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1.
细胞黏附在细胞生理功能中起着重要的调控作用,对细胞黏附行为进行定量研究有助于理解生命活动内在机制.原子力显微镜(AFM)的出现为研究溶液环境下微纳尺度生物系统的生物物理特性提供了强大工具,特别是AFM单细胞力谱(SCFS)技术可以对单细胞黏附力进行测量.但目前利用SCFS技术进行的研究主要集中在贴壁细胞,对于动物悬浮细胞黏附行为进行的研究还较为缺乏.本文利用AFM单细胞力谱技术(SCFS)对淋巴瘤细胞黏附行为进行了定量测量.研究了淋巴瘤细胞与其单克隆抗体药物利妥昔(利妥昔单抗与淋巴瘤细胞表面的CD20结合后激活免疫攻击)之间的黏附力,分析了利妥昔浓度及SCFS测量参数对黏附力的影响,并对淋巴瘤细胞之间的黏附力进行了测量.实验结果证明了SCFS技术探测动物悬浮细胞黏附行为的能力,加深了对淋巴瘤细胞黏附作用的认识,为单细胞尺度下生物力学探测提供了新的可能.  相似文献   

2.
We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.  相似文献   

3.
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.  相似文献   

4.
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 (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min 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.  相似文献   

5.
Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4+ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4+ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4+ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4+ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

6.
The impact of hyperglycemia on adhesion between lung carcinoma cells (A549) and pulmonary human aorta endothelial cells (PHAEC) was studied using the single‐cell force spectroscopy. Cancer cells were immobilized on a tipless Atomic Force Microscopy (AFM) cantilever and a single layer of endothelial cells was prepared on a glass slide. The measured force‐distance curves provided information about the detachment force and about the frequency of specific ligand‐receptor rupture events. Measurements were performed for different times of short term (up to 2 h) and prolonged hyperglycemia (3 h ‐ 24 h). Single‐cell force results were correlated with the expression of cell adhesion molecules (intercellular adhesion molecule, P‐selectin) and with the length and density of the PHAECs glycocalyx layer, which were measured by AFM nanoindentation. For short‐term hyperglycemia, we observed a statistically significant increase of the adhesion parameters that was accompanied by an increase of the glycocalyx length and expression of P‐selectin. Removal of hyaluronic acid from PHAECs glycocalyx significantly decreased the adhesion parameters, which indicates that hyaluronic acid has a strong impact on adhesion in A549/PHAEC system in short term of hyperglycemia. For prolonged hyperglycemia, the most significant increase of adhesion parameters was observed for 24 hours and this phenomenon correlated with the expression of adhesion molecules and a decrease of the glycocalyx length. Taking together, presented data indicate that both mechanical and structural properties of the endothelial glycocalyx strongly modulate the adhesion in the A549/PHAEC system.  相似文献   

7.
Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.  相似文献   

8.
The most life‐threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood‐brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high‐resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi‐physiological conditions. Hereby, we present our latest results by using this melanoma‐decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single‐cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic‐related processes.  相似文献   

9.
To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single‐cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen‐binding integrin α1β1 and fibronectin‐binding integrin α5β1 in HeLa cells. This crosstalk was unidirectional, from integrin α1β1 to integrin α5β1, and functioned by regulating the endocytosis of integrin α5β1. The single‐cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.  相似文献   

10.
Cryo‐scanning electron microscopy (cryo‐SEM) and atomic force microscopy (AFM) offer new avenues for the study of the morphology of tree frog adhesive toe pads. Using these techniques, we compare toe pad microstructure in two distantly related species of tree frog, Litoria caerulea, White (Hylidae) and Rhacophorus prominanus, Smith (Rhacophoridae), in which the toe pads are considered to be convergent. AFM demonstrates the extraordinary similarity of both surface microstructures (largely hexagonal epithelial cells surrounded by deep channels) and nanostructures (an array of nanopillars, ca. 350 nm in diameter, all with a small dimple at the apex). The cryo‐SEM studies examined the distribution of the fibrillar cytoskeleton within the different layers of the stratified toe pad epithelium, demonstrating that the cytoskeletal elements (keratin tonofilaments) that lie at an angle to the surface are relatively poorly developed in L. caerulea, clearly so in comparison to R. prominanus. Cryo‐SEM also enabled the visualization of the fluid layer that is critical to a toe pad's adhesive function. This was achieved by examination of the frozen fluid residues left behind after removal of a toe within the cryo‐SEM's experimental chamber. Such ‘toeprints’ demonstrated the presence of a wedge of fluid surrounding each toe pad, as well as fluid filling the channels that surround each epithelial cell. Cryo‐SEM was used to examine epithelial cell shape. In a sample of 582 cells, 59.5% were hexagonal, the remainder being mainly pentagonal (23.1%) or heptagonal (16.1%). The distribution of differently‐shaped cells was not random, but was not associated with either pad curvature or the distribution of mucous pores that provide fluid for the frogs' wet adhesion mechanism. Our main finding, the great similarity of toe pad structure in these two species, has important implications for biomimetics, for such convergent evolution suggests a good starting point for attempts to develop adhesives that will function in wet conditions. J. Morphol. 274:1384–1396, 2013. © 2013 Wiley Periodicals, Inc.  相似文献   

11.
Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip–sample interactions in time (microseconds) and controlling force in the low pico‐Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM‐Live Cell probe, having a short cantilever with a 17‐µm‐long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico‐Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.  相似文献   

12.
A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10?3 to 1.9 × 10?2 Hz in old and 8.4 × 10?4 to 1.5 × 10?2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.  相似文献   

13.
Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell–cell and cell–substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino‐acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C‐terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c‐type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell‐substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco‐elastic properties and interaction of the mucilage–substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.  相似文献   

14.
In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin‐like growth factor‐II (IGF‐II) and activation of the IGF‐I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5‐azacytidine (5‐aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm2 was in contact with a cell surface. The forces required to rupture the interactions of IGF‐IIs on a cell and anti mouse IGF‐II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody–ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5‐aza to the medium. This method could be used in non‐invasive and successive evaluation of a living cell's behavior. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

15.
叶志义  范霞 《生命科学》2009,(1):156-162
细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜(atomic force microscope,AFM)能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。  相似文献   

16.
《Journal of biomechanics》2014,47(16):3855-3861
Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell–cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm2 areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.  相似文献   

17.
The mechanical properties of single cells have been recently identified as the basis of an emerging approach in medical applications since they are closely related to the biological processes of cells and human health conditions. The problem in hand is how to measure mechanical properties in order to obtain them more accurately and applicably. Some of the cell’s properties such as elasticity module and adhesion have been measured before using various methods; nevertheless, comprehensive tests for two healthy and cancerous cells have not been performed simultaneously. As a Nanoscale device, AFM has been used for some biological cells, however for breast cells, it has been utilized just to measure elasticity module. To provide a more accurate comparison for the healthy and the malignant cancer cells of breast, mechanical properties of MCF-10A cells such as topography, elasticity module, adhesion force, viscoelastic characteristics, bending and axial rigidity were determined and compared to the MCF-7 cells results obtained in previous works. Results revealed that the healthy breast cells are stiffer and less adhesive in comparison with the cancerous ones. Topography images revealed that cancerous cells have bigger radii. These results can help with the diagnosis of malignant cancer cells and even the level of the disease.  相似文献   

18.
The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.  相似文献   

19.
Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.  相似文献   

20.
As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro‐projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro‐projection systems currently available, we introduced the assist of the photo‐responsive culture substrates functionalized with a photo‐acid‐generating polymer. In clear contrast to the existing laser‐based methods requiring point scanning, areal micro‐prjection of blue light with the wavelength 436 nm killed many CHO‐K1 cells at a time in the irradiated area on the substrate. The effect of the photo‐generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo‐selective killing of the adherent cells after preliminarily patterning through the photo‐induced removal of cell adhesion‐inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348–352. © 2012 Wiley Periodicals, Inc.  相似文献   

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