Bulletin of the American Physical Society
APS March Meeting 2014
Volume 59, Number 1
Monday–Friday, March 3–7, 2014; Denver, Colorado
Session J12: Focus Session: AFM in Studying Cell Mechanics and Biointerfaces |
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Sponsoring Units: DBIO Chair: Igor Sokolov, Tufts University Room: 205 |
Tuesday, March 4, 2014 2:30PM - 3:06PM |
J12.00001: Mechanical properties of metastatic breast cancer cells invading into collagen I matrices Invited Speaker: Robert Ros Mechanical interactions between cells and the extracellular matrix (ECM) are critical to the metastasis of cancer cells. To investigate the mechanical interplay between the cells and ECM during invasion, we created thin bovine collagen I hydrogels ranging from 0.1-5 kPa in Young's modulus that were seeded with highly metastatic MDA-MB-231 breast cancer cells. Significant population fractions invaded the matrices either partially or fully within 24 h. We then combined confocal fluorescence microscopy and indentation with an atomic force microscope to determine the Young's moduli of individual embedded cells and the pericellular matrix using novel analysis methods for heterogeneous samples. In partially embedded cells, we observe a statistically significant correlation between the degree of invasion and the Young's modulus, which was up to an order of magnitude greater than that of the same cells measured in 2D. ROCK inhibition returned the cells' Young's moduli to values similar to 2D and diminished but did not abrogate invasion. This provides evidence that Rho/ROCK-dependent acto-myosin contractility is employed for matrix reorganization during initial invasion, and suggests the observed cell stiffening is due to an attendant increase in actin stress fibers. [Preview Abstract] |
Tuesday, March 4, 2014 3:06PM - 3:18PM |
J12.00002: Mechanical Properties of Human Cells Change during Neoplastic Processes Martin Guthold, Xinyi Guo, Keith Bonin, Karin Scarpinato Using an AFM with a spherical probe of 5.3 $\mu$m, we determined mechanical properties of individual human mammary epithelial cells that have progressed through four stages of neoplastic transformation: normal, immortal, tumorigenic, and metastatic. Measurements on cells in all four stages were taken over both the nucleus and the cytoplasm. Moreover, the measurements were made for cells outside of a colony (isolated), on the periphery of a colony, and inside a colony. By fitting the AFM force vs. indentation curves to a Hertz model, we determined the Young's modulus, E. We found a distinct contrast in the influence a cell's colony environment has on its stiffness depending on whether the cells are normal or cancer cells. We also found that cells become softer as they advance to the tumorigenic stage and then stiffen somewhat in the final step to metastatic cells. For cells averaged over all locations the stiffness values of the nuclear region for normal, immortal, tumorigenic, and metastatic cells were (mean +/- sem) 880 +/- 50, 940+/-50, 400 +/- 20, and 600 +/-20 Pa respectively. Cytoplasmic regions followed a similar trend. These results point to a complex picture of the mechanical changes that occur as cells undergo neoplastic transformation. [Preview Abstract] |
Tuesday, March 4, 2014 3:18PM - 3:30PM |
J12.00003: Causes of retrograde flow in fish keratocytes Thomas Fuhs, Michael Goegler, Claudia A. Brunner, Charles W. Wolgemuth, Josef A. Kaes Confronting motile cells with AFM-cantilevers serving as obstacles and doubling as force sensors we tested the limits of the driving actin and myosin machinery. We could directly measure the force necessary to stop actin polymerization as well as the force present in the retrograde actin flow. Combined with detailed measurements of the retrograde flow velocity and specific manipulation of actin and myosin we found that actin polymerization and myosin contractility are not enough to explain the cells behavior. We show that ever-present depolymerization forces, a direct entropic consequence of actin filament recycling, are sufficient to fill this gap, even under heavy loads. [Preview Abstract] |
Tuesday, March 4, 2014 3:30PM - 4:06PM |
J12.00004: High-resolution elasticity maps and cytoskeletal dynamics of neurons measured by combined fluorescence and atomic force microscopy Invited Speaker: Cristian Staii Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here I present results obtained in my research group, which combine Atomic Force Microscopy and Fluorescence Microscopy measurements to produce systematic, high-resolution elasticity maps for different types of live neuronal cells cultured on glass or biopolymer-based substrates. We measure how the stiffness of neurons changes both during neurite outgrowth and upon chemical modification (disruption of the cytoskeleton) of the cell. We find a reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules in the cell soma. We also report a reversible shift in the elastic modulus of the cortical neurons cytoskeleton with temperature, from tubulin dominated regions at 37C to actin dominated regions at 25C. We demonstrate that the dominant mechanism by which the elasticity of the neuronal soma changes in response to temperature is the contractile stiffening of the actin component of the cytoskeleton induced by the activity of myosin II motors. [Preview Abstract] |
Tuesday, March 4, 2014 4:06PM - 4:18PM |
J12.00005: Analysis of Load Rate Dependence of Neuronal Soma Using Atomic Force Microscopy Elise Spedden, Maxim Dokukin, Igor Sokolov, Cristian Staii Surfaces of biological cells are covered with a layer of molecules (glycocalyx) and membrane protrusions (microvilli and microridges). This so-called ``brush'' layer plays a distinct role in the measured elastic modulus of cells. We utilize atomic force microscopy (AFM) to study mechanical properties of the soma and brush layer of live rat cortical neurons. The elastic modulus of the soma and brush are measured for cells indented at different AFM probe loading rates, ranging from 1-10 $\mu$m/s. The cells were studied at both 37 $^{\circ}$C (near-physiological temperature at which microtubules dominate high stiffness regions in the soma) and at 25 $^{\circ}$C (reduced temperature state at which actin components dominate high stiffness regions in the soma). If one uses a model with no brush taken into account, the derived elastic modulus shows the rate dependence similar to the one reported previously in the literature. Using the model with brush, we observed no statistically significant rate dependence of the elastic modulus of the soma, whereas the effective brush length demonstrates strong rate dependence. These measurements yield insight into the mechanical reaction of living neurons to externally applied stresses. [Preview Abstract] |
Tuesday, March 4, 2014 4:18PM - 4:30PM |
J12.00006: If mechanics of cells can be described by elastic modulus in AFM indentation experiments? Igor Sokolov, Maxim Dokukin, Nataliia Guz, Vivekanand Kalaparthi We study the question if cells, being highly heterogeneous objects, can be described with an elastic modulus (the Young's modulus) in a self-consistent way. We analyze the elastic modulus using indentation done with AFM of human cervical epithelial cells. Both sharp (cone) and dull AFM probes were used. The indentation data collected were processed through different elastic models. The cell was considered as a homogeneous elastic medium which had either smooth spherical boundary (Hertz/Sneddon models) or the boundary covered with a layer of glycocalyx and membrane protrusions (``brush'' models). Validity of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrate that only one model shows consistency with treating cells as homogeneous elastic medium, the bush model when processing the indentation data collected with the dull probe. The elastic modulus demonstrates strong depth dependence in all other three models. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus in a self-consistent way when using the brush model to analyze data collected with a dull AFM probe. [Preview Abstract] |
Tuesday, March 4, 2014 4:30PM - 4:42PM |
J12.00007: High spatiotemporal resolution imaging of mechanical processes in live cells using T- shaped cantilevers Nicola Mandriota, Ozgur Sahin Mechanical properties of cells are paramount regulators of a plethora of physiological processes, such as cell adhesion, motility and proliferation. Yet, their knowledge is currently hampered by the lack of techniques with sufficient spatiotemporal resolution to monitor the dynamics of such biological processes. We introduce an atomic force microscopy-based imaging platform based on newly-designed cantilevers with increased force sensitivity, while minimizing viscous drag. This allows us to uncover mechanical properties of a wide variety of living cells - including fibroblasts, neurons and Human Umbilical Vein Endothelial Cells - with an unprecedented spatiotemporal resolution. Our mechanical maps approach 50nm resolution and monitor cellular features within a minute's timescale. To identify the counterparts of our mechanical maps' features we perform simultaneous fluorescence microscopy and recognize cytoskeletal elements as the main molecular contributors of cellular stiffness at the nanoscale. Furthermore, the enhanced resolution and speed of our method allows the recognition of dynamic changes in the mechanics of fine cellular structures, which occurred independently of changes within optical images of fluorescently-labeled actin. [Preview Abstract] |
Tuesday, March 4, 2014 4:42PM - 4:54PM |
J12.00008: Poking vesicles in silico Ben Barlow, Martin Bertrand, Bela Joos The Atomic Force Microscope (AFM) is used to poke cells and study their mechanical properties. Using Coarse-Grained Molecular Dynamics simulations, we study the deformation and relaxation of lipid bilayer vesicles, when poked with a constant force. The relaxation time, equilibrium area expansion, and surface tension of the vesicle membrane are studied over a range of applied forces. The relaxation time exhibits a strong force-dependence. Our force-compression curves show a strong similarity with results from a recent experiment by Schafer et al. (Langmuir, 2013). They used an AFM to ``poke'' adherent giant liposomes with constant nanonewton forces and observed the resulting deformation with a Laser Scanning Confocal Microscope. Results of such experiments, whether on vesicles or cells, are often interpreted in terms of dashpots and springs. This simple approach used to describe the response of a whole cell ---complete with cytoskeleton, organelles etc.--- can be problematic when trying to measure the contribution of a single cell component. Our modeling is a first step in a ``bottom-up'' approach where we investigate the viscoelastic properties of an in silico cell prototype with constituents added step by step. [Preview Abstract] |
Tuesday, March 4, 2014 4:54PM - 5:06PM |
J12.00009: Morphology And Local Mechanical Properties Of A Block Copolymer Cell Substrate Craig Wall, Ivan Yermolenko, G. Rajesh Krishnan, Debanjan Sarkar, John Alexander Atomic force microscopy (AFM) was applied for the characterization of morphology and mechanical properties of a block copolymer coating designed for biomaterials applications. The material is a block-copolymer with poly(ethylene glycol) as one block and a peptide as second block, which are connected through urethane bonds. The AFM images obtained in amplitude modulation mode revealed the morphology is characterized by micron-scale sheaf-like structures embedded in a more homogeneous and, presumably, amorphous matrix. The self-assembly of the peptide segments is responsible for the formation of the ordered sheaf structures and this phenomenon was common for different variations of the components. Maps of elastic modulus and work of adhesion of the block copolymer, which also differentiate the matrix and ordered regions, were obtained with Hybrid mode at different tip-force levels. The quantitative estimates show that elastic modulus varies in the MPa range and work of adhesion in the hundreds of mJ/m$^{\mathrm{2}}$ range. These data are compared with AFM-based nanoindentation that was performed at higher tip-force level. The results indicate that material surface is more complicated and they suggest in-depth morphology variations. A tentative model of the structural organization is proposed. [Preview Abstract] |
Tuesday, March 4, 2014 5:06PM - 5:18PM |
J12.00010: Toolkit for the Automated Characterization of Optical Trapping Forces on Microscopic Particles Joseph Glaser, David Hoeprich, Andrew Resnick Optical traps have been in use in microbiological studies for the past 40 years to obtain noninvasive control of microscopic particles. However, the magnitude of the applied forces is often unknown. Therefore, we have developed an automated data acquisition and processing system which characterizes trap properties for known particle geometries. Extensive experiments and measurements utilizing well-characterized objects were performed and compared to literature to confirm the system's performance. This system will enable the future analysis of a trapped primary cilium, a slender rod-shaped organelle with aspect ratio L/R \textgreater 30, where `L' is the cilium length and `R' the cilium diameter. The trapping of cilia is of primary importance, as it will lead to the precise measurements of mechanical properties of the organelle and its significance to the epithelial cell. [Preview Abstract] |
Tuesday, March 4, 2014 5:18PM - 5:30PM |
J12.00011: A Simplified Model for the Optical Force exerted on a Vertically Oriented Cilium by an Optical Trap and the Resulting Deformation Ian Lofgren, Andrew Resnick Eukaryotic cilia are essentially whiplike structures extending from the cell body. Although their existence has been long known, their mechanical and functional properties are poorly understood. Optical traps are a non-contact method of applying a localized force to microscopic objects and an ideal tool for the study of ciliary mechanics. Starting with the discrete dipole approximation, a common means of calculating the optical force on an object that is not spherical, we tackle the problem of the optical force on a cilium. Treating the cilium as a homogeneous nonmagnetic cylinder and the electric field of the laser beam as linearly polarized results in a force applied in the direction of polarization. The force density in the polarization direction is derived from the force on an individual dipole within the cilium, which can be integrated over the volume of the cilium in order to find the total force. Utilizing Euler--Bernoulli beam theory, we integrate the force density over a cross section of the cilium and numerically solve a fourth order differential equation to obtain the final deformation of the cilium. This prediction will later be compared with experimental results to infer the mechanical stiffness of the cilium. [Preview Abstract] |
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