Johannes Rheinlaender

Comparison of Scanning Ion Conductance Microscopy with Atomic Force Microscopy for Cell Imaging

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.

Platelet-derived HMGB1 is a critical mediator of thrombosis

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

BackgroundCorynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.ResultsMicroscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these.ConclusionsOur data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

Evaluation of lamina cribrosa and peripapillary sclera stiffness in pseudoexfoliation and normal eyes by atomic force microscopy.

PURPOSE Pseudoexfoliation (PEX) syndrome is a systemic disorder of the elastic fiber system that can lead to PEX glaucoma. Elastotic alterations in the lamina cribrosa (LC) of PEX eyes suggested biomechanical implications predisposing to pressure-induced optic nerve damage. In this pilot study, the stiffness of LC and peripapillary sclera (ppSC) in eyes with and without PEX syndrome were analyzed by atomic force microscopy (AFM) nanoindentation. METHODS Unfixed cryosections (5-μm thick) were prepared from the optic nerve heads (ONH) of three donor eyes with PEX syndrome and three age-matched control eyes. AFM force mapping was performed in selected regions of the central, midperipheral, and peripheral LC and the ppSC using a spherical cantilever tip. To determine the local Youngs modulus of elasticity (YME) as a measure of tissue stiffness, force curves were acquired and analyzed using the spherical Hertz model. RESULTS For the LC, the median YME values calculated from single stiffness maps averaged 17.2 (±2.7) kPa in normal eyes and 10.1 (±1.4) kPa in PEX eyes, indicating a significant PEX-related decrease in stiffness by over 40% (P < 0.01). The corresponding YME values for the ppSC, which revealed a 9-fold higher tissue stiffness than in the LC, averaged 158.3 (±59.8) kPa for control and 85.8 (±16.9) kPa for PEX samples. CONCLUSIONS AFM was proven suitable for determining the stiffness of ONH tissues, encouraging further large-scale analyses. The marked decrease in stiffness, implying an increased deformability of the ONH in PEX eyes, may reflect an inherent tissue weakness rendering these eyes more vulnerable to glaucomatous damage.

Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc), is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs) and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM) and cell morphology analysed by scanning ion conductance microscopy (SICM). Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

Distribution of Young's modulus in porcine corneas after riboflavin/UVA-induced collagen cross-linking as measured by atomic force microscopy.

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Youngs modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Youngs modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Youngs modulus of the posterior 100 µm. The mean Youngs modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Youngs modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Youngs modulus in soft tissues in general.

Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells

BackgroundCorynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates.ResultsAdhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages.ConclusionsThe results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging.

Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cells elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM.

Mapping the mechanical stiffness of live cells with the scanning ion conductance microscope

Mapping the mechanical properties of living cells with high spatial and temporal resolution is important for the exploration of cell function. Widely used imaging techniques such as the atomic force microscope are generally based on direct mechanical contact between the probe and the cell, thereby involving the risk of damaging the cell. Here, we present a noncontact method for fast and quantitative stiffness mapping of living cells with sub-micrometer lateral resolution. This was achieved by repeatedly moving a pressurized nanopipette toward and away from the sample in a scanning ion conductance microscope (SICM). The pressure-induced microfluidic flow through the nanopipette produced a time-varying force on the sample surface, thereby locally indenting it without direct mechanical contact. Maps of sample stiffness (quantified by the Youngs modulus) were then determined from ion current approach curves using a finite element model. To demonstrate the capability of the method we visualized the dynamics of individual cytoskeleton fibers in living cells over several hours. Additionally, we found that spreading extensions of migrating fibroblast cells tend to be softer than their lamellum, which is consistent with a mechanism of cell migration by osmotic swelling.