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Scattering scanning near-field optical microscopy (s-SNOM) is a powerful technique for mid-infrared spectroscopy at nanometer length scales. By investigating objects in aqueous environments through ultrathin membranes, s-SNOM has recently been extended toward label-free nanoscopy of the dynamics of living cells and nanoparticles, assessing both the optical and the mechanical interactions between the tip, the membrane and the liquid suspension underneath. Here, the study reports that the tapping AFM tip induces a reversible nanometric deformation of the membrane manifested as either an indentation or protrusion. This mechanism depends on the driving force of the tapping cantilever, which is exploited to minimize topographical deformations of the membrane to improve optical measurements. Furthermore, it is shown that the tapping phase delay between driving signal and tip oscillation is a highly sensitive observable to study the mechanics of adhering objects, exhibiting highest contrast at low tapping amplitudes where the membrane remains nearly flat. Mechanical responses are correlated with simultaneously recorded spectroscopy data to reveal the thickness of nanometric water layers between membrane and adhering objects. Besides a general applicability of depth profiling, the technique holds great promise for studying mechano-active biopolymers and living cells, biomaterials that exhibit complex behaviors when under a mechanical load.
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BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.
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Aterosclerose/metabolismo , Adesão Celular/fisiologia , Endotoxemia/metabolismo , Monócitos/metabolismo , Eletricidade Estática , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Adesão Celular/efeitos dos fármacos , Endotoxemia/induzido quimicamente , Endotoxemia/patologia , Armadilhas Extracelulares/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/patologiaRESUMO
Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin-streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.
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Dobramento de Proteína , Fator de von Willebrand/química , Aminoácidos , Cálcio/metabolismo , Reagentes de Ligações Cruzadas/química , Elastina/química , Magnetismo , Fenômenos Mecânicos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.
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Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Dimerização , Microscopia de Força Atômica , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Fator de von Willebrand/químicaRESUMO
Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is activated for hemostasis by increased hydrodynamic forces at sites of vascular injury. Here, we present data from atomic force microscopy-based single-molecule force measurements, atomic force microscopy imaging, and small-angle x-ray scattering to show that the structure and mechanics of VWF are governed by multiple pH-dependent interactions with opposite trends within dimeric subunits. In particular, the recently discovered strong intermonomer interaction, which induces a firmly closed conformation of dimers and crucially involves the D4 domain, was observed with highest frequency at pH 7.4, but was essentially absent at pH values below 6.8. However, below pH 6.8, the ratio of compact dimers increased with decreasing pH, in line with a previous transmission electron microscopy study. These findings indicated that the compactness of dimers at pH values below 6.8 is promoted by other interactions that possess low mechanical resistance compared with the strong intermonomer interaction. By investigating deletion constructs, we found that compactness under acidic conditions is primarily mediated by the D4 domain, i.e., remarkably by the same domain that also mediates the strong intermonomer interaction. As our data suggest that VWF has the highest mechanical resistance at physiological pH, local deviations from physiological pH (e.g., at sites of vascular injury) may represent a means to enhance VWF's hemostatic activity where needed.
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Fenômenos Mecânicos , Multimerização Proteica , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Fenômenos Biomecânicos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
BACKGROUND: The discharge of the Cnidarian stinging organelle, the nematocyst, is one of the fastest processes in biology and involves volume changes of the highly pressurised (150 bar) capsule of up to 50%. Hitherto, the molecular basis for the unusual biomechanical properties of nematocysts has been elusive, as their structure was mainly defined as a stress-resistant collagenous matrix. RESULTS: Here, we characterise Cnidoin, a novel elastic protein identified as a structural component of Hydra nematocysts. Cnidoin is expressed in nematocytes of all types and immunostainings revealed incorporation into capsule walls and tubules concomitant with minicollagens. Similar to spider silk proteins, to which it is related at sequence level, Cnidoin possesses high elasticity and fast coiling propensity as predicted by molecular dynamics simulations and quantified by force spectroscopy. Recombinant Cnidoin showed a high tendency for spontaneous aggregation to bundles of fibrillar structures. CONCLUSIONS: Cnidoin represents the molecular factor involved in kinetic energy storage and release during the ultra-fast nematocyst discharge. Furthermore, it implies an early evolutionary origin of protein elastomers in basal metazoans.
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Elastômeros/química , Nematocisto/fisiologia , Seda/química , Sequência de Aminoácidos , Animais , Western Blotting , Colágeno/metabolismo , Elasticidade , Regulação da Expressão Gênica , Hydra/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Imuno-Histoquímica , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Agregados Proteicos , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Seda/ultraestrutura , Fatores de TempoRESUMO
Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking ß1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that ß1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.
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Adesão Celular/fisiologia , Colágeno/metabolismo , Citoesqueleto/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Integrina alfa1beta1/antagonistas & inibidores , Integrina alfa1beta1/metabolismo , Masculino , Microscopia de Força Atômica , Neoplasias da Próstata , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Quantitative analysis of cellular interactions with the extracellular environment is necessary to gain an understanding of how cells regulate adhesion in the development and maintenance of multicellular organisms, and how changes in cell adhesion contribute to diseases. We provide a practical guide to quantify the adhesive strength of living animal cells to various substrates using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). We describe how to control cell state and attachment to the AFM cantilever, how to functionalize supports for SCFS measurements, how to conduct cell adhesion measurements, and how to analyze and interpret the recorded SCFS data. This guide is intended to assist newcomers in the field to perform AFM-based SCFS measurements.
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Moléculas de Adesão Celular/química , Adesão Celular , Microscopia de Força Atômica/métodos , Animais , Calibragem , Técnicas de Cultura de Células , Células Cultivadas , Elasticidade , Proteínas Imobilizadas/químicaRESUMO
Lung transplantation (LT) constitutes the last therapeutic option for selected patients with end-stage respiratory disease. Primary graft dysfunction (PGD) is a form of severe lung injury, occurring in the first 72 h following LT and constitutes the most common cause of early death after LT. The presence of pulmonary hypertension (PH) has been reported to favor PGD development, with a negative impact on patients' outcomes while complicating medical management. Although several studies have suggested a potential association between pre-LT left ventricular diastolic dysfunction (LVDD) and PGD occurrence, the underlying mechanisms of such an association remain elusive. Importantly, the heterogeneity of the study protocols and the various inclusion criteria used to define the diastolic dysfunction in those patients prevents solid conclusions from being drawn. In this review, we aim at summarizing PGD mechanisms, risk factors, and diagnostic criteria, with a further focus on the interplay between LVDD and PGD development. Finally, we explore the predictive value of several diastolic dysfunction diagnostic parameters to predict PGD occurrence and severity.
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This comprehensive review explores the intricate aspects of left ventricular thrombus (LVT), a potential complication in both ischemic and non-ischemic cardiomyopathies. It provides a thorough understanding of left ventricular thrombus, revealing its uncommon incidence in the general population (7 cases per 10,000 patients), predominantly linked to ischemic heart diseases (ICMs) at an 80% prevalence rate. Diagnostic tools, notably transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR), demonstrate varying sensitivity but remain indispensable in specific clinical contexts related to LVT as non-invasive diagnostic modalities. A detailed comparison between ICM patients and those with non-ischemic cardiomyopathy (NICM) who have left ventricular thrombus reveals subtle distinctions with significant clinical implications. This analysis underscores the importance of these imaging techniques in distinguishing between the two conditions. Additionally, we explored the occurrence of LVT in specific non-ischemic cardiomyopathies, including Takotsubo syndrome, hypertrophic cardiomyopathy, eosinophilic myocarditis, Chagas disease, cardiac amyloidosis, and several other conditions. The article further delves into anticoagulation strategies, thoroughly examining their impact on LVT regression and patient outcomes. Pharmacological interventions, with a focus on direct oral anticoagulants, emerge as promising alternatives; however, there is insufficient information on their efficiency and safety, especially in NICM population. In conclusion, this review highlights the complex nature of LVT, incorporating a range of etiopathogenic factors, diagnostic complexities, and evolving therapeutic approaches. It emphasizes the pressing need for ongoing research in this field.
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Von Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications.
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Fator de von Willebrand/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Humanos , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteólise , Espectrometria de Fluorescência , Fator de von Willebrand/metabolismoRESUMO
Performing transesophageal imaging of the left atrial appendage is key before cardioversion for atrial fibrillation. Ultrasound artifacts may induce misinterpretation and decrease in confidence for thrombus exclusion.
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At the beginning of 2020, coronavirus disease 2019 (COVID-19) emerged as a new pandemic, leading to a worldwide health crisis and overwhelming healthcare systems due to high numbers of hospital admissions, insufficient resources, and a lack of standardized therapeutic protocols. Multiple genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been detected since its first public declaration in 2020, some of them being considered variants of concern (VOCs) corresponding to several pandemic waves. Nevertheless, a growing number of COVID-19 patients are continuously discharged from hospitals, remaining symptomatic even months after their first episode of COVID-19 infection. Long COVID-19 or 'post-acute COVID-19 syndrome' emerged as the new pandemic, being characterized by a high variability of clinical manifestations ranging from cardiorespiratory and neurological symptoms such as chest pain, exertional dyspnoea or cognitive disturbance to psychological disturbances, e.g., depression, anxiety or sleep disturbance with a crucial impact on patients' quality of life. Moreover, Long COVID is viewed as a new cardiovascular risk factor capable of modifying the trajectory of current and future cardiovascular diseases, altering the patients' prognosis. Therefore, in this review we address the current definitions of Long COVID and its pathophysiology, with a focus on cardiovascular manifestations. Furthermore, we aim to review the mechanisms of acute and chronic cardiac injury and the variety of cardiovascular sequelae observed in recovered COVID-19 patients, in addition to the potential role of Long COVID clinics in the medical management of this new condition. We will further address the role of future research for a better understanding of the actual impact of Long COVID and future therapeutic directions.
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Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.
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Complexo de Golgi , Fator de von Willebrand , Sítios de Ligação , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Domínios Proteicos , Fator de von Willebrand/metabolismoRESUMO
The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.
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Adesão Celular , Integrina alfa4/metabolismo , Integrina alfa4beta1/metabolismo , Paxilina/metabolismo , Estresse Mecânico , Moléculas de Adesão Celular , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Imunoglobulinas/metabolismo , Integrina alfa4/farmacologia , Células Jurkat , Ligantes , Mucoproteínas/metabolismo , Mutação , Paxilina/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/metabolismo , Talina , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the SA-biotin linkage is frequently used in single-molecule force spectroscopy (SMFS) experiments. Recent data suggest that SA-biotin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezers assay. We observed orders-of-magnitude differences in the lifetimes under force, which we attribute to the distinct force-loading geometries in the different SA variants. Lifetimes showed exponential dependencies on force, with extrapolated lifetimes at zero force that are similar for the different SA variants and agree with parameters determined from constant-speed dynamic SMFS experiments. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments.
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Biotina , Imagem Individual de Molécula , Fenômenos Mecânicos , Microscopia de Força Atômica , EstreptavidinaRESUMO
Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.
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Calcificação Fisiológica , Adesão Celular , Neoplasias da Próstata/metabolismo , Animais , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Colágeno/metabolismo , Citoesqueleto/metabolismo , Dentina/química , Elefantes , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Alicerces Teciduais/química , Vitamina D/metabolismoRESUMO
Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.
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Actinas/fisiologia , Angiotensina II/fisiologia , Contração Muscular/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Amidas/farmacologia , Azepinas/farmacologia , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Técnicas Citológicas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Módulo de Elasticidade , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Rim/embriologia , Microscopia de Força Atômica , Microscopia Confocal , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina/efeitos dos fármacosRESUMO
The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.