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Dynamic cellular biomechanics in responses to chemotherapeutic drug in hypoxia probed by atomic force spectroscopy.

ABSTRACT: The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12-24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55-75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

SUBMITTER: Alhalhooly L

PROVIDER: S-EPMC8202777 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cell Adhesion on Dynamic Supramolecular Surfaces Probed by Fluid Force Microscopy-Based Single-Cell Force Spectroscopy.

Project description:Biomimetic and stimuli-responsive cell-material interfaces are actively being developed to study and control various cell-dynamics phenomena. Since cells naturally reside in the highly dynamic and complex environment of the extracellular matrix, attempts are being made to replicate these conditions in synthetic biomaterials. Supramolecular chemistry, dealing with noncovalent interactions, has recently provided possibilities to incorporate such dynamicity and responsiveness in various types of architectures. Using a cucurbit[8]uril-based host-guest system, we have successfully established a dynamic and electrochemically responsive interface for the display of the integrin-specific ligand, Arg-Gly-Asp (RGD), to promote cell adhesion. Due to the weak nature of the noncovalent forces by which the components at the interface are held together, we expected that cell adhesion would also be weaker in comparison to traditional interfaces where ligands are usually immobilized by covalent linkages. To assess the stability and limitations of our noncovalent interfaces, we performed single-cell force spectroscopy studies using fluid force microscopy. This technique enabled us to measure rupture forces of multiple cells that were allowed to adhere for several hours on individual substrates. We found that the rupture forces of cells adhered to both the noncovalent and covalent interfaces were nearly identical for up to several hours. We have analyzed and elucidated the reasons behind this result as a combination of factors including the weak rupture force between linear Arg-Gly-Asp and integrin, high surface density of the ligand, and increase in effective concentration of the supramolecular components under spread cells. These characteristics enable the construction of highly dynamic biointerfaces without compromising cell-adhesive properties.

| S-EPMC5406783 | biostudies-literature

Mechanics of spreading cells probed by atomic force microscopy.

Project description:Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.

| S-EPMC3728925 | biostudies-literature

Atomic force microscopy-coupled microcoils for cellular-scale nuclear magnetic resonance spectroscopy.

Project description:We present the coupling of atomic force microscopy (AFM) and nuclear magnetic resonance (NMR) technologies to enable topographical, mechanical, and chemical profiling of biological samples. Here, we fabricate and perform proof-of-concept testing of radiofrequency planar microcoils on commercial AFM cantilevers. The sensitive region of the coil was estimated to cover an approximate volume of 19.4 × 10(3) μm(3) (19.4 pl). Functionality of the spectroscopic module of the prototype device is illustrated through the detection of (1)Η resonance in deionized water. The acquired spectra depict combined NMR capability with AFM that may ultimately enable biophysical and biochemical studies at the single cell level.

| S-EPMC3637277 | biostudies-other

Subnanometre enzyme mechanics probed by single-molecule force spectroscopy.

Project description:Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.

| S-EPMC4770092 | biostudies-literature

Effect of CM15 on Supported Lipid Bilayer Probed by Atomic Force Microscopy.

Project description:Antimicrobial peptides are key components of the immune system. These peptides affect the membrane in various ways; some form nano-sized pores, while others only produce minor defects. Since these peptides are increasingly important in developing antimicrobial drugs, understanding the mechanism of their interactions with lipid bilayers is critical. Here, using atomic force microscopy (AFM), we investigated the effect of a synthetic hybrid peptide, CM15, on the membrane surface comprising E. coli polar lipid extract. Direct imaging of supported lipid bilayers exposed to various concentrations of the peptide revealed significant membrane remodeling. We found that CM15 interacts with supported lipid bilayers and forms membrane-spanning defects very quickly. It is found that CM15 is capable of remodeling both leaflets of the bilayer. For lower CM15 concentrations, punctate void-like defects were observed, some of which re-sealed themselves as a function of time. However, for CM15 concentrations higher than 5 µM, the defects on the bilayers became so widespread that they disrupted the membrane integrity completely. This work enhances the understanding of CM15 interactions with the bacterial lipid bilayer.

| S-EPMC10672887 | biostudies-literature

Modular, Nondegenerate Polyprotein Scaffolds for Atomic Force Spectroscopy.

Project description: Not available

| S-EPMC4940236 | biostudies-literature

Minimizing pulling geometry errors in atomic force microscope single molecule force spectroscopy.

Project description:In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.

| S-EPMC2553119 | biostudies-literature

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.

Project description:Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.

| S-EPMC3397402 | biostudies-literature

Single-molecule atomic force microscopy force spectroscopy study of Aβ-40 interactions.

Project description:Misfolding and aggregation of amyloid β-40 (Aβ-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aβ-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aβ-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.

| S-EPMC3113552 | biostudies-literature

Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy.

Project description:Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.

| S-EPMC3588056 | biostudies-literature

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