Enrico Klotzsch

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

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 Youngs 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 domains 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

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.

Binding-Activated Localization Microscopy of DNA Structures

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

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.

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

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 Is 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.

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

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.

A critical survey of methods to detect plasma membrane rafts

The plasma membrane is still one of the enigmatic cellular structures. Although the microscopic structure is getting clearer, not much is known about the organization at the nanometre level. Experimental difficulties have precluded unambiguous approaches, making the current picture rather fuzzy. In consequence, a variety of different membrane models has been proposed over the years, on the basis of different experimental strategies. Recent data obtained via high-resolution single-molecule microscopy shed new light on the existing hypotheses. We thus think it is a good time for reviewing the consistency of the existing models with the new data. In this paper, we summarize the available models in ten propositions, each of which is discussed critically with respect to the applied technologies and the strengths and weaknesses of the approaches. Our aim is to provide the reader with a sound basis for his own assessment. We close this chapter by exposing our picture of the membrane organization at the nanoscale.

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

Background: The serotonin transporter (SERT) terminates synaptic signaling by reuptake of the neurotransmitter serotonin. Results: Interaction kinetics and number of subunits are elucidated by single molecule brightness analysis of SERT complexes. Conclusion: The oligomeric state of SERT complexes is stably determined before being integrated into the plasma membrane. Significance: The results reveal the first evidence for kinetic trapping of preformed neurotransmitter transporter oligomers. 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.

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

Significance The question as to how the proton motive force in mitochondria is distributed among the proteins that require a proton gradient for their work is one of the central unresolved questions in mitochondrial physiology and important for the mechanistic insight in the function of mitochondrial proteins. Our results suggest that the local separation of the proteins on the inner mitochondrial membrane makes it impossible for uncoupling protein 4 (UCP4) to uncouple phosphorylation from proton pumping. Nonetheless, UCP4 should be well able to shortcut excessive transmembrane proton gradients to thereby regulate reactive oxygen species production. It explains how the proton transporter may fulfill that function without being a real UCP like UCP1. 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.

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

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.