Bulletin of the American Physical Society
2007 APS March Meeting
Volume 52, Number 1
Monday–Friday, March 5–9, 2007; Denver, Colorado
Session W35: Dynamic Forces at Cell and Subcell Levels |
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Sponsoring Units: DBP Chair: Shane Hutson, Vanderbilt University Room: Colorado Convention Center 405 |
Thursday, March 8, 2007 2:30PM - 2:42PM |
W35.00001: Correlation of Force Production with Apoptosis in Tissue Dynamics Yusuke Toyama, Xomalin Peralta, Stephanos Venakides, Daniel Kiehart, Glenn Edwards To understand embryo morphogenesis, it is necessary to know the force distribution in the various tissues. Since cells are largely inaccessible to mechanical probes \textit{in vivo}, measurements of the net forces exerted by cells are challenging. The combination of experimental and theoretical approaches has proven to improve our understanding of these forces. A steerable UV-laser microbeam was used to probe the forces and the resulting kinematics were monitored with confocal microscopy. Dorsal closure is a developmental stage in \textit{Drosophila} embryogenesis, where the dynamics are a consequence of four biological processes [1]. During this stage, cells that have outlived their usefulness undergo apoptosis, a biological process also known as programmed cell death for cells. Apoptotic events were decreased with genetic techniques or increased by irradiation with a UV-C lamp. We present experimental evidence for force generation correlating with apoptosis. This research has been supported by the NIH (GM33830 and GM61240). [1] M. S. Hutson, et al. Science, \textbf{300}, 145 (2003). [Preview Abstract] |
Thursday, March 8, 2007 2:42PM - 2:54PM |
W35.00002: Recoil Dynamics after Laser Ablation of Single Cell Edges in Embyronic Epithelia Xiaoyan Ma, M. Shane Hutson In order to determine the interfacial tensions along cell-cell boundaries in living fruit fly (\textit{Drosophila}) embryos, we have developed a microsurgical method based on laser ablation and laser-scanning confocal microscopy. Following ablation of one cell edge, we follow the recoil dynamics (strain relaxation) of adjacent GFP-labeled cell edges (with time resolution down to 2 ms). The recoils are consistently fit best by a double exponential decay with one time constant around 80 ms and the other around 1.2 s. The initial recoil velocities are in the range of 10-20 $\mu $m/s. We observe the same biphasic strain relaxation in multiple (N = 60) embryos at different developmental stages. Both recoil time constants are much longer than either the plasma lifetime or the duration of cavitation. [Preview Abstract] |
Thursday, March 8, 2007 2:54PM - 3:06PM |
W35.00003: Confounding Effect of Spot-Size on the Wavelength-Dependence of Tissue Ablation Metrics M. Shane Hutson, Gilma Adunas, Yaowu Xiao Tunable free-electron lasers have been used in several previous studies to investigate the mid-IR wavelength-dependence of tissue ablation. These studies gave conflicting results on an important question: do the ablation metrics depend on targeting the laser energy to a water or protein vibration? Here, we investigate the effects of two parameters that varied widely in previous studies -- fluence and focused spot-size. We measured ablation threshold, etch depth and collateral damage in porcine corneas for a set of five matched wavelengths -- same absorption coefficients, but different primary chromophores. Although the ablation thresholds are similar, the slope of etch depth versus fluence (ablation efficiency) differs by up to a factor of five. These differences are most strongly dependent on the focused spot diameter, not wavelength. When spot sizes are matched, protein-targeting wavelengths still leave less collateral damage, but they remove tissue less efficiently. The confounding roles of fluence and spot size have strong implications for the interpretation of previous wavelength-dependent results. [Preview Abstract] |
Thursday, March 8, 2007 3:06PM - 3:18PM |
W35.00004: Nanosecond Infrared Laser for Tissue Ablation G.S. Edwards, R.D. Pearlstein, M.L. Copeland, M.S. Hutson, K. Latone, A. Spiro, G. Pasmanik The Mark-III Free-Electron Laser (FEL), operating at the 6.45$\mu $m wavelength, has been used successfully in human surgery. Due to the FEL's size and cost, there has been interest in the development of a compact, inexpensive infrared laser for human surgical applications. We have investigated the role of the FEL superpulse, leading to the prediction that nanosecond pulses can satisfy the dynamic criteria for tissue ablation. We have developed a laser based on difference frequency mixing and stimulated Raman scattering with four stages of frequency conversion, emitting at a wavelength of 6.45$\mu $m with 3-5ns pulse duration, pulse energies of up to 2mJ, and a pulse repetition rate of 3MHz. The laser system successfully ablated tissue, where collateral thermal damage was limited to several microns. In the future, it will be necessary to increase the pulse repetition rate to achieve an ablation rate acceptable for human surgery. We acknowledge the grant support: R43 RR018435, N00014-99-1-0891, and F49620-00-1-0370. [Preview Abstract] |
Thursday, March 8, 2007 3:18PM - 3:30PM |
W35.00005: Morphogenic asymmetries in tissue dynamics Xomalin G. Peralta, Y. Toyama, R. Montague, S. Venakides, D.P. Kiehart, G.S. Edwards Structural and kinematic symmetries in living organisms arise from the forces responsible for tissue movements during development. Tissue dynamics during dorsal closure, a stage of {\it Drosophila} development, provide a model system for cell sheet morphogenesis. It is characterized by tissue movements, driven by four biological processes which are coordinated in space and synchronized in time. Quantifying morphogenic asymmetries is essential for understanding the spatial and temporal differences in the contributing processes, the extent to which they can vary and still result in successful closure. They also provide a basis for understanding dynamic changes that occur to compensate for perturbations. We measured spatial, kinematic and dynamic asymmetries to biophysically characterize natural asymmetries in unperturbed closure, resiliency to laser perturbations and failure of closure in some mutant embryos. We found an asymmetric upregulation of a biological process in response to laser perturbations. In the mutants, there is a reversed asymmetry. Supported by NIH (GM33830 and GM61240). [Preview Abstract] |
Thursday, March 8, 2007 3:30PM - 3:42PM |
W35.00006: The detection of cancer in living tissue with single-cell precision and the development of a system for targeted drug delivery to cancer Adam Fields, Sean Pi, Alex Ramek, Taylor Bernheim, Jessica Fields, Nadine Pernodet, Miriam Rafailovich The development of innovations in the field of cancer diagnostics is imperative to improve the early identification of malignant cells within the human body. Two novel techniques are presented for the detection of cancer cells in living tissue. First, shear modulation force microscopy (SMFM) was employed to measure cell mechanics of normal and cancer cells in separate and mixed tissue cultures. We found that the moduli of normal keratinocytes were twice as high as the moduli of SCC cancerous keratinocytes, and that the cancer cells were unambiguously identifiable from a mixture of both kinds of cells. Second, confocal microscopy and the BIAcore 2000 were used to demonstrate the preferential adhesion of glass micro-beads impregnated with fluorescent dye to the membranes of cancer cells as compared to those of normal cells. In addition to their use as a cancer detection system, these hollow and porous beads present a model system for targeted drug delivery in the treatment of cancer. [Preview Abstract] |
Thursday, March 8, 2007 3:42PM - 3:54PM |
W35.00007: Characterizing Cell Mechanics with AFM and Microfluidics N. Walter, A. Micoulet, S. Suresh, J.P. Spatz Cell mechanical properties and functionality are mainly determined by the cytoskeleton, besides the cell membrane, the nucleus and the cytosol, and depend on various parameters e.g. surface chemistry and rigidity, surface area and time available for cell spreading, nutrients and drugs provided in the culture medium. Human epithelial pancreatic and mammary cancer cells and their keratin intermediate filaments are the main focus of our work. We use Atomic Force Microscopy (AFM) to study cells adhering to substrates and Microfluidic Channels to probe cells in suspension, respectively. Local and global properties are extracted by varying AFM probe tip size and the available adhesion area for cells. Depth-sensing, instrumented indentation tests with AFM show a clear difference in contact stiffness for cells that are spread of controlled substrates and those that are loosely attached. Microfluidic Channels are utilized in parallel to evaluate cell deformation and ``flow resistance'', which are dependent on channel cross section, flow rate, cell nucleus size and the mechanical properties of cytoskeleton and membrane. The results from the study are used to provide some broad and quantitative assessments of the connections between cellular/subcellular mechanics and biochemical origins of disease states. [Preview Abstract] |
Thursday, March 8, 2007 3:54PM - 4:06PM |
W35.00008: Measurement of the adhesion and elasticity of single cells using a novel micropipette-based technique Marie-Josee Colbert, Adam N. Raegen, Cecile Fradin, Kari Dalnoki-Veress Numerous biological processes have to go through cell adhesion, which makes the fundamental study of the adhesion of cells on solid substrates a key research topic in cellular biophysics. We will present our work on the elasticity and adhesion of a single liposome on a substrate. A vesicle is held at the end of a micropipette mounted on a micromanipulator and put into contact with a surface. We developed a technique to directly measure adhesion using the spring-constant of an L-shaped micropipette when pulling the vesicle from the substrate. The deflection is used to determine the adhesion force of cells as well as a cell’s elasticity. Since the force applied on the cell is known at every moment of the experiment, this technique enables dynamical measurements. The links between the adhesion strength and the surface tension will also be discussed. [Preview Abstract] |
Thursday, March 8, 2007 4:06PM - 4:18PM |
W35.00009: Measuring the Interaction between Cell-Surface Markers and Substrate-Coupled Proteins as a Means of Determining Cell Membrane Fluidity Andrea Carbonaro, Lucy A. Godley, Lydia L. Sohn We have analyzed the detailed interaction between cell-surface markers and substrate-coupled proteins by measuring the transit time of individual cells as they pass through a functionalized pore. Cells that have a specific cell-surface marker will transiently interact with the walls of a pore that are functionalized with a correspondingly specific protein. This interaction results in the cell moving slowly through the pore. In contrast, cells that do not express the specific marker will not interact with the functionalized walls and will pass quickly through the pore. The distribution of transit times measured for interacting cells can be explained in terms of the number of ligand-receptor bonds created between the immobilized proteins on the pore wall and the cell-surface receptors. We will show that this number is a function of both the ligand and receptor densities on the pore and cell membrane, respectively, as well as the fluidity of the cell membrane. [Preview Abstract] |
Thursday, March 8, 2007 4:18PM - 4:30PM |
W35.00010: Cooperativity of Integrin-mediated Adhesion on Nanopatterned Substrates Christine Selhuber, Thorsten Erdmann, Ulrich Schwarz, Horst Kessler, Joachim Spatz Surfaces of defined adhesion properties are required for a physical understanding of cell adhesion in vivo. In this work, biofunctional nanopatterns are employed, which allow adhesion ligands to be positioned in a quasi-hexagonal lattice. Such nanopatterns are used to investigate integrin-mediated cell adhesion, which is a highly complex biological process and essential for numerous cell functions. With nanopatterns the distance between adjacent integrin binding sites is precisely defined. Cell culture experiments have revealed that this distance strongly affects cell adhesion and the formation of adhesion clusters, known as focal contacts. To quantify the adhesion cluster formation for different integrin binding site spacings, cell adhesion forces were studied using atomic force microscopy (AFM). The experiments demonstrate that an integrin binding site spacing of 70 nm and more prevents the cooperative formation of early adhesion clusters. In long-term adhesion studies, after several hours of cell adhesion, it turned out that focal contact formation cooperatively increases the local adhesion strength. The obtained results were related to theoretical models on adhesion cluster stability. [Preview Abstract] |
Thursday, March 8, 2007 4:30PM - 4:42PM |
W35.00011: Dynamics of Cell Migration for cells embedded in Collagen using a multimodal platform of Optical Coherence Tomography, Multi-Photon excitation and Second Harmonic Generation Kandice Tanner, Shuo Tang, Enrico Gratton We developed Raster Image Correlation Spectroscopy (RICS) to analyze the dynamics of cell migration from data obtained on a confocal multi-photon microscope. We assembled a microscope that can simultaneously measure the scattering signal from optical coherence tomography (OCT), multi-photon excited emission (TPEF) and second harmonic signals (SHG) with comparable spatial resolution and the same time resolution. We present data here showing the combined 3-D images of the cells embedded in a collagen matrix. The OCT signal adds fine structural information of the cellular morphology and collagen which is enhanced by the SHG image. The RICS analysis of the TPEF signal gives the dynamics of the GFP --style proteins. We show that the cell morphology and the distribution of cell organelles are different in the collagen matrix than what is observed in cells growing on flat surfaces. Using the three modalities of cell imaging we could reach a more realistic interpretation of cell dynamics in tissue. [Preview Abstract] |
Thursday, March 8, 2007 4:42PM - 4:54PM |
W35.00012: Coordinated Buckling of Microtubule Bundles Produces the Long Wavelength of Microtubule Birefringent Pattern Yongxing Guo, Yifeng Liu, Allan Bower, Jay Tang, James Valles Aligned microtubule (MT) bundles spontaneously form, elongate and buckle in high concentration tubulin solutions that are subjected to a field that initially aligns the microtubules. The nesting of the buckled bundles produces a macroscopic birefringence pattern of stripes. Of interest here is the buckling wavelength, which controls the stripe width. It is shorter than the fundamental wavelength expected in classic Euler buckling and longer than the wavelength expected for the buckling of a single MT bundle within the elastic network formed by the dispersed MTs. We present a mechanical buckling model that accounts for this intermediate wavelength. It shows that the wavelength is shorter than the fundamental one because of the lateral reinforcement by the MT network, and longer than the wavelength expected for a single laterally reinforced bundle due to the coordinated buckling of the neighboring bundles. [Preview Abstract] |
Thursday, March 8, 2007 4:54PM - 5:06PM |
W35.00013: Cell elasticity as a function of actin expression Carsten St\"uber, Josef K\"as The deformation response to an external force of an eurkaryotic cell mainly depends on its cytoskeletal composition. Theoretical models have been introduced to quantify the concentration dependence of the different cytoskeletal components to the elastic strength of cells. Verifying the models experimentally, the optical stretcher, a two beam optical trap, is used to elongate fibroblast cells. These fibroblasts are transfected with GFP-actin, which leads to an overexpression of actin within the cell and allows to determine the actin concentration using fluorescence image analysis. The dependence of the elasticity on the actin concentration of fibroblasts shows a softening of the cell with increasing number of actin filaments. [Preview Abstract] |
Thursday, March 8, 2007 5:06PM - 5:18PM |
W35.00014: Measuring mitotic spindle formation through connection graphs Stuart Schaffner, Jorge Jose The mitotic spindle, an important structure formed during biological cell division, consists of a pattern of stiff fibers called microtubules and crosslinking molecular motor complexes. The spindle, consisting of objects interacting through pairwise interactions, is well suited to study via its connection graph. Thermal motion is important in this system; molecular motors attach and detach randomly from the microtubules, but only where the geometry allows. We have found the connection graph approach to be helpful in several ways for analyzing spindle properties. Monitoring the number and size of connected components in the graph allows us to quantify the development of the spindle bipolar pattern. Minimum cut-sets in components measure spindle pole robustness. These computations not only allow us to measure the dynamics of initial pattern formation, but also the structural rearrangements within spindles that have already formed. Our results are compared to known experimental results. [Preview Abstract] |
Thursday, March 8, 2007 5:18PM - 5:30PM |
W35.00015: ABSTRACT WITHDRAWN |
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