ABSTRACT
Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.
Subject(s)
Neurons , Neurons/drug effects , Neurons/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/chemistry , Light , Lipid Bilayers/chemistry , Molecular Dynamics Simulation , Rats , Mice , OptogeneticsABSTRACT
Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.
Subject(s)
Laminin , Laminin/metabolism , Microscopy, Atomic Force , Protein Isoforms/metabolism , Protein Domains , Cell AdhesionABSTRACT
Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.
Subject(s)
Anoctamins , Ion Channels , Anoctamins/metabolism , Ion Channels/metabolism , Electrophysiological Phenomena , Phospholipid Transfer Proteins/metabolism , Lipids , Calcium/metabolismABSTRACT
S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.
Subject(s)
Actomyosin , Focal Adhesions , Humans , Focal Adhesions/metabolism , Actomyosin/metabolism , Calcium/metabolism , Cytoskeletal Proteins/metabolism , Myosin Type II/metabolism , S100 Proteins/genetics , S100 Proteins/metabolismABSTRACT
Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, ß) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.
Subject(s)
Microscopy , Monocytes , Monocytes/metabolism , Macrophages/metabolism , Elastic Modulus , Cell Differentiation , Cell AdhesionABSTRACT
Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.
Subject(s)
Fibroblasts , Focal Adhesions , Animals , Mice , Cell Adhesion/physiology , Fibroblasts/metabolism , Focal Adhesions/metabolism , Integrins/metabolism , Talin/genetics , Talin/chemistry , Talin/metabolism , Vinculin/genetics , Vinculin/chemistry , Vinculin/metabolismABSTRACT
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.
Subject(s)
Nanotechnology , Proteins , Computer Simulation , Microscopy, Atomic Force/methods , Proteins/chemistry , SoftwareABSTRACT
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.
ABSTRACT
Integrin α2ß1 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 α2ß1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2ß1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2ß1 as a receptor only for collagen in these cells. Inversely, the α2ß1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2ß1 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 α2ß1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2ß1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2ß1 expression downregulated transcription and expression of integrin subunits α6 and ß4, components of the main laminin-111 binding receptors integrin α6ß1 and α6ß4 in these cells. Integrin α6 and ß4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2ß1 expression negatively regulates integrin α6ß1 and α6ß4-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 ß4 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, α2ß1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.
Subject(s)
Integrin alpha2beta1 , Receptors, Laminin , Animals , CHO Cells , Cell Adhesion , Cricetinae , Cricetulus , Humans , Integrin alpha2beta1/genetics , Integrin alpha6beta1 , LamininABSTRACT
We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.
ABSTRACT
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.
Subject(s)
Adipose Tissue/chemistry , Cell Culture Techniques/methods , Molecular Imaging , Optical Phenomena , Stem Cells/cytology , Tissue Engineering , Tissue Scaffolds/chemistry , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation/drug effects , Humans , Polyesters/pharmacology , Porosity , Stem Cells/drug effects , Stem Cells/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
αVß3 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 αVß3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVß3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVß3 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 αVß3-Fn binding. Thus, force-dependent conformational changes in αVß3 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.
Subject(s)
Fibronectins , Integrins , Cell Adhesion , Extracellular Matrix Proteins , Fibronectins/genetics , Integrin alphaVbeta3/genetics , Ligands , Mechanical Phenomena , Vitronectin/geneticsABSTRACT
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 ß-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.
Subject(s)
Breast Neoplasms/metabolism , Cadherins/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Intraductal, Noninfiltrating/metabolism , Carcinoma, Lobular/metabolism , Tetraspanins/metabolism , Animals , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Carcinoma, Lobular/pathology , Cell Line, Tumor , Extracellular Vesicles , Female , Humans , Neoplasm Metastasis , Rats , Signal TransductionABSTRACT
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.
Subject(s)
Cadherins/metabolism , Cell Adhesion , Cell Movement , Microscopy, Atomic Force , Neural Stem Cells/cytology , Neural Stem Cells/ultrastructure , Cadherins/ultrastructure , Cell Size , Humans , Neural Stem Cells/metabolismABSTRACT
Cadherin receptors have a well-established role in cell-cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with ß1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell-matrix adhesion during cell migration.
Subject(s)
Cadherins/metabolism , Cell Adhesion , Cells/cytology , Focal Adhesions/metabolism , Xenopus laevis/metabolism , Animals , Cadherins/genetics , Cell Line , Cell Movement , Cells/metabolism , Fibronectins/metabolism , Focal Adhesions/genetics , Humans , Mice , Neural Crest/growth & development , Neural Crest/metabolism , Protein Transport , Xenopus laevis/embryology , Xenopus laevis/geneticsABSTRACT
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 <5 min of cell-matrix contact and subsequently extend at a rate of 0.25 µm/min at sites of cell membrane retraction. FN nanofibrils display a complex linear array of globular features spaced at varying distances, indicating the coexistence of different conformational states within the fibril. In some cases, initial fibrils extended in discrete increments of â¼ 800 nm during a series of cyclical membrane retractions, indicating a stepwise fibrillar extension mechanism. In presence of Mn(2+), a known activator of integrin adhesion to FN, fibrillogenesis was accelerated almost threefold to 0.68 µm/min and fibrillar dimensions were increased, underlining the importance of integrin activation for early FN fibrillogenesis. FN fibrillogenesis visualized by time-lapse AFM thus provides new structural and mechanistic insight into initial steps of cell-driven FN fibrillogenesis.
Subject(s)
Fibronectins/metabolism , Animals , Cell Membrane/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Integrins/metabolism , Microscopy, Atomic Force/methods , Microscopy, Fluorescence/methods , Rats , Time-Lapse Imaging/methodsABSTRACT
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, ß1-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.
Subject(s)
Cell Culture Techniques/instrumentation , Epithelial Cells/cytology , Fibroblasts/cytology , Tissue Scaffolds/chemistry , Animals , Cell Adhesion , Cell Line , Cell Proliferation , Cell Size , Equipment Design , Extracellular Matrix/chemistry , Lasers , Mice , RatsABSTRACT
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.
Subject(s)
Cell Surface Extensions/physiology , Cell Surface Extensions/ultrastructure , Membrane Fluidity/physiology , Microscopy, Atomic Force/instrumentation , Neural Crest/physiology , Neural Crest/ultrastructure , Animals , Cell Adhesion/physiology , Cells, Cultured , Elastic Modulus/physiology , Equipment Design , Equipment Failure Analysis , Hardness/physiology , Micromanipulation/instrumentation , Micromanipulation/methods , Microscopy, Atomic Force/methods , Stress, Mechanical , Xenopus laevisABSTRACT
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.
Subject(s)
Collagen Type I/metabolism , Collagen Type I/ultrastructure , Kinetics , Microscopy, Atomic Force/methods , Molecular Imaging/methodsABSTRACT
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.