RESUMEN
Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.
Asunto(s)
Centrómero , Proteínas Cromosómicas no Histona , Autoantígenos/genética , Autoantígenos/metabolismo , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN/genética , HumanosRESUMEN
In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.
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Cromosomas Humanos , Cromosomas , Cromosomas/genética , Cromosomas/metabolismo , Cromosomas Humanos/metabolismo , ADN/química , ADN-Topoisomerasas de Tipo II/genética , Humanos , Mitosis , Óptica y FotónicaRESUMEN
Membrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.
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Fusión de Membrana , Membrana Dobles de Lípidos , Microscopía de Fuerza Atómica , Pinzas ÓpticasRESUMEN
Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.
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Actinas/química , Fibroblastos/química , Fibroblastos/citología , Modelos Biológicos , Actinas/fisiología , Animales , Fenómenos Biomecánicos , Línea Celular , Membrana Celular/química , Membrana Celular/fisiología , Fuerza Compresiva , Perros , Elasticidad , Microscopía de Fuerza Atómica , Miosinas/química , Miosinas/fisiología , Reología/métodos , ViscosidadRESUMEN
Biomineralization of CaCO3 commonly involves the formation of amorphous CaCO3 precursor particles that are produced in a confined space surrounded by a lipid bilayer. While the influence of confinement itself has been investigated with different model systems, the impact of an enclosing continuous lipid bilayer on CaCO3 formation in a confined space is still poorly understood as appropriate model systems are rare. Here, we present a new versatile method based on droplet-based microfluidics to produce fluid-phase giant unilamellar vesicles (GUVs) in the presence of high CaCl2 concentrations. These GUVs can be readily investigated by means of confocal laser scanning microscopy in combination with bright-field microscopy, demonstrating that the formed CaCO3 particles are in conformal contact with the fluid-phase lipid bilayer and thus suggesting a strong interaction between the particle and the membrane. Atomic force microscopy adhesion studies with membrane-coated spheres on different CaCO3 crystals corroborated this notion of a strong interaction between the lipids and CaCO3.
RESUMEN
Cellular adhesion is an intricate physical process controlled by ligand-receptor affinity, density, mobility, and external forces transmitted through the elastic properties of the cell. As a model for cellular adhesion we study the detachment of cell-sized liposomes and membrane-coated silica beads from supported bilayers using atomic force microscopy. Adhesion between the two surfaces is mediated by the interaction between the adhesive lipid anchored saccharides lactosylceramide and the ganglioside GM3. We found that force-distance curves of liposome detachment have a very peculiar, partially concave shape, reminiscent of the nonlinear extension of polymers. By contrast, detachment of membrane coated beads led to force-distance curves similar to the detachment of living cells. Theoretical modelling of the enforced detachment suggests that the non-convex force curve shape arises from the mobility of ligands provoking a switch of shapes from spherical to unduloidal during detachment.
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Liposomas , Adhesión Celular , Ligandos , Membranas , Microscopía de Fuerza AtómicaRESUMEN
Intermediate filaments (IFs) are part of the cytoskeleton of eukaryotic cells and, therefore, are largely responsible for the cell's mechanical properties. IFs are characterized by a pronounced extensibility and remarkable resilience that enable them to support cells in extreme situations. Previous experiments showed that, under strain, α-helices in vimentin IFs might unfold to ß-sheets. Upon repeated stretching, the filaments soften; however, the remaining plastic strain is negligible. Here, we observe that vimentin IFs do not recover their original stiffness on reasonable time scales, and we explain these seemingly contradicting results by introducing a third, less well-defined conformational state. Reversibility on the nanoscale can be fully rescued by introducing cross-linkers that prevent transition to the ß-sheet. Our results classify IFs as a nanomaterial with intriguing mechanical properties, which is likely to play a major role for the cell's local adaption to external stimuli.
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Filamentos Intermedios/química , Vimentina/química , Fenómenos Biomecánicos , Humanos , Nanoestructuras/química , Pinzas Ópticas , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Estrés MecánicoRESUMEN
The cytoskeleton is a composite network of three types of protein filaments, among which intermediate filaments (IFs) are the most extensible ones. Two very important IFs are keratin and vimentin, which have similar molecular architectures but different mechanical behaviors. Here we compare the mechanical response of single keratin and vimentin filaments using optical tweezers. We show that the mechanics of vimentin strongly depends on the ionic strength of the buffer and that its force-strain curve suggests a high degree of cooperativity between subunits. Indeed, a computational model indicates that in contrast to keratin, vimentin is characterized by strong lateral subunit coupling of its charged monomers during unfolding of α helices. We conclude that cells can tune their mechanics by differential use of keratin versus vimentin.
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Citoesqueleto/química , Queratinas/química , Modelos Biológicos , Vimentina/química , Fenómenos Biomecánicos , Tampones (Química) , Citoesqueleto/metabolismo , Queratinas/metabolismo , Microscopía de Fuerza Atómica , Pinzas Ópticas , Concentración Osmolar , Conformación Proteica en Hélice alfa , Vimentina/metabolismoRESUMEN
Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.
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Fusión de Membrana , Proteínas SNARE/química , Membrana Dobles de Lípidos/química , Liposomas/química , Microscopía/métodos , Dióxido de Silicio/químicaRESUMEN
The thylakoid membrane of algae and land plants is characterized by its intricate architecture, comprising tightly appressed membrane stacks termed grana. The contributions of individual components to grana stack formation are not yet fully elucidated. As an in vitro model, we use supported lipid bilayers made of thylakoid lipid mixtures to study the effect of major light-harvesting complex (LHCII), different lipids, and ions on membrane stacking, seen as elevated structures forming on top of the planar membrane surface in the presence of LHCII protein. These structures were examined by confocal laser scanning microscopy, atomic force microscopy, and fluorescence recovery after photobleaching, revealing multilamellar LHCII-membrane stacks composed of connected lipid bilayers. Both native-like and non-native interactions between the LHCII complexes may contribute to membrane appression in the supported bilayers. However, applying in vivo-like salt conditions to uncharged glycolipid membranes drastically increased the level of stack formation due to enforced LHCII-LHCII interactions, which is in line with recent crystallographic and cryo-electron microscopic data [Wan, T., et al. (2014) Mol. Plant 7, 916-919; Albanese, P., et al. (2017) Sci. Rep. 7, 10067-10083]. Furthermore, we observed the nonbilayer lipid MGDG to strongly promote membrane stacking, pointing to the long-term proposed function of MGDG in stabilizing the inner membrane leaflet of highly curved margins in the periphery of each grana disc because of its negative intrinsic curvature [Murphy, D. J. (1982) FEBS Lett. 150, 19-26].
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Diglicéridos/química , Complejos de Proteína Captadores de Luz/química , Membrana Dobles de Lípidos/química , Pisum sativum/enzimología , Complejos de Proteína Captadores de Luz/ultraestructura , Microscopía de Fuerza Atómica , Microscopía Confocal , Pisum sativum/ultraestructuraRESUMEN
The mechanical properties of eukaryotic cells are to a great extent determined by the cytoskeleton, a composite network of different filamentous proteins. Among these, intermediate filaments (IFs) are exceptional in their molecular architecture and mechanical properties. Here we directly record stress-strain curves of individual vimentin IFs using optical traps and atomic force microscopy. We find a strong loading rate dependence of the mechanical response, supporting the hypothesis that IFs could serve to protect eukaryotic cells from fast, large deformations. Our experimental results show different unfolding regimes, which we can quantitatively reproduce by an elastically coupled system of multiple two-state elements.
RESUMEN
Weak noncovalent intermolecular interactions play a pivotal role in many biological processes such as cell adhesion or immunology, where the overall binding strength is controlled through bond association and dissociation dynamics as well as the cooperative action of many parallel bonds. Among the various molecules participating in weak bonds, carbohydrate-carbohydrate interactions are probably the most ancient ones allowing individual cells to reversibly enter the multicellular state and to tell apart self and nonself cells. Here, we scrutinized the kinetics and thermodynamics of small homomeric Lewis X-Lewis X ensembles formed in the contact zone of a membrane-coated colloidal probe and a solid supported membrane ensuring minimal nonspecific background interactions. We used an atomic force microscope to measure force distance curves at Piconewton resolution, which allowed us to measure the force due to unbinding of the colloidal probe and the planar membrane as a function of contact time. Applying a contact model, we could estimate the free binding energy of the formed adhesion cluster as a function of dwell time and thereby determine the precise size of the contact zone, the number of participating bonds, and the intrinsic rates of association and dissociation in the presence of calcium ions. The unbinding energy per bond was found to be on the order of 1 kBT. Approximately 30 bonds were opened simultaneously at an off-rate of koff = 7 ± 0.2 s(-1).
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Trisacáridos/química , Trisacáridos/metabolismo , Membrana Celular/metabolismo , Cinética , Lípidos de la Membrana/metabolismo , Microscopía de Fuerza Atómica , Modelos Moleculares , TermodinámicaRESUMEN
Copper, iron, and gallium coordination chemistries of the new pentadentate bis-sulfonamide ligand 2,6-bis(N-2-pyridylmethylsulfonamido)-4-methylphenol (psmpH3) were investigated. PsmpH3 is capable of varying degrees of deprotonation, and notably, complexes containing the fully trideprotonated ligand can be prepared in aqueous solutions using only divalent metal ions. Two of the copper(II) complexes, [Cu2(psmp)(OH)] and [Cu2(psmp)(OAc)2](-), demonstrate the anticipated 1:2 ligand/metal stoichiometry and show that the dimetallic binding site created for exogenous ligands possesses high inherent flexibility since additional one- and three-atom bridging ligands bridge the two copper(II) ions in each complex, respectively. This gives rise to a difference of 0.4 Å in the Cu···Cu distances. Complexes with 2:3 and 2:1 ligand/metal stoichiometries for the divalent and trivalent metal ions, respectively, were observed in [Cu3(psmp)2(H2O)] and [M(psmpH)(psmpH2)], where M = Ga(III), Fe(III). The deprotonated tridentate N-2-pyridylsulfonylmethylphenolato moieties chelate the metal ions in a meridional fashion, whereas in [Cu3(psmp)2(H2O)] the rare µ2-N-sulfonamido bridging coordination mode is observed. In the bis-ligand mononuclear complexes, one picolyl arm of each ligand is protonated and uncoordinated. Magnetic susceptibility measurements on the doubly and triply bridged dicopper(II) complexes indicate strong and medium strength antiferromagnetic coupling interactions, with J = 234 cm(-1) and 115 cm(-1) for [Cu2(psmp)(OH)] and [Cu2(psmp)(OAc)2](-), respectively (in HHDvV =...+JS1S2 convention). The trinuclear [Cu3(psmp)2(H2O)], in which the central copper ion is linked to two flanking copper atoms by two µ2-N-sulfonamido bridges and two phenoxide bridges shows an overall magnetic behavior of antiferromagnetic coupling. This is corroborated computationally by broken-symmetry density functional theory, which for isotropic modeling of the coupling predicts an antiferromagnetic coupling strength of J = 70.5 cm(-1).
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In the context of soft matter and cellular mechanics, microrheology - the use of micron-sized particles to probe the frequency-dependent viscoelastic response of materials - is widely used to shed light onto the mechanics and dynamics of molecular structures. Here we present the implementation of active microrheology in an Acoustic Force Spectroscopy setup (AFMR), which combines multiplexing with the possibility of probing a wide range of forces ( ~ pN to ~nN) and frequencies (0.01-100 Hz). To demonstrate the potential of this approach, we perform active microrheology on biological samples of increasing complexity and stiffness: collagen gels, red blood cells (RBCs), and human fibroblasts, spanning a viscoelastic modulus range of five orders of magnitude. We show that AFMR can successfully quantify viscoelastic properties by probing many beads with high single-particle precision and reproducibility. Finally, we demonstrate that AFMR to map local sample heterogeneities as well as detect cellular responses to drugs.
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Elasticidad , Eritrocitos , Fibroblastos , Reología , Humanos , Viscosidad , Fibroblastos/fisiología , Reología/métodos , Colágeno/química , AcústicaRESUMEN
Stimuli responsive polymer coatings are a common motive for designing surfaces for cell biological applications. In the present study, we have characterized temperature dependent adhesive properties of poly(N-isopropylacrylamide) (PNIPAm) microgel coated surfaces (PMS) using various atomic force microscopy based approaches. We imaged and quantified the material properties of PMS upon a temperature switch using quantitative AFM imaging but also employed single-cell force spectroscopy (SCFS) before and after decreasing the temperature to assess the forces and work of initial adhesion between cells and PMS. We performed a detailed analysis of steps in the force-distance curves. Finally, we applied colloid probe atomic force microscopy (CP-AFM) to analyze the adhesive properties of two major components of the extracellular matrix to PMS under temperature control, namely collagen I and fibronectin. In combination with confocal imaging, we could show that these two ECM components differ in their detachment properties from PNIPAm microgel films upon cell harvesting, and thus gained a deeper understanding of cell-sheet maturation and harvesting process and the involved partial ECM dissolution.
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Resinas Acrílicas/química , Adhesión Celular , Geles/química , Animales , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Perros , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Oro/química , Células de Riñón Canino Madin Darby , Microscopía de Fuerza Atómica , Propiedades de Superficie , TemperaturaRESUMEN
Force spectroscopy allows the manipulation of single molecules and the characterization of their properties and interactions thereby rendering it a powerful tool for biological sciences. Force spectroscopy at the level of individual molecules requires force resolution in the piconewton regime as achieved by optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM) with AFM providing the largest force range from tenth of piconewton to several micronewton. In membrane probe spectroscopy the commonly used sharp cantilever tip is replaced by a lipid-coated glass sphere. This technique expands the scope of force spectroscopy to processes at and between lipid bilayers, like the formation of coiled coils between SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins as well as subsequent membrane fusion. To this end, two solid-supported membranes equipped with SNARE proteins or fusion peptides are separately deposited on a flat glassy surface and on a micrometer glass sphere attached to the end of a tipless AFM cantilever. These two membranes are rapidly brought into contact until a defined force is reached. The AFM deflection readout is used to monitor the distance between the two bilayers, which allows to observe and identify fusion processes of the two lipid membranes, while the forces needed to separate the two surfaces give insights into the formation of SNARE complexes. By changing the contact pressure one can access fusion kinetics and to some extent reconstruct the energy landscape of membrane fusion. In this chapter we describe the preparation of membrane-coated colloidal probes attached to AFM cantilevers, experimental procedures, and necessary data analysis to perform membrane probe spectroscopy in the presence of fusogenic peptides or proteins.
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Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana , Proteínas SNARE/metabolismo , Análisis Espectral/métodos , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Pinzas Ópticas , Dominios Proteicos , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas SNARE/química , Análisis Espectral/instrumentaciónRESUMEN
Biological adhesion is essential for all motile cells and generally limits locomotion to suitably functionalized substrates displaying a compatible surface chemistry. However, organisms that face vastly varying environmental challenges require a different strategy. The model organism Dictyostelium discoideum (D.d.), a slime mould dwelling in the soil, faces the challenge of overcoming variable chemistry by employing the fundamental forces of colloid science. To understand the origin of D.d. adhesion, we realized and modified a variety of conditions for the amoeba comprising the absence and presence of the specific adhesion protein Substrate Adhesion A (sadA), glycolytic degradation, ionic strength, surface hydrophobicity and strength of van der Waals interactions by generating tailored model substrates. By employing AFM-based single cell force spectroscopy we could show that experimental force curves upon retraction exhibit two regimes. The first part up to the critical adhesion force can be described in terms of a continuum model, while the second regime of the curve beyond the critical adhesion force is governed by stochastic unbinding of individual binding partners and bond clusters. We found that D.d. relies on adhesive interactions based on EDL-DLVO (Electrical Double Layer-Derjaguin-Landau-Verwey-Overbeek) forces and contributions from the glycocalix and specialized adhesion molecules like sadA. This versatile mechanism allows the cells to adhere to a large variety of natural surfaces under various conditions.
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Adhesión Celular , Dictyostelium/citología , Microscopía de Fuerza Atómica , Adhesividad , Coloides/química , Glicocálix/química , Proteínas Fluorescentes Verdes/química , Interacciones Hidrofóbicas e Hidrofílicas , Iones , Fenómenos Mecánicos , Nanopartículas/química , Nanotecnología , Análisis Espectral , Electricidad Estática , Propiedades de Superficie , HumectabilidadRESUMEN
Structure and dynamics of living matter rely on design principles fundamentally different from concepts of traditional material science. Specialized intracellular filaments in the cytoskeleton permit living systems to divide, migrate, and grow with a high degree of variability and durability. Among the three filament systems, microfilaments, microtubules, and intermediate filaments (IFs), the physical properties of IFs and their role in cellular mechanics are the least well understood. We use optical trapping of individual vimentin filaments to investigate energy dissipation, strain history dependence, and creep behavior of stretched filaments. By stochastic and numerical modeling, we link our experimental observations to the peculiar molecular architecture of IFs. We find that individual vimentin filaments display tensile memory and are able to dissipate more than 70% of the input energy. We attribute these phenomena to distinct nonequilibrium folding and unfolding of α helices in the vimentin monomers constituting the filaments.
RESUMEN
In the photosynthetic apparatus of plants a high proportion of LHCII protein is needed to integrate 50% non-bilayer lipid MGDG into the lamellar thylakoid membrane, but whether and how the stability of the protein is also affected is not known. Here we use single-molecule force spectroscopy to map the stability of LHCII against mechanical unfolding along the polypeptide chain as a function of oligomerization state and lipid composition. Comparing unfolding forces between monomeric and trimeric LHCII demonstrates that the stability does not increase significantly upon trimerization but can mainly be correlated with specific contact sites between adjacent monomers. In contrast, unfolding of trimeric complexes in membranes composed of different thylakoid lipids reveals that the non-bilayer lipid MGDG substantially increases the mechanical stability of LHCII in many segments of the protein compared to other lipids such as DGDG or POPG. We attribute these findings to steric matching of conically formed MGDG and the hourglass shape of trimeric LHCII, thereby extending the role of non-bilayer lipids to the structural stabilization of membrane proteins in addition to the modulation of their folding, conformation and function.