Cortical cell stiffness is independent of substrate mechanics

AbstractCortical stiffness is an important cellular property that changes during migration, adhesion, and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM onto cells results in a significant deformation of the underlying substrate if it is softer than the cells. This ‘soft substrate effect’ leads to an underestimation of a cell’s elastic modulus when analyzing data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the ‘composite cell-substrate model’ (CoCS model). Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.

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Material Property Characterization of Costal Cartilage Using AFM Indentation

Volume 6: ASME Power Transmission and Gearing Conference; 3rd International Conference on Micro- and Nanosystems; 11th International Conference on Advanced Vehicle and Tire Technologies ◽

10.1115/detc2009-87462 ◽

2009 ◽

Author(s):

S. Tripathy ◽

E. J. Berger

Keyword(s):

Material Characterization ◽

Costal Cartilage ◽

Indentation Modulus ◽

Hertz Contact ◽

Force Microscopy ◽

Afm Indentation ◽

Atomic Force ◽

Macro Scale ◽

Property Characterization

Costal cartilage is one of the load bearing tissues of the rib cage. Literature on the material characterization of the costal cartilage is limited. Atomic force microscopy has been extremely successful in characterizing the elastic properties of articular cartilage, but no studies have been published on costal cartilage. In this study AFM indentations on human costal cartilage were performed and compared with macro scale indentation data. Spherical beaded tips of three sizes were used for the AFM indentations. The Hertz contact model for spherical indenter was used to analyze the data and obtain the Young’s modulus. The costal cartilage was found to be almost linearly elastic till 600 nm of indentation depth. It was also found that the modulus values decreased with the distance from the junction. The modulus values from macro indentations were found to be 2-fold larger than the AFM indentation modulus.

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LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments

Journal of the American Society of Nephrology ◽

10.1681/asn.2019121261 ◽

2020 ◽

Vol 31 (10) ◽

pp. 2372-2391 ◽

Cited By ~ 1

Author(s):

Xuhua Ge ◽

Tao Zhang ◽

Xiaoxia Yu ◽

Alecia N. Muwonge ◽

Nanditha Anandakrishnan ◽

...

Keyword(s):

Structural Integrity ◽

Rho Gtpase ◽

Focal Adhesions ◽

Super Resolution ◽

Actin Binding ◽

Puromycin Aminonucleoside ◽

Force Microscopy ◽

Afm Indentation ◽

Atomic Force ◽

Key Aspects

BackgroundMaintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood.MethodsCells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside–induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes.ResultsNebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation.ConclusionsLIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

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Conductive Filaments Produced by Aeromonas hydrophila

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.825.210 ◽

2013 ◽

Vol 825 ◽

pp. 210-213 ◽

Cited By ~ 5

Author(s):

Laura Castro ◽

Mario Vera ◽

Jesús Angel Muñoz ◽

María Luisa Blázquez ◽

Felisa González ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Aeromonas Hydrophila ◽

Oxygen Depletion ◽

Facultative Anaerobe ◽

Force Microscopy ◽

Bacterial Interactions ◽

Atomic Force ◽

Cell Substrate ◽

Scanning Electron

Aeromonas hydrophilais a facultative anaerobe which, under conditions of oxygen depletion, is able to respire iron (III). Scanning electron microscopy (SEM) and conducting-probe atomic force microscopy (AFM) revealed the presence of filaments between cells and cell-substrate and their conductive nature. These results indicate that the pili ofA. hydrophilamight serve as biological nanowires, transferring electrons from the cell to the surface of Fe (III) oxides. Conductive pili could also play a role in bacterial interactions and in inter/intra species signalling, and could lead to biotechnological approaches for novel materials.

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Investigating Cell Stiffness in Wild-Type Candida albicans and its Morphologies using Contact Atomic Force Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2018.11.2311 ◽

2019 ◽

Vol 116 (3) ◽

pp. 429a-430a

Author(s):

Michelle K. Ash ◽

Jeff D. Stephens

Keyword(s):

Atomic Force Microscopy ◽

Candida Albicans ◽

Cell Stiffness ◽

Wild Type ◽

Force Microscopy ◽

Atomic Force

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Analysis of Indentation: Implications for Measuring Mechanical Properties With Atomic Force Microscopy

Journal of Biomechanical Engineering ◽

10.1115/1.2835074 ◽

1999 ◽

Vol 121 (5) ◽

pp. 462-471 ◽

Cited By ~ 186

Author(s):

K. D. Costa ◽

F. C. P. Yin

Keyword(s):

Indentation Depth ◽

Constitutive Law ◽

Elastic Materials ◽

Force Microscopy ◽

Linear Elastic ◽

Afm Indentation ◽

Atomic Force ◽

Afm Probe ◽

Nonlinear Elastic ◽

Indenter Geometry

Indentation using the atomic force microscope (AFM) has potential to measure detailed micromechanical properties of soft biological samples. However, interpretation of the results is complicated by the tapered shape of the AFM probe tip, and its small size relative to the depth of indentation. Finite element models (FEMs) were used to examine effects of indentation depth, tip geometry, and material nonlinearity and heterogeneity on the finite indentation response. Widely applied infinitesimal strain models agreed with FEM results for linear elastic materials, but yielded substantial errors in the estimated properties for nonlinear elastic materials. By accounting for the indenter geometry to compute an apparent elastic modulus as a function of indentation depth, nonlinearity and heterogeneity of material properties may be identified. Furthermore, combined finite indentation and biaxial stretch may reveal the specific functional form of the constitutive law—a requirement for quantitative estimates of material constants to be extracted from AFM indentation data.

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Generalized Hertz model for bimodal nanomechanical mapping

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.7.89 ◽

2016 ◽

Vol 7 ◽

pp. 970-982 ◽

Cited By ~ 35

Author(s):

Aleksander Labuda ◽

Marta Kocuń ◽

Waiman Meinhold ◽

Deron Walters ◽

Roger Proksch

Keyword(s):

Contact Mechanics ◽

Small Amplitude ◽

Sample Surface ◽

Hertzian Contact ◽

Modes Of Operation ◽

Hertz Model ◽

Force Microscopy ◽

Atomic Force ◽

Resonant Modes ◽

Shape And Size

Bimodal atomic force microscopy uses a cantilever that is simultaneously driven at two of its eigenmodes (resonant modes). Parameters associated with both resonances can be measured and used to extract quantitative nanomechanical information about the sample surface. Driving the first eigenmode at a large amplitude and a higher eigenmode at a small amplitude simultaneously provides four independent observables that are sensitive to the tip–sample nanomechanical interaction parameters. To demonstrate this, a generalized theoretical framework for extracting nanomechanical sample properties from bimodal experiments is presented based on Hertzian contact mechanics. Three modes of operation for measuring cantilever parameters are considered: amplitude, phase, and frequency modulation. The experimental equivalence of all three modes is demonstrated on measurements of the second eigenmode parameters. The contact mechanics theory is then extended to power-law tip shape geometries, which is applied to analyze the experimental data and extract a shape and size of the tip interacting with a polystyrene surface.

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Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

AJP Cell Physiology ◽

10.1152/ajpcell.00014.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. C160-C172 ◽

Cited By ~ 39

Author(s):

Francesca Sbrana ◽

Chiara Sassoli ◽

Elisabetta Meacci ◽

Daniele Nosi ◽

Roberta Squecco ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Myosin Ii ◽

Cell Stiffness ◽

C2c12 Myoblasts ◽

Tension Development ◽

Force Microscopy ◽

Atomic Force ◽

Sphingosine 1 Phosphate ◽

Membrane Stretching

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, Cm) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, Cm, used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce Cm and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

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Chemotherapy exposure increases leukemia cell stiffness

Blood ◽

10.1182/blood-2006-08-043570 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3505-3508 ◽

Cited By ~ 179

Author(s):

Wilbur A. Lam ◽

Michael J. Rosenbluth ◽

Daniel A. Fletcher

Keyword(s):

Cell Death ◽

Leukemia Cell ◽

Vascular Complications ◽

Cell Types ◽

Cell Stiffness ◽

Microfluidic Channels ◽

Cell Deformability ◽

Force Microscopy ◽

Atomic Force ◽

Acute Myeloid Leukemia Cells

Abstract Deformability of blood cells is known to influence vascular flow and contribute to vascular complications. Medications for hematologic diseases have the potential to modulate these complications if they alter blood cell deformability. Here we report the effect of chemotherapy on leukemia cell mechanical properties. Acute lymphoblastic and acute myeloid leukemia cells were incubated with standard induction chemotherapy, and individual cell stiffness was tracked with atomic force microscopy. When exposed to dexamethasone or daunorubicin, leukemia cell stiffness increased by nearly 2 orders of magnitude, which decreased their passage through microfluidic channels. This stiffness increase occurred before caspase activation and peaked after completion of cell death, and the rate of stiffness increase depended on chemotherapy type. Stiffening with cell death occurred for all cell types investigated and may be due to dynamic changes in the actin cytoskeleton. These observations suggest that chemotherapy itself may increase the risk of vascular complications in acute leukemia.

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