilized and remobilized. We identify the crucial role that environment morphology and swimmer directionality play on these highly dynamic chain behaviors. These activity-induced interactions open the door toward exploiting cooperation for increasing the efficiency of microswimmer motion, with temporal and spatial control, thereby enabling them to perform intricate tasks inside complex environments.
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Hidrodinâmica , Movimento (Física)RESUMO
Colloidal molecules are designed to mimic their molecular analogues through their anisotropic shape and interactions. However, current experimental realizations are missing the structural flexibility present in real molecules thereby restricting their use as model systems. We overcome this limitation by assembling reconfigurable colloidal molecules from silica particles functionalized with mobile DNA linkers in high yields. We achieve this by steering the self-assembly pathway toward the formation of finite-sized clusters by employing high number ratios of particles functionalized with complementary DNA strands. The size ratio of the two species of particles provides control over the overall cluster size, i.e., the number of bound particles N, as well as the degree of reconfigurability. The bond flexibility provided by the mobile linkers allows the successful assembly of colloidal clusters with the geometrically expected maximum number of bound particles and shape. We quantitatively examine the self-assembly dynamics of these flexible colloidal molecules by a combination of experiments, agent-based simulations, and an analytical model. Our "flexible colloidal molecules" are exciting building blocks for investigating and exploiting the self-assembly of complex hierarchical structures, photonic crystals, and colloidal metamaterials.
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Coloides , Fótons , Anisotropia , Coloides/químicaRESUMO
Reliably distinguishing between cells based on minute differences in receptor density is crucial for cell-cell or virus-cell recognition, the initiation of signal transduction, and selective targeting in directed drug delivery. Such sharp differentiation between different surfaces based on their receptor density can only be achieved by multivalent interactions. Several theoretical and experimental works have contributed to our understanding of this "superselectivity." However, a versatile, controlled experimental model system that allows quantitative measurements on the ligand-receptor level is still missing. Here, we present a multivalent model system based on colloidal particles equipped with surface-mobile DNA linkers that can superselectively target a surface functionalized with the complementary mobile DNA-linkers. Using a combined approach of light microscopy and Foerster resonance energy transfer (FRET), we can directly observe the binding and recruitment of the ligand-receptor pairs in the contact area. We find a nonlinear transition in colloid-surface binding probability with increasing ligand or receptor concentration. In addition, we observe an increased sensitivity with weaker ligand-receptor interactions, and we confirm that the timescale of binding reversibility of individual linkers has a strong influence on superselectivity. These unprecedented insights on the ligand-receptor level provide dynamic information into the multivalent interaction between two fluidic membranes mediated by both mobile receptors and ligands and will enable future work on the role of spatial-temporal ligand-receptor dynamics on colloid-surface binding.
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Coloides/química , Sistemas de Liberação de Medicamentos , DNA/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ligantes , Modelos Químicos , Ligação Proteica , Propriedades de SuperfícieRESUMO
Microswimmers typically move near walls, which can strongly influence their motion. However, direct experimental measurements of swimmer-wall separation remain elusive to date. Here, we determine this separation for model catalytic microswimmers from the height dependence of the passive component of their mean-squared displacement. We find that swimmers exhibit "ypsotaxis," a tendency to assume a fixed height above the wall for a range of salt concentrations, swimmer surface charges, and swimmer sizes. Our findings indicate that ypsotaxis is activity induced, posing restrictions on future modeling of their still-debated propulsion mechanism.
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Synthetic microswimmers are widely employed model systems in the studies of out-of-equilibrium phenomena. Unlike biological microswimmers which naturally occur in various shapes and forms, synthetic microswimmers have so far been limited almost exclusively to spherical shapes. Here, we exploit 3D printing to produce microswimmers with complex shapes in the colloidal size regime. We establish the flexibility of 3D printing by two-photon polymerisation to produce particles smaller than 10 microns with a high-degree of shape complexity. We further demonstrate that 3D printing allows control over the location of the active site through orienting the particles in different directions during printing. We verify that particles behave colloidally by imaging their motion in the passive and active states and by investigating their mean square displacement. In addition, we find that particles exhibit shape-dependant behavior, thereby demonstrating the potential of our method to launch a wide-range of in-depth studies into shape-dependent active motion and behaviour.
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Previous studies on the phase behaviour of multicomponent lipid bilayers found an intricate interplay between membrane geometry and its composition, but a fundamental understanding of curvature-induced effects remains elusive. Thanks to a combination of experiments on lipid vesicles supported by colloidal scaffolds and theoretical work, we demonstrate that the local geometry and global chemical composition of the bilayer determine both the spatial arrangement and the amount of mixing of the lipids. In the mixed phase, a strong geometrical anisotropy can give rise to an antimixed state, where the lipids are mixed, but their relative concentration varies across the membrane. After phase separation, the bilayer organizes in multiple lipid domains, whose location is pinned in specific regions, depending on the substrate curvature and the bending rigidity of the lipid domains. Our results provide critical insights into the phase separation of cellular membranes and, more generally, two-dimensional fluids on curved substrates.
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Membrana Celular/química , Bicamadas Lipídicas/química , Microdomínios da Membrana , Lipossomos/químicaRESUMO
Experiments on supported lipid bilayers featuring liquid ordered/disordered domains have shown that the spatial arrangement of the lipid domains and their chemical composition are strongly affected by the curvature of the substrate. Furthermore, theoretical predictions suggest that both these effects are intimately related with the closed topology of the bilayer. In this work, we test this hypothesis by fabricating supported membranes consisting of colloidal particles of various shapes lying on a flat substrate. A single lipid bilayer coats both colloids and substrate, allowing local lipid exchange between them, thus rendering the system thermodynamically open, i.e. able to exchange heat and molecules with an external reservoir in the neighborhood of the colloid. By reconstructing the Gibbs phase diagram for this system, we demonstrate that the free-energy landscape is directly influenced by the geometry of the colloid. In addition, we find that local lipid exchange enhances the pinning of the liquid disordered phase in highly curved regions. This allows us to provide estimates of the bending moduli difference of the domains. Finally, by combining experimental and numerical data, we forecast the outcome of possible experiments on catenoidal and conical necks and show that these geometries could greatly improve the precision of the current estimates of the bending moduli.
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Bicamadas Lipídicas/química , Coloides/química , TermodinâmicaRESUMO
Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation.