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Dive into the research topics where Katja Tönsing is active.

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Featured researches published by Katja Tönsing.


Plant Physiology | 2010

Nucleocytoplasmic Distribution of the Arabidopsis Chromatin-Associated HMGB2/3 and HMGB4 Proteins

Dorthe S. Pedersen; Thomas Merkle; Dorte Launholt Lildballe; Martin Antosch; Thorsten Bergmann; Katja Tönsing; Dario Anselmetti; Klaus D. Grasser

High mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that as architectural factors are involved in the regulation of transcription and other DNA-dependent processes. HMGB proteins are generally considered nuclear proteins, although mammalian HMGB1 can also be detected in the cytoplasm and outside of cells. Plant HMGB proteins studied so far were found exclusively in the cell nucleus. Using immunofluorescence and fluorescence microscopy of HMGB proteins fused to the green fluorescent protein, we have examined the subcellular localization of the Arabidopsis (Arabidopsis thaliana) HMGB2/3 and HMGB4 proteins, revealing that, in addition to a prominent nuclear localization, they can be detected also in the cytoplasm. The nucleocytoplasmic distribution appears to depend on the cell type. By time-lapse fluorescence microscopy, it was observed that the HMGB2 and HMGB4 proteins tagged with photoactivatable green fluorescent protein can shuttle between the nucleus and the cytoplasm, while HMGB1 remains nuclear. The balance between the basic amino-terminal and the acidic carboxyl-terminal domains flanking the central HMG box DNA-binding domain critically influences the nucleocytoplasmic distribution of the HMGB proteins. Moreover, protein kinase CK2-mediated phosphorylation of the acidic tail modulates the intranuclear distribution of HMGB2. Collectively, our results show that, in contrast to other Arabidopsis HMGB proteins such as HMGB1 and HMGB5, the HMGB2/3 and HMGB4 proteins occur preferentially in the cell nucleus, but to various extents also in the cytoplasm.


Biophysical Journal | 2009

Binding Kinetics of Bisintercalator Triostin A with Optical Tweezers Force Mechanics

Christoph Kleimann; Andy Sischka; Andre Spiering; Katja Tönsing; Norbert Sewald; Ulf Diederichsen; Dario Anselmetti

The binding kinetics of the intercalative binding of Triostin A to lambda-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.


Review of Scientific Instruments | 2008

Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores

Andy Sischka; Christoph Kleimann; Wiebke Hachmann; Marcus M. Schäfer; Ina Seuffert; Katja Tönsing; Dario Anselmetti

We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference.


Bioelectrochemistry | 2000

Perspectives for microelectrode arrays for biosensing and membrane electroporation

Eberhard Neumann; Katja Tönsing; Peter M. Siemens

Electrochemical microelectrode devices are among the great challenges for bioelectrochemistry, cell biology and recently also for biomedical research and new clinical electrotherapies. Two representative cases in cell biology and medical research for new trends in the technical devices are selected, heading at new diagnostic and therapeutic clinical applications. One example is from the field of biosensing cholinergic neurotransmitter substances by the nicotinic acetylcholine receptor (AcChoR) in solid-supported lipid bilayer membrane and the other one refers to new developments of electrode systems for the electrochemical delivery of drugs and genes to biological cell aggregates and tissue by the powerful method of membrane electroporation. In both cases addressed to, the new developments include the use of electrical feedback control of electrode arrays for biosensing processes as well as for the extent and duration of tissue electroporation. In line with the impressive advances in medical microsurgery, where increasingly smaller organ targets become accessible, microelectrode systems have become a continuous technical challenge for bioanalytical purposes and, as discussed here in some detail, for the new field of the electroporative delivery of effector substances like drugs and genes, using miniaturized electrochemical electrode arrays.


Circulation-cardiovascular Genetics | 2013

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

Andreas Brodehl; Mareike Dieding; Baerbel Klauke; Eric Dec; Shrestha Madaan; Taosheng Huang; John Jay Gargus; Azra Fatima; Tomo Saric; Hamdin Cakar; Volker Walhorn; Katja Tönsing; Tim Skrzipczyk; Ramona Cebulla; Désirée Gerdes; Uwe Schulz; Jan Gummert; Jesper Hastrup Svendsen; M.S. Olesen; Dario Anselmetti; Alex Horby Christensen; Virginia E. Kimonis; Hendrik Milting

Background—The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. Methods and Results—We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. Conclusions—Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.


Biomedical optics | 2006

Two-photon laser scanning microscopy on native cartilage and collagen membranes for tissue engineering

Jörg Martini; Katja Tönsing; Michael Dickob; Ronald Schade; Klaus Liefeith; Dario Anselmetti

In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-106/cm3 and 20-106/cm3, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.


Biophysical Journal | 2016

Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers

Ying Wang; Andy Sischka; Volker Walhorn; Katja Tönsing; Dario Anselmetti

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.


Review of Scientific Instruments | 2012

Video-based and interference-free axial force detection and analysis for optical tweezers

Sebastian Knust; Andre Spiering; Henning Vieker; André Beyer; Armin Gölzhäuser; Katja Tönsing; Andy Sischka; Dario Anselmetti

For measuring the minute forces exerted on single molecules during controlled translocation through nanopores with sub-piconewton precision, we have developed a video-based axial force detection and analysis system for optical tweezers. Since our detection system is equipped with a standard and versatile CCD video camera with a limited bandwidth offering operation at moderate light illumination with minimal sample heating, we integrated Allan variance analysis for trap stiffness calibration. Upon manipulating a microbead in the vicinity of a weakly reflecting surface with simultaneous axial force detection, interference effects have to be considered and minimized. We measured and analyzed the backscattering light properties of polystyrene and silica microbeads with different diameters and propose distinct and optimized experimental configurations (microbead material and diameter) for minimal light backscattering and virtually interference-free microbead position detection. As a proof of principle, we investigated the nanopore threading forces of a single dsDNA strand attached to a microbead with an overall force resolution of ±0.5 pN at a sample rate of 123 Hz.


Biochimica et Biophysica Acta | 2017

Biophysical characterization of the association of histones with single-stranded DNA

Ying Wang; Luis van Merwyk; Katja Tönsing; Volker Walhorn; Dario Anselmetti; Xavier Fernàndez-Busquets

BACKGROUND Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. METHODS Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. RESULTS Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. CONCLUSIONS At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. GENERAL SIGNIFICANCE In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.


Nanopores for Bioanalytical Applications | 2012

Single-molecule DNA Translocation Through Si3N4- and Graphene Solid-state Nanopores

Andre Spiering; Sebastian Knust; Sebastian Getfert; André Beyer; Karsten Rott; L. Redondo; Katja Tönsing; Peter Reimann; Andy Sischka; Dario Anselmetti

The controlled translocation of a single, double-stranded DNA (dsDNA) through a solid-state nanopore (NP) with optical tweezers (OT) is described in the presence of an electric field under buffer conditions. Upon threading dsDNA complexed by single proteins through a NP in 20 nm thick Si3N4-membranes, we find distinct asymmetric and retarded force signals that critically depend on the overall charge of the protein, the molecular elasticity of the dsDNA and the counter-ionic shielding of the polyelectrolyte (dsDNA) in the buffer3. This force response can be quantitatively simulated and understood within a theoretical model where an isolated charge on an elastic, polyelectrolyte strand experiences a harmonic nanopore potential during translocation. In order to extend these experiments to atomically thin solid-state NP, dsDNA was threaded through single nanolayer graphene NP by a transmembrane voltage.

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Klaus Liefeith

University of Düsseldorf

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