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Three-dimensional (3D) culture is expected to reproduce biological tissues more representatively than monolayer culture, which is important for in vitro research such as drug screening. Recently, various cell culture substrates for spheroid engineering have been developed based on the prevention of cell adhesion. However, despite the expanded usability these substrates provide, they remain limited in terms of optical microscopy imaging of spheroids with high magnification lenses. This is because almost all substrates generated by nanoimprinting hamper the light passing through them owing to their low optical transparency caused by the thickness and surface structure. In this study, we achieved the preparation of spheroids from adipose-tissue derived stem cells (ASCs) on free-standing porous polymeric ultrathin films ("porous nanosheets") consisting of poly(d,l-lactic acid) (PDLLA) with thickness of 120 nm and average pore diameter of 4 μm. ASCs migrated on the porous nanosheet, leading to the spontaneous organization of spheroids anchored via a cell monolayer. The porous nanosheet also provided more than twice the optical transparency in confocal and holographic microscopy observation compared to conventional nanoimprinted substrates for 3D cell culture (NanoCulture Dish). The internal structure of the organized spheroids could be clearly observed with 40× magnification. In addition, the engineered spheroids showed bioactivities indicated by mRNA expression of fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). Thus, porous nanosheets offer a unique cell culture substrate, not only for engineering 3D cellular organization from stem cells, but also for imaging detailed structure using light microscopy.

Monocytes in the blood torrent, when activated by pro-inflammatory signals, adhere to the vascular endothelium and migrate into the tissue for ultimately differentiate into macrophages. Mechanics and adhesion play a crucial role in macrophage functions, such as migration and phagocytosis. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood.In this work, we combined atomic force microscopy (AFM) viscoelastic mapping with interference contrast microscopy (ICM) to simultaneously probe, at the single-cell level, viscoelasticity and adhesion during monocyte differentiation. THP-1 monocytic cells were differentiated into macrophages through phorbol 12-myristate 13-acetate (PMA). Morphological quantification was achieved using holographic tomography imaging and the expression of integrin subunit CD11b was tracked as a marker of differentiation.Holographic tomography proved to be a quantitative in vivo technique, revealing a dramatic increase in macrophage volume and surface area and two subpopulations, spread and round macrophages. AFM viscoelastic mapping revealed an increased stiffness and more solid-like behavior of differentiated macrophages, especially in the lamellipodia and microvilli regions. Differentiated cells revealed an important increase of the apparent Young’s modulus (E0) and a decrease of cell fluidity (β) on differentiated cells, which correlated with an increase in adhesion area. Macrophages with a spreading phenotype enhanced these changes. Remarkably, when adhesion was eliminated, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activity, such as phagocytosis, making it more efficient. Our proposed approach revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

Atomic force microscopy (AFM) can visualize the dynamics of single biomolecules under near-physiological conditions. However, the scanning tip probes only the molecular surface with limited resolution, missing details required to fully deduce functional mechanisms from imaging alone. To overcome such drawbacks, we developed a computational framework to reconstruct 3D atomistic structures from AFM surface scans, employing simulation AFM and automatized fitting to experimental images. We provide applications to AFM images ranging from single molecular machines, protein filaments, to large-scale assemblies of 2D protein lattices, and demonstrate how the obtained full atomistic information advances the molecular understanding beyond the original topographic AFM image. We show that simulation AFM further allows for quantitative molecular feature assignment within measured AFM topographies. Implementation of the developed methods into the versatile interactive interface of the BioAFMviewer software, freely available at www.bioafmviewer.com, presents the opportunity for the broad Bio-AFM community to employ the enormous amount of existing structural and modeling data to facilitate the interpretation of resolution-limited AFM images.

We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20–100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm2) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Integrin α

αVβ3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVβ3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVβ3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVβ3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVβ3-Fn binding. Thus, force-dependent conformational changes in αVβ3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin.This article has an associated First Person interview with the first author of the paper.

Integrin α2β1 is a widely expressed collagen I receptor which also mediates laminin-111 binding in some cell types, but the functional relevance of collagen versus laminin binding for different cell types is poorly understood. Here we use AFM-based singe-cell force spectroscopy (SCFS) to compare α2β1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2β1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2β1 as a receptor only for collagen in these cells. Inversely, the α2β1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2β1 expression suppresses laminin binding. Analogous results were obtained in a pair of SAOS-2 human osteosarcoma cell lines. Again, wildtype cells (SAOS-WT) adhered strongly to laminin and poorly to collagen, while expression of integrin α2β1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2β1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2β1 expression downregulated transcription and expression of integrin subunits α6 and β4, components of the main laminin-111 binding receptors integrin α6β1 and α6β4 in these cells. Integrin α6 and β4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2β1 expression negatively regulates integrin α6β1 and α6β4-mediated adhesion, spreading and invasion on laminin in different cancer cell types. In contrast to SAOS-WT, but similar to SAOS-A2 osteosarcoma cells, primary Human osteoblasts (HOB) cells express α2 but only low levels of β4 integrin, preferentially adhere to and spread on collagen over laminin and show suppressed laminin-dependent transmigration. By enhancing collagen binding directly and suppressing laminin binding indirectly through laminin receptor downregulation, α2β1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution.

Time-lapse atomic force microscopy imaging is used to visualize the initial stages of fibronectin fibrillogenesis directly in living cells with high resolution. This approach provides new structural and mechanistic details, such as a stepwise extension mechanism and an accelerating effect of extracellular Mn2+ on early FN fibrillogenesis., Fibronectin (FN) is an extracellular matrix protein that can be assembled by cells into large fibrillar networks, but the dynamics of FN remodeling and the transition through intermediate fibrillar stages are incompletely understood. Here we used a combination of fluorescence microscopy and time-lapse atomic force microscopy (AFM) to visualize initial stages of FN fibrillogenesis in living fibroblasts at high resolution. Initial FN nanofibrils form within

αVβ3 integrin recognizes multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn). However, cell experiments are frequently performed on homogenously coated substrates with only one integrin ligand present. Here, we employed binary-choice substrates of Fn and Vn to dissect αVβ3 integrin-mediated binding to both ligands on the subcellular scale. Superresolution imaging revealed that αVβ3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required mechanical load on αVβ3 integrin. Integrin mutations, structural analysis, and molecular dynamics simulations established a model where the extended-closed conformation of αVβ3 integrin binds Vn but not Fn. Force-mediated hybrid domain swing-out characterizes the extended-open conformation needed for efficient Fn binding. Thus, force-dependent conformational changes in αVβ3 integrin increase the number of available ligands and therefore the ligand promiscuity of this integrin. These findings for αVβ3 integrin were shown to regulate cell migration and mechanotransduction differentially on Fn compared to Vn and therefore to regulate cell behavior.

During development cranial neural crest cells (NCCs) display a striking transition from collective to single-cell migration, but the mechanisms enabling individual NCCs to separate from the neural crest tissue are still incompletely understood. In this study we have employed atomic force microscopy (AFM) to investigate potential adhesive and mechanical changes associated with the dissociation of individual cells from cohesive Xenopus NCC explants at early stages of migration. AFM-based single-cell force spectroscopy (SCFS) revealed a uniform distribution of cell-cell adhesion forces within NCC explants, including semi-detached leader cells in the process of delaminating from the explant edge. This suggested that dissociation from the cell sheet may not require prior weakening of cell-cell contacts. However, mapping NCC sheet elasticity by AFM microbead indentation demonstrated strongly reduced cell stiffness in semi-detached leader cells compared to neighbouring cells in the NCC sheet periphery. Reduced leader cell stiffness coincided with enhanced cell spreading and high substrate traction, indicating a possible mechano-regulation of leader cell delamination. In support, AFM elasticity measurements of individual NCCs in optical side view mode demonstrated that reducing cell tension by inhibiting actomyosin contractility induces rapid spreading, possibly maximizing cell-substrate interactions as a result. Depletion of cadherin-11, a classical cadherin with an essential role in NCC migration and substrate adhesion, prevented the tension reduction necessary for NCC spreading, both in individual cells and at the edge of explanted sheets. In contrast, overexpression of cadherin-11 accelerated spreading of both individual cells and delaminating leader cells. As cadherin-11 expression increases strongly during NCC migration, this suggests an important role of cadherin-11 in regulating NCC elasticity and spreading at later stages of NCC migration. We therefore propose a model in which high tension at the NCC sheet periphery prevents premature NCC spreading and delamination during early stages of migration, while a cadherin-11-dependent local decrease in cell tension promotes leader cell spreading and delamination at later stages of migration.

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (60min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.

Tissue-embedded cells are often exposed to a complex mixture of extracellular matrix (ECM) molecules, to which they bind with different cell adhesion receptors and affinities. Differential cell adhesion to ECM components is believed to regulate many aspects of tissue function, such as the sorting of specific cell types into different tissue compartments or ECM niches. In turn, aberrant switches in cell adhesion preferences may contribute to cell misplacement, tissue invasion, and metastasis. Methods to determine differential adhesion profiles of single cells are therefore desirable, but established bulk assays usually only test cell population adhesion to a single type of ECM molecule. We have recently demonstrated that atomic force microscopy-based single-cell force spectroscopy (SCFS), performed on bifunctional, microstructured adhesion substrates, provides a useful tool for accurately quantitating differential matrix adhesion of single Chinese hamster ovary cells to laminin and collagen I. Here, we have extended this approach to include additional ECM substrates, such as bifunctional collagen I/collagen IV surfaces, as well as adhesion-passivated control surfaces. We investigate differential single cell adhesion to these substrates and analyze in detail suitable experimental conditions for comparative SCFS, including optimal cell-substrate contact times and the impact of force cycle repetitions on single cell adhesion force statistics. Insight gained through these experiments may help in adapting this technique to other ECM molecules and cell systems, making directly comparative SCFS a versatile tool for comparing receptor-mediated cell adhesion to different matrix molecules in a wide range of biological contexts. Copyright © 2013 John Wiley & Sons, Ltd.

Producing sterile glass surfaces is of great importance for a wide range of laboratory and medical applications, including in vitro cell culture and tissue engineering. However, sterilization may change the surface properties of glass and thereby affect its use for medical applications, for instance as a substrate for culturing cells. To investigate potential effects of sterilization on glass surface topography, borosilicate glass coverslips were left untreated or subjected to several common sterilization procedures, including low-temperature plasma gas, gamma irradiation and steam. Imaging by atomic force microscopy demonstrated that the surface of untreated borosilicate coverslips features a complex landscape of microislands ranging from 1000 to 3000 nm in diameter and 1 to 3 nm in height. Steam treatment completely removes these microislands, producing a nanosmooth glass surface. In contrast, plasma treatment partially degrades the microisland structure, while gamma irradiation has no effect on microisland topography. To test for possible effects of the nanotopographic structures on cell adhesion, human gingival fibroblasts were seeded on untreated or sterilized glass surfaces. Analyzing fibroblast adhesion 3, 6, and 24 h after cell seeding revealed significant differences in cell attachment and spreading depending on the sterilization method applied. Furthermore, single-cell force spectroscopy revealed a connection between the nanotopographic landscape of glass and the formation of cellular adhesion forces, indicating that fibroblasts generally adhere weakly to nanosmooth but strongly to nanorough glass surfaces. Nanotopographic changes induced by different sterilization methods may therefore need to be considered when preparing sterile glass surfaces for cell culture or biomedical applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2755–2766, 2014.

Cell populations often display heterogeneous behavior, including cell-to-cell variations in morphology, adhesion and spreading. However, better understanding the significance of such cell variations for the function of the population as a whole requires quantitative single-cell assays. To investigate adhesion variability in a CHO cell population in detail, we measured integrin-mediated adhesion to laminin and collagen, two ubiquitous ECM components, by AFM-based single-cell force spectroscopy (SCFS). CHO cells generally adhered more strongly to laminin than collagen but population adhesion force distributions to both ECM components were broad and partially overlapped. To determine the levels of laminin and collagen binding in individual cells directly, we alternatingly measured single cells on adjacent microstripes of collagen and laminin arrayed on the same adhesion substrate. In repeated measurements (≥60) individual cells showed a stable and ECM type-specific adhesion response. All tested cells bound laminin more strongly, but the scale of laminin over collagen binding varied between cells. Together, this demonstrates that adhesion levels to different ECM components are tightly yet differently set in each cell of the population. Adhesion variability to laminin was non-genetic and cell cycle-independent but scaled with the range of α6 integrin expression on the cell surface. Adhesive cell-to-cell variations due to varying receptor expression levels thus appear to be an inherent feature of cell populations and should to be considered when fully characterizing population adhesion. In this approach, SCFS performed on multifunctional adhesion substrates can provide quantitative single-cell information not obtainable from population-averaging measurements on homogeneous adhesion substrates.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O(6)-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (microCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.

The atomic force microscope (AFM), a member of the scanning probe family of microscopes, generates height maps of sample surfaces with subnanometer resolution. Importantly, AFM offers the opportunity to image samples with little or no treatment and under physiologically-relevant conditions, making it well-suited for investigating the structure of biological samples, including fixed or living cells and tissues. In addition to its high-resolution imaging capability, AFM used in force spectroscopy mode is a sensitive force measuring device, able to detect or exert forces ranging from the pico- to the nanonewton scale. Here we review a broad range of cell biological applications of AFM, including high resolution imaging of adherent cells, measuring cell adhesion down to the single-receptor level and characterizing the mechanical properties of cells. Furthermore, we present recent examples of how the combined use of AFM and advanced light microscopy techniques can provide complementary structural information.

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.

We have characterized early steps of α2β1integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Atomic force microscopy (AFM) can produce high-resolution topographic images of biological samples in physiologically relevant environments and is therefore well suited for the imaging of cellular surfaces. In this work we have investigated focal adhesion complexes by combined fluorescence microscopy and AFM. To generate high-resolution AFM topographs of focal adhesions, REF52 (rat embryo fibroblast) cells expressing YFP-paxillin as a marker for focal adhesions were de-roofed and paxillin-positive focal adhesions subsequently imaged by AFM. The improved resolution of the AFM topographs complemented the optical images and offered ultrastructural insight into the architecture of focal adhesions. Focal adhesions had a corrugated dorsal surface formed by microfilament bundles spaced 127+/-50 nm (mean+/-s.d.) apart and protruding 118+/-26 nm over the substratum. Within focal adhesions microfilaments were sometimes branched and arranged in horizontal layers separated by 10 to 20 nm. From the AFM topographs focal adhesion volumes could be estimated and were found to range from 0.05 to 0.50 microm(3). Furthermore, the AFM topographs show that focal adhesion height increases towards the stress-fiber-associated end at an angle of about 3 degrees . Finally, by correlating AFM height information with fluorescence intensities of YFP-paxillin and F-actin staining, we show that the localization of paxillin is restricted to the ventral half of focal adhesions, whereas F-actin-containing microfilaments reside predominantly in the membrane-distal half.

The simulation of manufacturing processes is more and more becoming an important tool in simultaneous engineering. The aim is to cut the time necessary for development and to optimise processes by simulation of the complete manufacturing chain. The field of heat treatment offers a large variety of applications for the use of simulation tools. Heat treatment of steels always includes the development of residual stresses and distortions. The geometry of the part, the composition of the material, the heat treatment process as well as the initial state of the part interact with each other in complex ways and have an influence on the distortion of the part. Using simulation the temporal development of temperature, phases, stresses and distortions while quenching as well as the residual stress distribution and distortion after quenching can be calculated. Transformation plasticity has been proved to be very important for heat treatment simulation. Three steels with identical contents of alloying elements but different carbon contents of 0.2, 0.5 and 0.8 wt.-% were analysed. The transformation plasticity constants for the martensitic transformation under tensile as well as compressive stresses were determined by quenching hollow specimen with nitrogen. Distortions and residual stresses were examined experimentally with cylinders made out of the three steels. Additionally, simulations of the quenching process of the cylinders were taken into account in the analysis of the experimental findings.

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

p120 catenin (p120ctn), an armadillo protein and component of the cadherin adhesion complex, has been found recently to induce a dendritic morphology by regulating Rho family GTPases. We have identified specific serines within the Arm repeat domain that, when mutated to alanine, promote p120ctn association with interphase microtubules, leading to microtubule reorganization and stabilization. The mutant p120ctn also localized to the mitotic spindle and centrosomes. In contrast to wild-type p120ctn, the microtubule-associated p120ctn mutant did not activate Rac1 and did not induce a dendritic morphology. In addition, we show that a basic motif within the p120ctn Arm repeat domain known to be required for the inhibition of RhoA is also required for binding to microtubules. We therefore propose that binding of p120ctn to microtubules is inversely related to its ability to regulate Rho GTPases.

Die Simulation von Fertigungsprozessen ist ein entscheidendes Werkzeug im Rahmen des Simultaneous Engineering. Ziel ist es, durch Simulation der kompletten Fertigungskette Entwicklungszeiten zu verkurzen und Verfahren zu optimieren. Der Bereich der Warmebehandlung bietet dem Einsatz von Simulationswerkzeugen ein breites Anwendungsgebiet. Die Bauteilgeometrie, die Werkstoffzusammensetzung, das Warmebehandlungsverfahren und dessen Ablauf sowie der Ausgangszustand mit den herstell- und fertigungsbedingten Einflussen auf das Bauteil sind miteinander in komplexer Weise verknupft und beeinflussen das Mas- und Formanderungsverhalten der Bauteile wahrend der Warmebehandlung. Die Berechnung von Gefuge- und Harteverteilungen kann konkrete Hinweise auf geeignete Abschreckbedingungen, Anlagentechnik oder Chargierbedingungen geben, und berechnete Eigenspannungen sowie Mas- und Formanderungen konnen bei der Entwicklung und Konstruktion der Bauteile berucksichtigt werden. Am Institut fur Werkstoffkunde I der Universitat Karlruhe (TH) wird seit fast 30 Jahren auf diesem Gebiet sowohl numerisch als auch experimentell geforscht.

A recent meeting at the Max Delbrück Center in Berlin, Germany provided a forum to discuss the molecular mechanisms of cell migration in a broad range of contexts including chemotaxis, development, immunity, and cancer.

During development cell–cell adhesion is not only crucial to maintain tissue morphogenesis and homeostasis, it also activates signalling pathways important for the regulation of different cellular processes including cell survival, gene expression, collective cell migration and differentiation. Importantly, gene mutations of adhesion receptors can cause developmental disorders and different diseases. Quantitative methods to measure cell adhesion are therefore necessary to understand how cells regulate cell–cell adhesion during development and how aberrations in cell–cell adhesion contribute to disease. Different in vitro adhesion assays have been developed in the past, but not all of them are suitable to study developmentally-related cell–cell adhesion processes, which usually requires working with low numbers of primary cells. In this review, we provide an overview of different in vitro techniques to study cell–cell adhesion during development, including a semi-quantitative cell flipping assay, and quantitative single-cell methods based on atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) or dual micropipette aspiration (DPA). Furthermore, we review applications of Förster resonance energy transfer (FRET)-based molecular tension sensors to visualize intracellular mechanical forces acting on cell adhesion sites. Finally, we describe a recently introduced method to quantitate cell-generated forces directly in living tissues based on the deformation of oil microdroplets functionalized with adhesion receptor ligands. Together, these techniques provide a comprehensive toolbox to characterize different cell–cell adhesion phenomena during development.

The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems.

Lumican and decorin, two members of the small leucine-rich repeat proteoglycan (SLRP) family, have been implicated as regulators of collagen I fibril structure in different tissues. Both proteoglycans consist of a core protein and a glycosaminoglycan (GAG) chain, but quantitative information regarding the precise role of the protein and GAG moieties in regulating collagen structure is still limited. In this study, we used AFM imaging and a model system of aligned collagen I nanofibrils to investigate the role of lumican and decorin on collagen I fibril structure with high resolution. When co-assembled with collagen I, recombinant lumican or decorin proteins lacking the GAG chains decreased collagen fibril width to values below

Transmigrating cells often need to deform cell body and nucleus to pass through micrometer-sized pores in extracellular matrix scaffolds. Furthermore, chemoattractive signals typically guide transmigration, but the precise interplay between mechanical constraints and signaling mechanisms during 3D matrix invasion is incompletely understood and may differ between cell types. Here, we used Direct Laser Writing to fabricate 3D cell culture scaffolds with adjustable pore sizes (2-10 μm) on a microporous carrier membrane for applying diffusible chemical gradients. Mouse embryonic fibroblasts invade 10 μm pore scaffolds even in absence of chemoattractant, but invasion is significantly enhanced by knockout of lamin A/C, a known regulator of cell nucleus stiffness. Nuclear stiffness thus constitutes a major obstacle to matrix invasion for fibroblasts, but chemotaxis signals are not essential. In contrast, epithelial A549 cells do not enter 10 μm pores even when lamin A/C levels are reduced, but readily enter scaffolds with pores down to 7 μm in presence of chemoattractant (serum). Nuclear stiffness is therefore not a prime regulator of matrix invasion in epithelial cells, which instead require chemoattractive signals. Microstructured scaffolds with adjustable pore size and diffusible chemical gradients are thus a valuable tool to dissect cell-type specific mechanical and signaling aspects during matrix invasion.

Tissue-embedded cells are often exposed to a complex mixture of extracellular matrix (ECM) molecules, to which they bind with different cell adhesion receptors and affinities. Differential cell adhesion to ECM components is believed to regulate many aspects of tissue function, such as the sorting of specific cell types into different tissue compartments or ECM niches. In turn, aberrant switches in cell adhesion preferences may contribute to cell misplacement, tissue invasion, and metastasis. Methods to determine differential adhesion profiles of single cells are therefore desirable, but established bulk assays usually only test cell population adhesion to a single type of ECM molecule. We have recently demonstrated that atomic force microscopy-based single-cell force spectroscopy (SCFS), performed on bifunctional, microstructured adhesion substrates, provides a useful tool for accurately quantitating differential matrix adhesion of single Chinese hamster ovary cells to laminin and collagen I. Here, we have extended this approach to include additional ECM substrates, such as bifunctional collagen I/collagen IV surfaces, as well as adhesion-passivated control surfaces. We investigate differential single cell adhesion to these substrates and analyze in detail suitable experimental conditions for comparative SCFS, including optimal cell-substrate contact times and the impact of force cycle repetitions on single cell adhesion force statistics. Insight gained through these experiments may help in adapting this technique to other ECM molecules and cell systems, making directly comparative SCFS a versatile tool for comparing receptor-mediated cell adhesion to different matrix molecules in a wide range of biological contexts.

Producing sterile glass surfaces is of great importance for a wide range of laboratory and medical applications, including in vitro cell culture and tissue engineering. However, sterilization may change the surface properties of glass and thereby affect its use for medical applications, for instance as a substrate for culturing cells. To investigate potential effects of sterilization on glass surface topography, borosilicate glass coverslips were left untreated or subjected to several common sterilization procedures, including low-temperature plasma gas, gamma irradiation and steam. Imaging by atomic force microscopy demonstrated that the surface of untreated borosilicate coverslips features a complex landscape of microislands ranging from 1000 to 3000 nm in diameter and 1 to 3 nm in height. Steam treatment completely removes these microislands, producing a nanosmooth glass surface. In contrast, plasma treatment partially degrades the microisland structure, while gamma irradiation has no effect on microisland topography. To test for possible effects of the nanotopographic structures on cell adhesion, human gingival fibroblasts were seeded on untreated or sterilized glass surfaces. Analyzing fibroblast adhesion 3, 6, and 24 h after cell seeding revealed significant differences in cell attachment and spreading depending on the sterilization method applied. Furthermore, single-cell force spectroscopy revealed a connection between the nanotopographic landscape of glass and the formation of cellular adhesion forces, indicating that fibroblasts generally adhere weakly to nanosmooth but strongly to nanorough glass surfaces. Nanotopographic changes induced by different sterilization methods may therefore need to be considered when preparing sterile glass surfaces for cell culture or biomedical applications.

We have recently demonstrated strong nuclear deformation of SaOs-2 osteosarcoma cells on poly-L-lactic acid (PLLA) micropillar substrates. In the present study, we first demonstrated that chemical and mechanical properties of the micropillar substrates have no dominant effect on deformation. However, SaOs-2 nucleus deformation could be strongly modulated by varying the pillar size and spacing, highlighting the importance of geometric constraints for shaping the nucleus. Furthermore, comparing the capacity for nuclear deformation in three different osteosarcoma cell lines (SaOs-2, MG-63 and OHS-4) revealed strong cell-type specific differences. Surprisingly, the highly-deformable SaOs-2 cell line displayed the highest cell stiffness as assessed by AFM-based colloidal force spectroscopy and featured a more prominent array of actin fibres above the nucleus, suggesting a link between actin-mediated cell stiffness and cell nucleus deformation. In contrast, in MG-63 and OHS-4 cells dense microtubule and vimentin networks seem to facilitate some nuclear deformation even in the absence of a prominent actin cytoskeleton. Together these results suggest that an interaction of all three cytoskeletal elements is needed for efficient nuclear deformation. In conclusion, the dominant parameters influencing nuclear deformation on micropillar substrates are not their material properties but the substrate geometry together with cell phenotype and cytoskeleton organization.

Interactions with the extracellular matrix (ECM) govern a wide range of cellular functions, including survival, migration and invasion. However, in adherent cells these interactions occur primarily on the basal cell side, making them inaccessible to high-resolution, surface-scanning imaging techniques such as atomic force microscopy (AFM) or scanning electron microscopy (SEM). Here we describe a fast and reliable method for inverting adherent cells, exposing the basal cell membrane for direct analysis by AFM or SEM in combination with fluorescence microscopy. Cells including their matrix adhesion sites remain intact during the inversion process and are transferred together with the complete array of basally associated ECM proteins. Molecular features of ECM proteins, such as the characteristic 67 nm collagen D-periodicity, are well preserved after inversion. To demonstrate the versatility of the method, we compared basal interactions of fibroblasts with fibrillar collagen I and fibronectin matrices. While fibroblasts remodel the fibronectin layer exclusively from above, they actively invade even thin collagen layers by contacting individual collagen nanofibrils both basally and apically through a network of cellular extensions. Cell-matrix entanglement coincides with enhanced cell spreading and flattening, indicating that nanoscale ECM interactions govern macroscopic changes in cell morphology. The presented cell inversion technique can thus provide novel insight into nanoscale cell-matrix interactions at the basal cell side.

The biofunctionalization of materials creates interfaces on which proteins, cells, or tissues can fulfill native or desired tasks. Here we report how to control the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Collagen fibrils in these ultrathin (approximately 3 nm) matrices maintained their native structure as observed in vivo. This opens up the possibility to create programmable biofunctionalized matrices using collagen-binding proteins or proteins fused with collagen-binding domains. Applied to eukaryotic cells, these nanostructured matrices can direct cellular processes such as adhesion, orientation and migration.

Fibrillar collagens constitute a main component of many tissues, where they form a scaffold for cell attachment and provide mechanical strength. Gaining insight into molecular mechanisms of collagen self-assembly from in vitro experiments is important for better understanding the complex hierarchical processes involved in collagen fibril formation in vivo. In addition, such insight can be used to assemble collagen into desirable structures for the biofunctionalization of surfaces in different biotechnological and medical applications. Here, we describe a method to direct the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Within these matrices, the self-assembly of collagen molecules into fibrils can be directly observed by time-lapse atomic force microscopy (AFM). High-resolution AFM topographs reveal substructural details of the collagen fibril architecture and provide information about mechanisms and dynamics of fibril formation.

Fibrillar collagens constitute a main component of many tissues, where they form a scaffold for cell attachment and provide mechanical strength. Gaining insight into molecular mechanisms of collagen self-assembly from in vitro experiments is important for better understanding the complex hierarchical processes involved in collagen fibril formation in vivo. In addition, such insight can be used to assemble collagen into desirable structures for the biofunctionalization of surfaces in different biotechnological and medical applications. Here, we describe a method to direct the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Within these matrices, the self-assembly of collagen molecules into fibrils can be directly observed by time-lapse atomic force microscopy (AFM). High-resolution AFM topographs reveal substructural details of the collagen fibril architecture and provide information about mechanisms and dynamics of fibril formation.