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Maasi: A 3D printed spin coater with touchscreen

Carbonell Rubio D, Weber W, Klotzsch E

HardwareX, 2022 - Article

Spin coaters are widely used to apply thin films of a material uniformly over a flat substrate. Despite the simplicity of this technique the entry price for such machines might be prohibitive, ranging from few hundreds to thousands of Euros. Here we present Maasi, an affordable alternative that is easy to build and has all functional key features to be used in a wide range of applications. Our design has a price of less than hundred Euros and an assembly time of only two hours. One of the key design principles was to use only 3D printed parts in combination with affordable Commercial Off-The-Shelf (COTS) components [1]. Reducing the complexity we use an electronic speed controller (ESC) with telemetry, to eliminate the need for a rotor position sensor [2]. A touchscreen further improves its usability, thus setting a perfect startpoint for the design of other affordable lab tools. The Maasi project includes different 3D printable substrate holders allowing treatment of formats up to 80 mm in diameter. We furthermore validate the Maasi spin coater by measuring its speed accuracy and performance for coating polydimethylsiloxane (PDMS) on glass coverslips for mechanobiological assay.

Barring the gates to the battleground: DDR1 promotes immune exclusion in solid tumors

Wagner DL, Klotzsch E

Nature Sig. Transd. and Targ. Therapy, 2022 - Article

"Cancer is one of the leading causes of death worldwide. In the last decade, T cell-targeted immunotherapy has revolutionized the treatment of patients with extensive solid tumor burden who were previously considered “terminally ill“. A particularly successful example of immunotherapy is immune checkpoint inhibitors (ICI), which reinvigorate the potent anti-tumor efficacy of endogenous T cells.2 In general, the response to immunotherapy is dependent on the presence and activation of effector T cells in the tumor microenvironment (TME) and subsequent lysis of cancerous cells. However, the TME of large solid tumors can create physical barriers and block immune cell infiltration. To date, we lack specific drugs that allow us to remodel the TME and allow effective migration of T cells into the tumors."

The solid tumor microenvironment-Breaking the barrier for T cells: How the solid tumor microenvironment influences T cells: How the solid tumor microenvironment influences T cells

Simsek H, Klotzsch E

Bioessays, 2022 - Article

The tumor microenvironment (TME) plays a pivotal role in the behavior and development of solid tumors as well as shaping the immune response against them. As the tumor cells proliferate, the space they occupy and their physical interactions with the surrounding tissue increases. The growing tumor tissue becomes a complex dynamic structure, containing connective tissue, vascular structures, and extracellular matrix (ECM) that facilitates stimulation, oxygenation, and nutrition, necessary for its fast growth. Mechanical cues such as stiffness, solid stress, interstitial fluid pressure (IFP), matrix density, and microarchitecture influence cellular functions and ultimately tumor progression and metastasis. In this fight, our body is equipped with T cells as its spearhead against tumors. However, the altered biochemical and mechanical environment of the tumor niche affects T cell efficacy and leads to their exhaustion. Understanding the mechanobiological properties of the TME and their effects on T cells is key for developing novel adoptive tumor immunotherapies.

Understanding immune signaling using advanced imaging techniques

Brameshuber M, Klotzsch E, Ponjavic A, Sezgin E

Biochemical Society Transactions , 2022 - Article

Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity.

Nanoconfinement of Microvilli Alters Gene Expression and Boosts T cell Activation

Aramesh MS, Stoycheva D, Sandu I, Ihle S, Zünd T, Shiu J, Forro C, Asghari M, Bernero M, Lickert S, Oxenius A, Vogel V, Klotzsch E.S

Proc. Natl. Acad. Science U.S.A., 2021 - Article

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation

An H, Richardson A, Rajasekariah P, Zhong L, Fernando BSM, Macmillan A, Klotzsch E, Bryant K, Kaakoush KO, Tedla NT

Journal of Proteome Research, 2021 - Article

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.

Temporal Analysis of T cell Receptor-Imposed Forces via Quantitative Single Molecule FRET Measurements

Göhring J, Kellner F, Schrangl L, Platzer R, Klotzsch E, Stockinger H, Huppa JB, Schütz GJ

Nature Communications, 2021 - Article

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation

Emmanouilidis L, Esteban-Hofer L, Damberger F, de Vries T, Nguyen C, Fábregas Ibáñez I, Mergenthal S, Klotzsch E, Yulikov M, Jeschke G and Allain F

Nature Chem. Biol., 2021 - Article

Many RNA-binding proteins undergo liquid–liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)—a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients—can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron–electron resonance measurements revealed a compaction of FUS in the condensed phase.

External cues to drive B cell function towards immunotherapy. Microscope

Stoycheva D, Simsek H, Weber W, Hauser A and Klotzsch E

Acta Biomaterialia, 2021 - Review

Immunotherapy stands out as a powerful and promising therapeutic strategy in the treatment of cancer, infections, and autoimmune diseases. Adoptive immune therapies are usually centered on modified T cells and their specific expansion towards antigen-specific T cells against cancer and other diseases. However, despite their unmatched features, the potential of B cells in immunotherapy is just beginning to be explored. The main role of B cells in the immune response is to secrete antigen-specific antibodies and provide long-term protection against foreign pathogens. They further function as antigen-presenting cells (APCs) and secrete pro- and anti-inflammatory cytokines and thus exert positive and negative regulatory stimuli on other cells involved in the immune response such as T cells. Therefore, while hyperactivation of B cells can cause autoimmunity, their dysfunctions lead to severe immunodeficiencies. Only suitably activated B cells can play an active role in the treatment of cancers, infections, and autoimmune diseases. As a result, studies have focused on B cell-targeted immunotherapies in recent years. For this, the development, functions, interactions with the microenvironment, and clinical importance of B cells should be well understood. In this review, we summarize the main events during B cell activation. From the viewpoint of mechanobiology we discuss the translation of external cues such as surface topology, substrate stiffness, and biochemical signaling into B cell functions. We further dive into current B cell-targeted therapy strategies and their clinical applications.

Functionalized Bead Assay to Measure Three-dimensional Traction Forces during T-cell Activation

Aramesh M, Mergenthal S, Issler M, Plochberg B, Weber F, Qin XH, Liska R, Huppa J, Duda GN, Huppa JB, Ries J, Schuetz GJ and Klotzsch E

Nano Letters, 2021 - Article

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Here, we developed a traction force microscopy platform which allows for quantifying the pulls and pushes exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. We immobilized specific T-cell activating antibodies on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR, and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas CD45 is excluded from bead–microvilli contacts.

Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope

Aramesh M, Forró C, Dorwling-Carter L, Lüchtefeld I, Schlotter T, Ihle SJ, Shorubalko I, Hosseini V, Momotenko D, Zambelli T, Klotzsch E, Vörös J

Nature Nanotechnology, 2019 - Article

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Tissue transglutaminase in fibrosis - more than an ECM crosslinker

Benn MC, Weber W, Klotzsch E, Vogel V, Pot SA

Current Opinion in Biomedical Engineering, 2019 - Review

Tissue transglutaminase (TG2) is upregulated in the pathogenesis of a wide variety of chronic diseases. In this review, special emphasis will be placed on fundamental mechanisms underlying the critical role of TG2 in fibroproliferative disorders. TG2 is best known for its cross-linking capacities in the extracellular space but has many critical and multifaceted roles beyond protein cross-linking, which are driven by the conformation and specific localization of the molecule. As extracellular cross-linker TG2 promotes fibrotic disease through the storage of latent transforming growth factor (TGF)-β1 in a stiffened extracellular matrix. As membrane-bound cell adhesion cofactor and signaling protein and intracellular cross-linker or G-protein, TG2 promotes fibrotic disease through cell survival and profibrotic pathway activation on a signaling, transcriptional, and translational level. Similarities between the roles that TG2 plays in scar tissue and in the tumor stroma suggest that a deeper understanding of key common pathways in disease pathogenesis and progression might lead to the identification of novel treatment targets and the development of new drugs and diagnostic methods.

Engineering T-cell Activation for Immunotherapy by Mechanical Forces

Aramesh M, Stoycheva D, Raaz L, Klotzsch E

Current Opinion in Biomedical Engineering, 2019 - Review

Adoptive immunotherapy relies on the isolation, modification, activation, and transfer of antigen-specific T-cells. With constant advances in bioengineering, each individual step in T-cell activation and expansion can be further improved. In this review, we discuss the current knowledge on factors influencing the mechanical forces applied on a T-cell during activation. We provide insights from ligand presentation and surface modification and present ideas of a mechanobiological signal transduction from extracellular receptor–ligand interaction to intranuclear gene expression changes as a determining factor of a T-cell response.

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Anderluh A,  Hofmaier T, Klotzsch E, Kudlacek O, Stockner T, Sitte HH, Schütz GJ

Nature Communications, 2017 - Article

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

Tuning membrane protein mobility by confinement into nanodomains

Karner A, Nimmervoll B, Plochberger B, Klotzsch E, Horner A, Knyazev D, Kuttner R, Winkler K, Winter L, Siligan C, Ollinger N, Pohl P, Preiner J

Nature Nanotechnology, 2016 - Article

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

Soluble LILRA3 promotes neurite outgrowth and synapses formation through a high-affinity interaction with Nogo 66

An H, Brettle M, Lee T, Heng B, Lim CK, Guillemin GJ, Lord MS, Klotzsch E, Geczy CL, Bryant K, Fath T, Tedla N

J. Cell Science, 2016 - Article

Inhibitory proteins, particularly Nogo 66, a highly conserved 66-amino-acid loop of Nogo A (an isoform of RTN4), play key roles in limiting the intrinsic capacity of the central nervous system (CNS) to regenerate after injury. Ligation of surface Nogo receptors (NgRs) and/or leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue the paired immunoglobulin-like receptor B (PIRB) by Nogo 66 transduces inhibitory signals that potently inhibit neurite outgrowth. Here, we show that soluble leukocyte immunoglobulin-like receptor A3 (LILRA3) is a high-affinity receptor for Nogo 66, suggesting that LILRA3 might be a competitive antagonist to these cell surface inhibitory receptors. Consistent with this, LILRA3 significantly reversed Nogo-66-mediated inhibition of neurite outgrowth and promoted synapse formation in primary cortical neurons through regulation of the ERK/MEK pathway. LILRA3 represents a new antagonist to Nogo-66-mediated inhibition of neurite outgrowth in the CNS, a function distinct from its immune-regulatory role in leukocytes. This report is also the first to demonstrate that a member of LILR family normally not expressed in rodents exerts functions on mouse neurons through the highly homologous Nogo 66 ligand.

Super-resolution microscopy reveals the spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

Klotzsch E, Smorodchenko A, Löfler L, Parkinson E, Moldzio R, Schütz GJ, Pohl EE

Proc. Natl. Acad. Science U.S.A., 2015 - Article

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix

Kubow KE, Vuikmirovic R, Zhe L, Klotzsch E, Smith ML, Gourdon D, Luna S, Vogel V

Nature Communications, 2015 - Article

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues—particularly in development, wound healing, tissue regeneration and cancer—the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I’s Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

Klotzsch E, Stiegler J, Ben-Ishay E, Gaus K

BBA Molecular Cell Research, 2015 - Review

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor–ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.

Pulling fibronectin fibers from a protein monolayer at the air-liquid interface: a sheet-to-fiber transition generating fibers with a densely packed, spiral, lamellar interior.

Mitsi M, Handschin S, Gerber I, Schwartländer R, Klotzsch E, Wepf R, Vogel V

Biomaterials, 2015 - Article

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Enzymatic synthesis of hyaluronic acid vinyl esters for two-photon microfabrication of biocompatible and biodegradable hydrogel constructs

Qin XH, Gruber P, Markovic M, Plochberger B, Klotzsch E, Stampfl J, Ovsianikov A, Liska R

Polymer Chemistry, 2014 - Article

Two-photon polymerization (2PP) allows 3D microfabrication of biomaterial scaffolds with user-defined geometry. This technique is highly promising for 3D cell culture and tissue engineering. However, biological applications of 2PP require photopolymerizable hydrogels with high reactivity and low cytotoxicity. This paper describes a novel hydrogel system based on hyaluronic acid vinyl esters (HA-VE), which enabled fast 2PP-fabrication of 3D hydrogel constructs with μm-scale accuracy. A series of HA-VE macromers with tunable degrees of substitution were synthesized by lipase-catalyzed transesterification. HA-VE gels were proved to be injectable, photocurable, enzymatically degradable and mechanically comparable to various soft tissues. Owing to the unique molecular design, degradation products of HA-VE gels through hydrolysis are non-toxic polyvinyl alcohol and adipic acid. Furthermore, HA-VE gels were systematically characterized and compared to HA-acrylates (HA-AC) and HA-methacrylates (HA-MA) gels including macromer cytotoxicity, photoreactivity, swelling, and gel stiffness. Cytotoxicity assay with L929 fibroblasts revealed that HA-VE was significantly less toxic than HA-AC (P < 0.01) and HA-MA (P < 0.05). Crosslinking efficiency of HA-VE was comparable to HA-AC and much higher than HA-MA. Although the reactivity of HA-VE for homopolymerization was insufficient for 2PP, it was demonstrated that thiol–ene chemistry could substantially improve its reactivity. This optimization led to 2PP-fabrication of a HA-VE hydrogel construct with μm-scale accuracy. Low cytotoxicity, high reactivity and good biodegradability makes HA-VE promising candidates for biological applications in cell culture and tissue engineering.

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membran

Anderluh A, Klotzsch E, Reismann AWAF, Brameshuber M, Kudlacek O, Newman AH, Sitte HH and Schütz GJ§ 

J.Biol.Chem., 2014 - Article

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.

Tracking Single Serotonin Transporter Molecules at the Endoplasmic Reticulum and Plasma Membrane

Anderluh A, Klotzsch E, Ries J, Reismann AWAF, Weber S, Fölser M, Koban F, Freissmuth M, Sitte HH and Schütz GJ§

Biophys J., 2014 - Article

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.

Single Molecule Localization Microscopy With Multiple Fluorophores To Explore the Conformation of Surface-Adsorbed Fibronectin Molecules

Klotzsch E, Schön I, Ries J, Renn A, Sandoghdar V & Vogel V. 

Biomaterials Science, 2014 - Article

Adsorbed proteins that promote cell adhesion mediate the response of cells to biomaterials and scaffolds. As proteins undergo conformational changes upon surface adsorption, their functional display may be significantly affected by surface chemistry or solution conditions during the adsorption process. A high-resolution localization microscopy technique is extended here to probe the conformation of individual fibronectin (Fn) molecules at the glass–water interface under physiological buffer conditions. To map distances, four available cysteines located on the modules FnIII7 and FnIII15 of dimeric Fn were site-specifically labeled with Cy3B, and their relative positions were determined by stepwise photobleaching with nanometer precision. The four labels on single Fn molecules did not show a uniform or linear arrangement. The distances between label positions were distributed asymmetrically around 33 nm with a tail towards higher distances. Exposure of Fn to denaturing solution conditions during adsorption increased the average distances up to 43 nm for 4 M guanidinium HCl, while changing the solution conditions after the adsorption had no effect, indicating that the observed intra-molecular distances are locked-in during the adsorption process. Also surface coatings of different hydrophobicity altered the conformational distribution, shifting label distances from a median of 24 nm on hydrophilic to 49 nm on hydrophobic surfaces. These results further highlight that the conformation of macromolecules at interfaces depends on the adsorption history. While illustrated here for surface adsorbed Fn, the power of localization-based microscopy extends the repertoire of techniques to characterize biomolecules at interfaces.

Improved ligand-discrimination by force-induced unbinding of the T cell receptor from peptide-MHC

Klotzsch E, Schütz GJ

Biophys J., 2013 - Article

T cell activation is mediated via the recognition of peptides by the T cell receptor (TCR). This receptor ligand interaction is highly specific, and the TCR has to discriminate between a huge number of peptides presented by the products of the major histocompatibility complexes (MHCs). Recent studies indicate that cells probe the TCR-pMHC interaction by imposing force on the interaction. Here we investigated in a theoretical analysis the consequences of such force-induced unbinding for T cell recognition. Our findings are as follows. First, the bond rupture under force is much faster, improving the time resolution of the discrimination process. Second, cells can access additional parameters characterizing the shape of the binding energy surface. Third, load-induced unbinding yields a reduced coefficient of variation of the bond lifetimes, which improves the discriminative power even between peptide/MHCs (pMHCs) with similar off-rates.

A critical survey of methods to detect plasma membrane rafts

Klotzsch E, Schütz GJ

Phil Trans R Soc B, 2013 - Review

The plasma membrane is still one of the enigmatic cellular structures.Although the microscopic structure is getting clearer, not much is knownabout the organization at the nanometre level. Experimental difficultieshave precluded unambiguous approaches, making the current picturerather fuzzy. In consequence, a variety of different membrane models hasbeen proposed over the years, on the basis of different experimentalstrategies. Recent data obtained via high-resolution single-moleculemicroscopy shed new light on the existing hypotheses. We thus think it isa good time for reviewing the consistency of the existing models with thenew data. In this paper, we summarize the available models in ten prop-ositions, each of which is discussed critically with respect to the appliedtechnologies and the strengths and weaknesses of the approaches. Ouraim is to provide the reader with a sound basis for his own assessment.We close this chapter by exposing our picture of the membrane organizationat the nanoscale.

GFP's mechanical intermediate states

Saeger J, Hytönen VP, Klotzsch E, & Vogel V

PLos One, 2012 - Article

Green fluorescent protein (GFP) mutants have become the most widely used fluorescence markers in the life sciences, and although they are becoming increasingly popular as mechanical force or strain probes, there is little direct information on how their fluorescence changes when mechanically stretched. Here we derive high-resolution structural models of the mechanical intermediate states of stretched GFP using steered molecular dynamics (SMD) simulations. These structures were used to produce mutants of EGFP and EYFP that mimic GFP's different mechanical intermediates. A spectroscopic analysis revealed that a population of EGFP molecules with a missing N-terminal α-helix was significantly dimmed, while the fluorescence lifetime characteristic of the anionic chromophore state remained unaffected. This suggests a mechanism how N-terminal deletions can switch the protonation state of the chromophore, and how the fluorescence of GFP molecules in response to mechanical disturbance might be turned off.

Binding-Activated Localization Microscopy of DNA Structures

Schoen I, Ries J, Klotzsch E, Ewers H & Vogel V

Nano Lett., 2011 - Article

Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.

Probing cellular traction forces by micropillar arrays: contribution of substrate warping to pillar deflection

Schoen I, Hu W, Klotzsch E, & Vogel V

Nano Lett., 2010 - Article

Quantifying cellular forces relies on accurate calibrations of the sensor stiffness. Neglecting deformations of elastic substrates to which elastic pillars are anchored systematically overestimates the applied forces (up to 40%). A correction factor considering substrate warping is derived analytically and verified experimentally. The factor scales with the dimensionless pillar aspect ratio. This has significant implications when designing pillar arrays or comparing absolute forces measured on different pillar geometries during cell spreading, motility or rigidity sensing.

Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells

Kubow KE, Klotzsch E, Smith ML, Gourdon D, Little WC, & Vogel

Integrative Biology , 2009 - Article

Elevated levels of tissue crosslinking are associated with numerous diseases (cancer stroma, organ fibrosis), and also eliminate the otherwise remarkable clinical successes of tissue-derived scaffolds, instead eliciting a foreign body reaction. Nevertheless, it is not well understood how the initial physical and biochemical properties of cellular microenvironments, stem cell niches, or of 3D tissue scaffolds guide the assembly and remodeling of new extracellular matrix (ECM) that is ultimately sensed by cells. Here, we incorporated FRET-based mechanical strain sensors, either into cell-derived ECM scaffolds or into the fibronectin (Fn) matrix assembled by reseeded fibroblasts, and demonstrated the following. Cell-generated tensile forces change the conformation of Fn in both 3D scaffolds and new matrix over time. The time course by which new matrix fibers are stretched by reseeded cells is accelerated by scaffold crosslinking. Importantly, stretching Fn fibers increases their elastic modulus (rigidity) and alters their biochemical display. Regulated by Fn fiber unfolding, more soluble Fn binds to the native than to the crosslinked scaffolds. Additionally, matrix assembly of fibroblasts is decreased by scaffold crosslinking. Taken together, scaffold crosslinking has a multifactorial impact on the microenvironment that reseeded cells assemble and respond to, with far-reaching implications for tissue engineering and disease physiology.

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sitesFibronectin forms the most extensible biological fibers displaying switchable force-exp

Klotzsch E, Smith ML, Kubow KE, Muntwyler S, Little WC, Beyeler F, Gourdon D, Nelson BJ, & Vogel V§ 

Proc. Natl. Acad. Science U.S.A., 2009 - Article

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Optimization strategies for electrospun silk fibroin tissue engineering scaffolds

Meinel AJ, Kubow KE, Klotzsch E, Garcia-Fuentes M, Smith ML, Vogel V, Merkle HP, & Meinel L

Biomaterials , 2009 - Article

As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well-established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSCs) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSCs showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds.

Interferometric detection and tracking of nanoparticles.

Jacobsen V, Klotzsch E & Sandoghdar V

Nano Biophotonics: Science and Technology, 2007 - Book Chapter

Chapter 9

Label-Free Optical Detection and Tracking of Single Virions Bound to Their Receptors in Supported Membrane Bilayers

Ewers H, Jacobsen V, Klotzsch E, Smith AE, Helenius A, & Sandoghdar V

Nano Lett., 2007 - Article

We apply an interferometric optical detection scheme to image and track unlabeled single virions. Individual simian virus 40 virions and uninfectious virus-like particles were imaged on a glass substrate and on a supported membrane bilayer. Moreover, single unlabeled virions were tracked when bound to supported membrane bilayers via the viral receptor, the glycolipid GM1. The technology presented here promises to be generally applicable to studying the motion of unlabeled macromolecules on membranes.

Optical Detection of Very Small Nonfluorescent Nanoparticles

Sandoghdar V, Klotzsch E, Jacobsen V, Renn A, Hakanson U, Agio M, Gerhardt I, Seelig J, & Wrigge G

CHIMIA, 2006 - Review

We discuss an interferometric method for the optical detection of very small nonfluorescent nanoparticles. In particular, we show that single gold nanoparticles with a diameter as small as 5 nm can be detected. We discuss the potential of such tiny particles as optical labels for biological studies. Furthermore, we show that our interferometric method can be also used for the detection and tracking of unlabelled biological nano-entities such as viruses or microtubuli.

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