Compressive nanomechanics of opposing aggrecan macromolecules

In this study, we have measured the nanoscale compressive interactions between opposing aggrecan macromolecules in near-physiological conditions, in order to elucidate the molecular origins of tissue-level cartilage biomechanical behavior. Aggrecan molecules from fetal bovine epiphyseal cartilage were chemically end-grafted to planar substrates, standard nanosized atomic force microscopy (AFM) probe tips (R(tip) approximately 50 nm), and larger colloidal probe tips (R(tip) approximately 2.5 microm). To assess normal nanomechanical interaction forces between opposing aggrecan layers, substrates with microcontact printed aggrecan were imaged using contact mode AFM, and aggrecan layer height (and hence deformation) was measured as a function of solution ionic strength (IS) and applied normal load. Then, using high-resolution force spectroscopy, nanoscale compressive forces between opposing aggrecan on the tip and substrate were measured versus tip-substrate separation distance in 0.001-1M NaCl. Nanosized tips enabled measurement of the molecular stiffness of 2-4 aggrecan while colloidal tips probed the nanomechanical properties of larger assemblies (approximately 10(4) molecules). The compressive stiffness of aggrecan was much higher when using a densely packed colloidal tip than the stiffness measured for using the nanosized tip with a few aggrecan, demonstrating the importance of lateral interactions to the normal nanomechanical properties. The measured stress at 0.1M NaCl (near-physiological ionic strength) increased sharply at aggrecan densities under the tip of approximately 40 mg/ml (physiological densities are approximately 20-80 mg/ml), corresponding to an average inter-GAG spacing of 4-5 Debye lengths (4-5 nm); this characteristic spacing is consistent with the onset of significant electrostatic interactions between GAG chains of opposing aggrecan molecules. Comparison of nanomechanical data to the predictions of Poisson-Boltzmann-based models further elucidated the regimes over which electrostatic and nonelectrostatic interactions affect aggrecan stiffness in compression. The most important aspects of this study include: the incorporation of experiments at two different length scales, the use of microcontact printing to enable quantification of aggrecan deformation and the corresponding nanoscale compressive stress vs. strain curve, the use of tips of differing functionality to provide insights into the molecular mechanisms of deformation, and the comparison of experimental data to the predictions of three increasingly refined Poisson-Boltzmann (P-B)-based theoretical models for the electrostatic double layer component of the interaction.     

Nanoscale shear deformation mechanisms of opposing cartilage aggrecan macromolecules

The nanoscale shear deformation behavior of two opposing end-grafted aggrecan layers was studied in aqueous solutions using atomic force microscopy, and was observed to depend markedly on bath ionic strength, the presence of calcium ions, and the applied lateral displacement rate. These results provide molecular-level insights into the contribution of aggrecan deformation mechanisms to cartilage tissue-level material properties.     

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.     

Nanomechanics of vascular endothelium

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function.     

Nanomechanics of adhesion proteins

Recent advances in molecular force measurements have resulted in the quantification of the nanomechanical properties of single molecular bonds, and elucidated novel relationships between molecular architecture and biomolecular adhesion. The measured forces to rupture single intermolecular bonds revealed novel and unexpected ways that proteins respond to mechanical force. Measurement of the magnitude of interprotein forces and the distances over which they act further determined how protein architecture may contribute to both the stability and structural organization of adhesive junctions.     

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.     

Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell–substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. 2013; 110: 2301–2310. © 2013 Wiley Periodicals, Inc.     

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity^1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures^1,6,7. We developed a new model¹ which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (K_d) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.     

Nanomechanics of the Endothelial Glycocalyx in Experimental Sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.     

Microbial glycosaminoglycan glycosyltransferases

Glycosaminoglycans, a class of linear polysaccharides composed of repeating disaccharide units containing a hexosamine, are important carbohydrates found in many organisms. Vertebrates utilize glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Certain pathogenic bacteria produce extracellular capsules composed of glycosaminoglycans or glycosaminoglycan-like polymers that enhance the microbes' ability to infect or to colonize the host. In the period from 1993 to 2001, bacterial enzymes were discovered that catalyze the polymerization of the repeating unit of hyaluronan, chondroitin, or N-acetylheparosan (unsulfated, unepimerized heparin). Depending on the specific carbohydrate and the microorganism, either a dual-action enzyme (synthase) that transfers two distinct monosaccharides or a pair of single-action transferases are utilized to synthesize the glycosaminoglycan polymer. Current views on the enzymology, structures, potential evolution, and the roles of the known glycosyltransferases from Streptococcus, Pasteurella, and Escherichia are discussed.     

Nanomechanics of Native Thick Filaments from Indirect Flight Muscles

During flight, the wings of Drosophila melanogaster beat nearly 200 times per second. The indirect flight muscle fibers that power this movement have evolved to resist the repetitive mechanical stress that results from the 5-ms wing beat cycle at a strain amplitude of 3.5%. In order to understand how this is achieved at the sarcomere level, we have analyzed the mechanical properties of native thick filaments isolated from indirect flight muscle. Single filaments adsorbed onto a solid support were manipulated in physiological buffer using an atomic force microscope. Images taken after the manipulation revealed that segments were stretched, on average, to 150%, with a maximum at 385% extension. The lateral-force-versus-displacement curve associated with each manipulation contained information about the bending and tensile properties of each filament. The bending process was dominated by shearing between myosin dimers and yielded a shear modulus between 3 and 13 MPa. Maximum tension along the stretched filaments was observed at ∼ 200% extension and varied between 8 and 17 nN. Based on current models of thick filament structure, these variations can be attributed to cross-links between myosin dimers distributed along the filament.     

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.     

Severe Myopathy Mutations Modify the Nanomechanics of Desmin Intermediate Filaments

Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of bona fide IFs in vitro and in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.     

Dielectrophoresis of macromolecules

Dilute solutions of rigid helical macromolecules with permanent dipole moments have been studied with dielectrophoresis. The polymers used were poly-γ-benzyl-L -glutmate and poly-n-butyl isocyanate, both fractions of average molecular weight 120,000. In this phenomenon dipolar molecules migrate in an inhomogeneous electric field towards the region of maximum field strength. The field gradient is produced by a 10-μm diameter Pt wire serving as the inner electrode of a cell, and a 2.5-mm diameter cylinder as the outer electrode. An equilibrium exponential concentration gradient is achieved within a few wire radii of the inner electrode in a matter of minutes, making both kinetic and equilibrium measurements feasible. The concentration gradient is monitored by measuring the change in capacitance ΔC of the cell from its value for a uniform solute concentration. The time dependences of ΔC for the build-up of the concentration gradient towards the equilibrium value, and the decay to a uniform concentration after the field is removed have been calculated, using a frictional coefficient and a Boltzmann factor, β₁, as the sole adjustable parameters. For low voltages (β₁ small), the measured build-up and decay are completely symmetrical, in agreement with theory, and yield the same frictional coefficient. For larger β₁, the build-up is faster, reflecting a reduced frictional coefficient due to partial alignment of the long axes of the polymers in the direction of migration. In either case, the decay determines a diffusion constant of randomly aligned molecules, identical to classical diffusion measurement. The value of ΔC at equilibrium, for β₁ small, determines μ²F², where μ is the dipole moment and F is the internal field at the polymer. Combined with a classical dielectric constant measurement, which gives μ²F, both μ and F may be determined, independent of assumptions about cavity shapes. Dielectrophoresis works in alternating as well as constant fields, provided the dipolar orientation can follow the field reversals. The dispersion of dielectrophoresis with frequency is a sensitive measure of the molecular-weight distribution of a given sample.     

Sulfotransferases in glycosaminoglycan biosynthesis

Most of the sulfotransferases participating in glycosaminoglycan biosynthesis have now been identified. Their essential role in generating binding sites for proteins interacting with glycosaminoglycans is apparent. These interactions may influence important biological processes such as growth control, signal transduction, cell adhesion and lipid metabolism. Gene targeting in mice as well as studies in Drosophila melanogaster and Caenorhabditis elegans have shown that dysfunction or lack of glycosaminoglycan sulfotransferases may result in severely disturbed embryonic development.     

Nanomechanics of superbugs and superdrugs: new frontiers in nanomedicine

The alarming rise in drug-resistant hospital 'superbugs' and the associated increase in fatalities is driving the development of technologies to search for new antibiotics and improve disease diagnostics. One of the most successful drug targets is the bacterial cell wall, an evolutionary feature of virtually all prokaryotes and vital for their survival by providing mechanical strength. The recent discovery of bacterial cytoskeletal proteins analogous to the key force-bearing machinery in eukaryotes also provides new opportunities for drug discovery, but little is known about their mechanical role in bacteria. In the present short article, I review recent developments in the field of nanotechnology to investigate the mechanical mechanisms of action of potent antibiotics on cell wall and cytoskeletal targets with unprecedented spatial, temporal and force resolution and the development of a new generation of nanomechanical devices to detect pathogens for point-of-care diagnostics.     

Regulatory aspects of glycosaminoglycan biosynthesis

The control of glycosaminoglycan biosynthesis was investigated by studying the kinetic and regulatory properties of some enzymes involved in the formation of UDP-sugar precursors: UDP-N-acetylglucosamine 4'-epimerase, catalyzing the interconversion of hexosamine precursors and UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase, utilizing UDP-glucose for the formation of uronic acid and galactose precursors. The study was carried out in tissues with different glycosaminoglycan production: bovine cornea, producing both chondroitin sulfate and keratan sulfate, and newborn-pig epiphysial-plate cartilage, producing mostly chondroitin sulfate. The biosynthesis of hexosamine precursors appeared to be regulated by the value of the NAD/NADH ratio. This control mechanism regulated also the activities of both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase and, therefore, it could correlate the biosynthesis of glycosaminoglycan precursors with the redox activity of the cell. At the level of UDP-glucose utilization two other control mechanisms were demonstrated: the different affinities of UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase for UDP-glucose in tissues with different glycosaminoglycan production and the cellular concentration of UDP-xylose. This sugar-nucleotide inhibited UDP-glucose dehydrogenase, but did not affect the UDP-glucose 4'-epimerase activity; therefore, and increase of its cellular concentration may result in a decreased chondroitin sulfate synthesis and in an increased keratan sulfate formation.     

Sequence of the halobacterial glycosaminoglycan

The cell-surface glycoprotein of halobacterium contains a sulfated repeating unit saccharide chain, similar to the mammalian glycosaminoglycans. The composition of a presumptive repeating pentasaccharide unit of this glycosaminoglycan is 1 GlcNAc, 1 GalNAc, 1 Gal, 1 GalA (where GalA represents galacturonic acid), 1 3-O-methyl-GalA, and 2 SO42-. Linkage to protein of this glycoconjugate involves the hitherto unique unit Asn-GalNAc, with the N-linked asparagine residue being the second NH2-terminal amino acid and part of the common N-linked glycosyl acceptor sequence Asn-X-Thr(Ser). Transfer of the completed, sulfated glycosaminoglycan from its lipid precursor to the protein occurs at the cell surface, and the presence of this sulfated saccharide chain in the cell-surface glycoprotein seems to be required to maintain the structural integrity of the rod-shaped halobacteria. In this paper, we report the complete saccharide structure of this N-linked glycosaminoglycan. This structure is deduced from chemical analyses of fragments that were isolated after hydrazinolysis and subsequent nitrous acid deamination or after mild acidic hydrolysis of purified Pronase-derived glycosaminoglycan-peptides. The halobacterial glycosaminoglycan consists, on the average, of 10 repeating pentasaccharide units of the following structure. (formula: see text) The reducing end N-acetylgalactosamine residue is linked directly to the asparagine, without a special saccharide linker region.