RESUMEN
We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly.
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Cinetocoros , Microtúbulos , Centrómero , Centrosoma , Segregación Cromosómica , Mitosis , Huso AcromáticoRESUMEN
Fibrin formation and mechanical stability are essential in thrombosis and hemostasis. To reveal how mechanical load impacts fibrin, we carried out optical trap-based single-molecule forced unbinding experiments. The strength of noncovalent A:a knob-hole bond stabilizing fibrin polymers first increases with tensile force (catch bonds) and then decreases with force when the force exceeds a critical value (slip bonds). To provide the structural basis of catch-slip-bond behavior, we analyzed crystal structures and performed molecular modeling of A:a knob-hole complex. The movable flap (residues γ295 to γ305) containing the weak calcium-binding site γ2 serves as a tension sensor. Flap dissociation from the B domain in the γ-nodule and translocation to knob 'A' triggers hole 'a' closure, resulting in the increase of binding affinity and prolonged bond lifetimes. The discovery of biphasic kinetics of knob-hole bond rupture is quantitatively explained by using a theory, formulated in terms of structural transitions in the binding pocket between the low-affinity (slip) and high-affinity (catch) states. We provide a general framework to understand the mechanical response of protein pairs capable of tension-induced remodeling of their association interface. Strengthening of the A:a knob-hole bonds at 30- to 40-pN forces might favor formation of nascent fibrin clots subject to hydrodynamic shear in vivo.
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Calcio/química , Fibrina/química , Complejos Multiproteicos/química , Sitios de Unión , Calcio/metabolismo , Fibrina/metabolismo , Humanos , Complejos Multiproteicos/metabolismoRESUMEN
We carried out dynamic force manipulations in silico on a variety of coiled-coil protein fragments from myosin, chemotaxis receptor, vimentin, fibrin, and phenylalanine zippers that vary in size and topology of their α-helical packing. When stretched along the superhelical axis, all superhelices show elastic, plastic, and inelastic elongation regimes and undergo a dynamic transition from the α-helices to the ß-sheets, which marks the onset of plastic deformation. Using the Abeyaratne-Knowles formulation of phase transitions, we developed a new theoretical methodology to model mechanical and kinetic properties of protein coiled-coils under mechanical nonequilibrium conditions and to map out their energy landscapes. The theory was successfully validated by comparing the simulated and theoretical force-strain spectra. We derived the scaling laws for the elastic force and the force for α-to-ß transition, which can be used to understand natural proteins' properties as well as to rationally design novel biomaterials of required mechanical strength with desired balance between stiffness and plasticity.
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Simulación de Dinámica Molecular , Péptidos/química , Cinética , Modelos Moleculares , Transición de Fase , Estructura Secundaria de ProteínaRESUMEN
The mechanical properties of virus capsids correlate with local conformational dynamics in the capsid structure. They also reflect the required stability needed to withstand high internal pressures generated upon genome loading and contribute to the success of important events in viral infectivity, such as capsid maturation, genome uncoating and receptor binding. The mechanical properties of biological nanoparticles are often determined from monitoring their dynamic deformations in Atomic Force Microscopy nanoindentation experiments; but a comprehensive theory describing the full range of observed deformation behaviors has not previously been described. We present a new theory for modeling dynamic deformations of biological nanoparticles, which considers the non-linear Hertzian deformation, resulting from an indenter-particle physical contact, and the bending of curved elements (beams) modeling the particle structure. The beams' deformation beyond the critical point triggers a dynamic transition of the particle to the collapsed state. This extreme event is accompanied by a catastrophic force drop as observed in the experimental or simulated force (F)-deformation (X) spectra. The theory interprets fine features of the spectra, including the nonlinear components of the FX-curves, in terms of the Young's moduli for Hertzian and bending deformations, and the structural damage dependent beams' survival probability, in terms of the maximum strength and the cooperativity parameter. The theory is exemplified by successfully describing the deformation dynamics of natural nanoparticles through comparing theoretical curves with experimental force-deformation spectra for several virus particles. This approach provides a comprehensive description of the dynamic structural transitions in biological and artificial nanoparticles, which is essential for their optimal use in nanotechnology and nanomedicine applications.
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Fenómenos Biomecánicos/fisiología , Proteínas de la Cápside/ultraestructura , Dinámicas no Lineales , Virión/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/fisiología , Biología Computacional , Simulación de Dinámica Molecular , Conformación Proteica , Virión/química , Virión/fisiologíaRESUMEN
Hydrodynamic interactions (HI) are incorporated into Langevin dynamics of the Cα -based protein model using the Truncated Expansion approximation (TEA) to the Rotne-Prager-Yamakawa diffusion tensor. Computational performance of the obtained GPU realization demonstrates the model's capability for describing protein systems of varying complexity (10(2) -10(5) residues), including biological particles (filaments, virus shells). Comparison of numerical accuracy of the TEA versus exact description of HI reveals similar results for the kinetics and thermodynamics of protein unfolding. The HI speed up and couple biomolecular transitions through cross-communication among protein domains, which result in more collective displacements of structure elements governed by more deterministic (less variable) dynamics. The force-extension/deformation spectra from nanomanipulations in silico exhibit sharper force signals that match well the experimental profiles. Hence, biomolecular simulations without HI overestimate the role of tension/stress fluctuations. Our findings establish the importance of incorporating implicit water-mediated many-body effects into theoretical modeling of dynamic processes involving biomolecules. © 2016 Wiley Periodicals, Inc.
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Hidrodinámica , Modelos Moleculares , Proteínas/química , Solventes/química , Algoritmos , Simulación por Computador , Pliegue de Proteína , Elementos Estructurales de las Proteínas , Programas Informáticos , TermodinámicaRESUMEN
Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs 'A' and 'B' in the central nodule of fibrin monomer to complementary holes 'a' and 'b' in the γ- and ß-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using molecular dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob 'A' mimetic) and GHRP (knob 'B' mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; however, the dissociation kinetics were sensitive to variations in acidity (pH 5-7) and temperature (T = 25-37 °C). There were similar structural changes in holes 'a' and 'b' during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of the interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an "all-or-none" transition as it occurred through distinct two-step or single step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH 7 than at pH 5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH 7 but stronger, tighter, and less compliant at pH 5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T = 37 °C) compared with T = 25 °C due to the helix-to-coil transition in loop I that helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.
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Fibrina/química , Termodinámica , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , TemperaturaRESUMEN
BACKGROUND: Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions. RESULTS: We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences. CONCLUSIONS: Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion.
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Malaria/parasitología , Plasmodium falciparum/genética , ADN Intergénico/genética , ADN Intergénico/metabolismo , Expresión Génica , Genoma de Protozoos , Humanos , Malaria/patología , Nucleosomas/metabolismo , Sistemas de Lectura Abierta/genética , Plasmodium falciparum/metabolismo , Poli dA-dT/química , Poli dA-dT/metabolismo , Regiones no Traducidas/genéticaRESUMEN
Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral noncovalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physicochemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversible dissociation of lateral bonds followed by irreversible dissociation of the longitudinal bonds. We have determined the free energies of dissociation of the lateral (6.9 ± 0.4 kcal/mol) and longitudinal (14.9 ± 1.5 kcal/mol) tubulin-tubulin bonds. These values in conjunction with the large flexural rigidity of tubulin protofilaments obtained (18,000-26,000 pN·nm(2)) support the idea that the disassembling microtubule is capable of generating a large mechanical force to move chromosomes during cell division. Our computational modeling offers a comprehensive quantitative platform to link molecular tubulin characteristics with the physiological behavior of microtubules. The developed in silico nanoindentation method provides a powerful tool for the exploration of biomechanical properties of other cytoskeletal and multiprotein assemblies.
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Microtúbulos/química , Nanoestructuras/química , Termodinámica , Tubulina (Proteína)/química , Modelos Moleculares , Polímeros/químicaRESUMEN
Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔHind = 11.5-12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔSind = 5.1-5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.
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Bromovirus/fisiología , Cápside/química , Cápside/fisiología , Simulación por Computador , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Modelos Moleculares , TermodinámicaRESUMEN
Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding.
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Miembro Posterior/cirugía , Queratinocitos/fisiología , Queratinas/genética , Regeneración/efectos de la radiación , Transcriptoma , Amputación Quirúrgica , Animales , Femenino , Sitios Genéticos , Genoma , Queratinocitos/metabolismo , Queratinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Regeneración/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Quartz crystal microbalances (QCMs) measure mass on the nanogram (ng) scale. We built novel QCMs as toxicity biosensors incorporating living cells. Human endothelial cells or canine macrophages were equilibrated on QCM crystal surfaces until stable oscillation frequencies occurred. Vehicle or sodium azide (NaN3) (25-100 mM) was added to these QCMs while continuously collecting crystal oscillation frequency data. At these doses, NaN3 alters mitochondrial membrane permeability and causes mitochondrial swelling and intrinsic apoptosis. Our studies demonstrated no frequency change in QCMs with untreated cells or without cells but NaN3. If NaN3 was added to either cell type within QCMs, 5 to 8 min later increases in oscillation frequency (Δf) occurred (400-1600 Hz) that correlated with dose. All frequency changes reverted to baseline by 15 min. In parallel, during the first 30 min, no change in cell or nuclear areas, or in actin or microtubule distributions, was detected. Yet, mitochondrial size and membrane permeability increased significantly during, but not after, 5 to 8 min. Viability studies confirmed dose-dependent toxicity that was predicted and proportionate to the 5- to 8-min Δf. These studies confirm that cell-based QCMs can detect early events in intrinsic apoptosis and reveal unique kinetic information about events occurring within subcellular structures in response to toxins.
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Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Tecnicas de Microbalanza del Cristal de Cuarzo , Azida Sódica/toxicidad , Animales , Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Perros , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Pulmón/citología , Tamaño Mitocondrial/efectos de los fármacos , Permeabilidad/efectos de los fármacosRESUMEN
The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to address two questions: how rapidly the microtubule-kinetochore connections are made, and how accurate these connections are. In most previous numerical simulations, the model geometry was drastically simplified. Using the CellDynaMo computational platform, we previously introduced a geometrically and mechanically realistic 3D model of the prometaphase mitotic spindle, and used it to evaluate thermal noise and microtubule kinetics effects on the capture of a single chromosome. Here, we systematically investigate how geometry and mechanics affect a spindle assembly's speed and accuracy, including nuanced distinctions between merotelic, mero-amphitelic, and mero-syntelic chromosomes. We find that softening of the centromere spring improves accuracy for short chromosome arms, but accuracy disappears for long chromosome arms. Initial proximity of chromosomes to one spindle pole makes assembly accuracy worse, while initial chromosome orientation matters less. Chromokinesins, added onto flexible chromosome arms, allow modeling of the polar ejection force, improving a spindle assembly's accuracy for a single chromosome. However, spindle space crowding by multiple chromosomes worsens assembly accuracy. Our simulations suggest that the complex microtubule network of the early spindle is key to rapid and accurate assembly.
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Centrómero , Cromosomas , Huso Acromático , Cinetocoros , Microtúbulos , Prometafase , Segregación Cromosómica , MitosisRESUMEN
Biological particles have evolved to possess mechanical characteristics necessary to carry out their functions. We developed a computational approach to "fatigue testing in silico", in which constant-amplitude cyclic loading is applied to a particle to explore its mechanobiology. We used this approach to describe dynamic evolution of nanomaterial properties and low-cycle fatigue in the thin spherical encapsulin shell, thick spherical Cowpea Chlorotic Mottle Virus (CCMV) capsid, and thick cylindrical microtubule (MT) fragment over 20 cycles of deformation. Changing structures and force-deformation curves enabled us to describe their damage-dependent biomechanics (strength, deformability, stiffness), thermodynamics (released and dissipated energies, enthalpy, and entropy) and material properties (toughness). Thick CCMV and MT particles experience material fatigue due to slow recovery and damage accumulation over 3-5 loading cycles; thin encapsulin shells show little fatigue due to rapid remodeling and limited damage. The results obtained challenge the existing paradigm: damage in biological particles is partially reversible owing to particle's partial recovery; fatigue crack may or may not grow with each loading cycle and may heal; and particles adapt to deformation amplitude and frequency to minimize the energy dissipated. Using crack size to quantitate damage is problematic as several cracks might form simultaneously in a particle. Dynamic evolution of strength, deformability, and stiffness, can be predicted by analyzing the cycle number (N) dependent damage, [Formula: see text] , where α is a power law and Nf is fatigue life. Fatigue testing in silico can now be used to explore damage-induced changes in the material properties of other biological particles. STATEMENT OF SIGNIFICANCE: Biological particles possess mechanical characteristics necessary to perform their functions. We developed "fatigue testing in silico" approach, which employes Langevin Dynamics simulations of constant-amplitude cyclic loading of nanoscale biological particles, to explore dynamic evolution of the mechanical, energetic, and material properties of the thin and thick spherical particles of encapsulin and Cowpea Chlorotic Mottle Virus, and the microtubule filament fragment. Our study of damage growth and fatigue development challenge the existing paradigm. Damage in biological particles is partially reversible as fatigue crack might heal with each loading cycle. Particles adapt to deformation amplitude and frequency to minimize energy dissipation. The evolution of strength, deformability, and stiffness, can be accurately predicted by analyzing the damage growth in particle structure.
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Fenómenos Mecánicos , Estrés Mecánico , Fenómenos Biomecánicos , Termodinámica , Ensayo de MaterialesRESUMEN
Elucidating the structure-function relationships for therapeutic RNA mimicking phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to the lack of information about their structures. While PMOs have been approved by the US Food and Drug Administration for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge-neutral, and stable molecules is available. We performed circular dichroism and solution viscosity measurements combined with molecular dynamics simulations and machine learning to resolve solution structures of 22-mer, 25-mer, and 30-mer length PMOs. The PMO conformational dynamics are defined by the competition between non-polar nucleobases and uncharged phosphorodiamidate groups for shielding from solvent exposure. PMO molecules form non-canonical, partially helical, stable folded structures with a small 1.4- to 1.7-nm radius of gyration, low count of three to six base pairs and six to nine base stacks, characterized by -34 to -51 kcal/mol free energy, -57 to -103 kcal/mol enthalpy, and -23 to -53 kcal/mol entropy for folding. The 4.5- to 6.2-cm3/g intrinsic viscosity and Huggins constant of 4.5-9.9 are indicative of extended and aggregating systems. The results obtained highlight the importance of the conformational ensemble view of PMO solution structures, thermodynamic stability of their non-canonical structures, and concentration-dependent viscosity properties. These principles form a paradigm to understand the structure-properties-function relationship for therapeutic PMOs to advance the design of new RNA-mimic-based drugs.
RESUMEN
Charge transport in biomolecules is crucial for many biological and technological applications, including biomolecular electronics devices and biosensors. RNA has become the focus of research because of its importance in biomedicine, but its charge transport properties are not well understood. Here, we use the Scanning Tunneling Microscopy-assisted molecular break junction method to measure the electrical conductance of particular 5-base and 10-base single-stranded (ss) RNA sequences capable of base stacking. These ssRNA sequences show single-molecule conductance values around [Formula: see text] ([Formula: see text]), while equivalent-length ssDNAs result in featureless conductance histograms. Circular dichroism (CD) spectra and MD simulations reveal the existence of extended ssRNA conformations versus folded ssDNA conformations, consistent with their different electrical behaviors. Computational molecular modeling and Machine Learning-assisted interpretation of CD data helped us to disentangle the structural and electronic factors underlying CT, thus explaining the observed electrical behavior differences. RNA with a measurable conductance corresponds to sequences with overall extended base-stacking stabilized conformations characterized by lower HOMO energy levels delocalized over a base-stacking mediating CT pathway. In contrast, DNA and a control RNA sequence without significant base-stacking tend to form closed structures and thus are incapable of efficient CT.
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ADN , ARN , ARN/metabolismo , ADN/química , ADN de Cadena Simple , Conformación Molecular , Modelos MolecularesRESUMEN
During transformation of a normal cell to a cell capable of forming a cancerous growth, cellular morphology, the cytoskeleton, and focal contacts undergo significant changes. These changes should be capable of being characterized via real-time monitoring of the dynamic cell adhesion process and viscoelastic properties of cells. Here, we describe use of the quartz crystal microbalance (QCM) to distinguish the dynamic cell adhesion signatures of human normal (HMEC) versus malignant (MCF-7) mammary epithelial cells. The significantly reduced QCM responses (changes in frequency [Δf] and motional resistance ΔR) of MCF-7 cells compared with those of HMECs mirror the cancer cells' morphological features as observed via optical microscope. We analyzed the initial 2-h cell adhesion kinetics, suggesting cell-cell cooperativity for HMECs and no or weak cell-cell interactions for MCF-7 cells. We propose that changes of the ΔR/Δf ratio, which we term the cell viscoelastic index (CVI), reflect the establishment of cytoskeleton structure and dynamic viscoelastic properties of living cells. The CVI decreases significantly on initiation of cell to surface interactions as cells establish their cytoskeletal structures. During the cell adhesion process, MCF-7 cells were consistently softer, exhibiting up to a 2.5-fold smaller CVI when compared with HMECs.
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Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Mama/citología , Mama/fisiología , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Adhesión Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Células Cultivadas , Elasticidad , Células Epiteliales/citología , Células Epiteliales/fisiología , Femenino , Humanos , ViscosidadRESUMEN
Microtubules (MTs), a cellular structure element, exhibit dynamic instability and can switch stochastically from growth to shortening; but the factors that trigger these processes at the molecular level are not understood. We developed a 3D Microtubule Assembly and Disassembly DYnamics (MADDY) model, based upon a bead-per-monomer representation of the αß-tubulin dimers forming an MT lattice, stabilized by the lateral and longitudinal interactions between tubulin subunits. The model was parameterized against the experimental rates of MT growth and shortening, and pushing forces on the Dam1 protein complex due to protofilaments splaying out. Using the MADDY model, we carried out GPU-accelerated Langevin simulations to access dynamic instability behavior. By applying Machine Learning techniques, we identified the MT characteristics that distinguish simultaneously all four kinetic states: growth, catastrophe, shortening, and rescue. At the cellular 25 µM tubulin concentration, the most important quantities are the MT length L , average longitudinal curvature κ long , MT tip width w , total energy of longitudinal interactions in MT lattice U long , and the energies of longitudinal and lateral interactions required to complete MT to full cylinder U long add and U lat add . At high 250 µM tubulin concentration, the most important characteristics are L , κ long , number of hydrolyzed αß-tubulin dimers n hyd and number of lateral interactions per helical pitch n lat in MT lattice, energy of lateral interactions in MT lattice U lat , and energy of longitudinal interactions in MT tip u long . These results allow greater insights into what brings about kinetic state stability and the transitions between states involved in MT dynamic instability behavior.
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BACKGROUND: Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs). RESULTS: We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 µg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 µg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 µg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays. CONCLUSION: These data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.
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Técnicas Biosensibles/métodos , Macrófagos/efectos de los fármacos , Monitoreo Fisiológico/métodos , Nanotubos de Carbono/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Macrófagos/patología , Macrófagos/fisiología , Fagocitosis/efectos de los fármacos , Poliestirenos/toxicidad , Cuarzo , Zimosan/toxicidadRESUMEN
Structural mechanisms underlying the mechanical properties of fibrin fibers are elusive. We combined tensile testing of uncrosslinked fibrin polymers in vitro and in silico to explore their material properties. The experimental stress (σ) - strain (ε) curves for fibrin fibers are characterized by elastic deformations with a weaker elastic response for ε<160% due to unraveling of αC tethers and straightening of fibrin protofibrils, and a stronger response for ε>160% owing to unfolding of the coiled coils and γ nodules in fibrin monomers. Fiber rupture for strains ε>212% is due to dissociation of the knob-hole bonds and rupture of D:D interfaces. We developed the Fluctuating Bilinear Spring model to interpret the σ-ε profiles in terms of the free energy for protofibril alignment ΔG0 = 10.1-11.5 kBT, Young's moduli for protofibril alignment Yu = 1.9-3.2 MPa and stretching Ya = 5.7-9.7 MPa, strain scale εË≈ 12-40% for fiber rupture, and protofibril cooperativity m= 3.6-8. We applied the model to characterize the fiber strength σcr≈ 12-13 MPa, deformability εcr≈ 222%, and rupture toughness U≈ 9 MJ/m3, and to resolve thermodynamic state functions, 96.9 GJ/mol entropy change for protofibril alignment (at room temperature) and 113.6 GJ/mol enthalpy change for protofibril stretching, which add up to 210.5 GJ/mol free-energy change. Fiber elongation is associated with protofibril dehydration and sliding mechanism to create an ordered protofibril array. Fibrin fibers behave like a hydrogel; protofibril dehydration and water expulsion account for â¼94-98% of the total free-energy changes for fiber elongation and rupture. STATEMENT OF SIGNIFICANCE: Structural mechanisms underlying the mechanical properties of fibrin fibers, major components of blood clots and obstructive thrombi, are elusive. We performed tensile testing of uncrosslinked fibrin polymers in vitro and in silico to explore their material properties. Fluctuating Bilinear Spring theory was developed to interpret the stress-strain profiles in terms of the energy for protofibril alignment, elastic moduli for protofibril alignment and stretching, and strain scale for fiber rupture, and to probe the limits of fiber strength, extensibility and toughness. Fibrin fibers behave like a hydrogel. Fiber elongation is defined by the protofibril dehydration and sliding. Structural rearrangements in water matrix control fiber elasticity. These results contribute to fundamental understanding of blood clot breakage that underlies thrombotic embolization.
Asunto(s)
Fibrina , Trombosis , Módulo de Elasticidad , Elasticidad , Humanos , TermodinámicaRESUMEN
We developed the Fluctuating Nonlinear Spring (FNS) model to describe the dynamics of mechanical deformation of biological particles, such as virus capsids. The theory interprets the force-deformation spectra in terms of the "Hertzian stiffness" (non-linear regime of a particle's small-amplitude deformations), elastic constant (large-amplitude elastic deformations), and force range in which the particle's fracture occurs. The FNS theory enables one to quantify the particles' elasticity (Young's moduli for Hertzian and bending deformations), and the limits of their strength (critical forces, fracture toughness) and deformability (critical deformations) as well as the probability distributions of these properties, and to calculate the free energy changes for the particle's Hertzian, elastic, and plastic deformations, and eventual fracture. We applied the FNS theory to describe the protein capsids of bacteriophage P22, Human Adenovirus, and Herpes Simplex virus characterized by deformations before fracture that did not exceed 10-19% of their size. These nanoshells are soft (~1-10-GPa elastic modulus), with low ~50-480-kPa toughness - a regime of material behavior that is not well understood, and with the strength increasing while toughness decreases with their size. The particles' fracture is stochastic, with the average values of critical forces, critical deformations, and fracture toughness comparable with their standard deviations. The FNS theory predicts 0.7-MJ/mol free energy for P22 capsid maturation, and it could be extended to describe uniaxial deformation of cylindrical microtubules and ellipsoidal cellular organelles.