Johannes S. Kanger

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.

Realization of a multichannel integrated Young interferometer chemical sensor

We report on the design, realization, and characterization of a four-channel integrated optical Young interferometer device that enables simultaneous and independent monitoring of three binding processes. The generated interference pattern is recorded by a CCD camera and analyzed with a fast-Fourier-transform algorithm. We present a thorough theoretical analysis of such a device. The realized device is tested by monitoring glucose solutions that induce well defined phase changes between output channels. The simultaneous measurement of three different glucose concentrations shows the multipurpose feature of such devices. The observed errors, caused by the mismatching of spatial frequencies of individual interference patterns with those determined from the CCD camera, are reduced with different reduction schemes. The phase resolution for different pairs of channels was approximately 1 x 10(-4) fringes, which corresponds to a refractive-index resolution of approximately 8.5 x 10(-8). The measured sensitivity coefficient of the phase change versus refractive-index change of approximately 1.22 x 10(3) x 2pi agrees well with the calculated coefficient of approximately 1.20 x 10(3) x 2pi.

Development of a multichannel integrated interferometer immunosensor

This report describes the design, realization and testing of a highly sensitive two-channel integrated optical (IO) Young interferometer (YI) as well as the design of a first multichannel YI. An integrated channel waveguide Y-splitter with two output parallel branches has been used as the basic optical component for building the YI. The generated interference pattern as result of the overlap of outgoing divergent beams is recorded by a CCD camera and is further analyzed by a computer program based on a Fast Fourier Transformation (FFT) algorithm. The integrated waveguide structure is fabricated in SiON technology. For testing the realized device the refractive indices of several glucose solutions have been measured. The realized sensor shows a phase resolution of 1×10−4 fringes corresponding to a refractive index resolution of 2×10−8. The long-term stability is about 1×10−3fringes×h−1 corresponding to a refractive index change of 2×10−7 h−1. The first design of the multichannel YI has four output parallel channels positioned at well-defined distances from each other, such that the phase shift between both channels of one pair can be monitored independently from the other channel pairs.

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.

Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

Intracellular manipulation of chromatin using magnetic nanoparticles

Magnetic tweezers are widely used for manipulating small magnetic beads inside the cell cytoplasm in order to gain insight into the structural and mechanical properties of the cytoskeleton. Here we discuss the use of magnetic tweezers for the study of nuclear architecture and the mechanical properties of chromatin in living cells. A custom-built, dedicated micro magnetic tweezer set-up is described. We review progress that has been made in applying this technology for the study of chromatin structure and discuss its prospects for the in situ analysis of nuclear architecture and chromatin function.

Biofunctionalized Lipid-Polymer Hybrid Nanocontainers with Controlled Permeability

We have successfully developed, for the first time, a novel polymer-lipid hybrid nanocontainer with controlled permeability functionality. The nanocontainer is made by nanofabricating holes with desired dimensions in an impermeable polymer scaffold by focused ion beam drilling and sealing them with lipid bilayers containing remote-controlled pore-forming channel proteins. This system allows exchange of solutions only after channel activation at will to form temporary pores in the container. Potential applications are foreseen in bionanosensors, nanoreactors, nanomedicine, and triggered delivery.

Integrin-dependent activation of the JNK signaling pathway by mechanical stress.

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells. We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization. These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch.

The influence of ionic strength on the adhesive bond stiffness of oral streptococci possessing different surface appendages as probed using AFM and QCM-D

Bacterial adhesion to surfaces poses threats to human-health, not always associated with adhering organisms, but often with their detachment causing contamination elsewhere. Bacterial adhesion mechanisms may not be valid for their detachment, known to proceed according to a visco-elastic mechanism. Here we aimed to investigate influences of ionic strength on the adhesive bond stiffness of two spherically shaped Streptococcus salivarius strains with different lengths of fibrillar surface appendages. The response of a Quartz-Crystal-Microbalance-with-Dissipation (QCM-D) upon streptococcal adhesion and changes in the ionic strength of the surrounding fluid indicated that the bond stiffness of S. salivarius HB7, possessing a dense layer of 91 nm long fibrils, was unaffected by ionic strength. Atomic-force-microscopic (AFM) imaging in PeakForce-QNM mode showed a small decrease in bond stiffness from 1200 to 880 kPa upon decreasing ionic strength from 57 to 5.7 mM, while Total-Internal-Reflection-Microscopy suggested a complete collapse of fibrils. S. salivarius HBV51, possessing a less dense layer of shorter (63 nm) fibrils, demonstrated a strong decrease in bond stiffness both from QCM-D and AFM upon decreasing the ionic strength, and a partial collapse of fibrils. Probably, the more hydrophobic and less negatively charged long fibrils on S. salivarius HB7 collapse side-on to the cell surface, while the more hydrophilic and negatively charged fibrils of S. salivarius HBV51 remain partially stretched. In summary, we demonstrate how a combination of different methods can yield a description of the structural changes occurring in the interfacial region between adhering, fibrillated streptococci and a substratum surface upon changing the ionic strength.