Martin L. Bennink

Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers

Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single λ phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of ∼65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20–40 pN, comparable to forces reported for RNA- and DNA-polymerases.

Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinsons disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method.

Mechanical testing of electrospun PCL fibers

Poly(ε-caprolactone) (PCL) fibers ranging from 250 to 700 nm in diameter were produced by electrospinning a polymer tetrahydrofuran/N,N-dimethylformamide solution. The mechanical properties of the fibrous scaffolds and individual fibers were measured by different methods. The Youngs moduli of the scaffolds were determined using macro-tensile testing equipment, whereas single fibers were mechanically tested using a nanoscale three-point bending method, based on atomic force microscopy and force spectroscopy analyses. The modulus obtained by tensile-testing eight different fiber scaffolds was 3.8±0.8 MPa. Assuming that PCL fibers can be described by the bending model of isotropic materials, a Youngs modulus of 3.7±0.7 GPa was determined for single fibers. The difference of three orders of magnitude observed in the moduli of fiber scaffolds vs. single fibers can be explained by the lacunar and random structure of the scaffolds.

Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy

We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.

Force detection in optical tweezers using backscattered light.

In force-measuring optical tweezers applications the position of a trapped bead in the direction perpendicular to the laser beam is usually accurately determined by measuring the deflection of the light transmitted through the bead. In this paper we demonstrate that this position and thus the force exerted on the bead can be determined using the backscattered light. Measuring the deflection for a 2.50 mum polystyrene bead with both a position sensitive detector (PSD) and a quadrant detector (QD) we found that the linear detection range for the PSD is approximately twice that for the QD. In a transmission-based setup no difference was found between both detector types. Using a PSD in both setups the linear detection range for 2.50 mum beads was found to be approximately 0.50 mum in both cases. Finally, for the reflection-based setup, parameters such as deflection sensitivity and linear detection range were considered as a function of bead diameter (in the range of 0.5-2.5 mum). 140pN was the largest force obtained using 2.50 mum beads.

Surface modification of electrospun fibre meshes by oxygen plasma for bone regeneration

Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 ± 0.96xa0µm for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30xa0min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30xa0min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7xa0days of culture. The amount of ALP positive cells increased between 7 and 21xa0days of culture on both untreated and 30xa0min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21xa0days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7xa0days of culture, while no differences were observed in the expression of other osteogenic markers. At 21xa0days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.

Direct integration of micromachined pipettes in a flow channel for single DNA molecule study by optical tweezers

We have developed a micromachined flow cell consisting of a flow channel integrated with micropipettes. The flow cell is used in combination with an optical trap setup (optical tweezers) to study mechanical and structural properties of /spl lambda/-DNA molecules. The flow cell was realized using silicon micromachining including the so-called buried channel technology to fabricate the micropipettes, the wet etching of glass to create the flow channel, and the powder blasting of glass to make the fluid connections. The volume of the flow cell is 2 /spl mu/l. The pipettes have a length of 130 /spl mu/m, a width of 5-10 /spl mu/m, a round opening of 1 /spl mu/m and can be processed with different shapes. Using this flow cell we stretched single molecules (/spl lambda/-DNA) showing typical force-extension curves also found with conventional techniques. These pipettes can be also used for drug delivery, for injection of small gas bubbles into a liquid flow to monitor the streamlines, and for the mixing of liquids to study diffusion effects. The paper describes the design, the fabrication and testing of the flow cell.

Micromechanical analysis of native and cross-linked collagen type I fibrils supports the existence of microfibrils

The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress-strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress-strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues.

Atomic force microscopy under controlled conditions reveals structure of C-terminal region of alpha-synuclein in amyloid fibrils

Atomic force microscopy (AFM) is widely used to measure morphological and mechanical properties of biological materials at the nanoscale. AFM is able to visualize and measure these properties in different environmental conditions. However, these conditions can influence the results considerably, rendering their interpretation a matter of some subtlety. We demonstrate this by imaging ~10 nm diameter α-synuclein amyloid fibrils, focusing specifically on the structure of the C-terminal part of the protein monomers incorporated into fibrils. Despite these influences leading to variations in fibril heights, we have shown that by maintaining careful control of AFM settings we can quantitatively compare the morphological parameters of fibrils imaged in air or in buffer conditions. From this comparison we were able to deduce the semiflexible character of this C-terminal region. Fibril height differences measured in air and liquid indicate that the C-terminal region collapses onto the fibril core upon drying. The fibril heights decrease upon increasing ion concentration in solution, suggesting that the C-terminal tails collapse into more compact structures as a result of charge screening. Finally, PeakForce QNM measurements show an apparent heterogeneity of C-terminal packing along the fibril length.