Bulletin of the American Physical Society
APS March Meeting 2013
Volume 58, Number 1
Monday–Friday, March 18–22, 2013; Baltimore, Maryland
Session Y44: Focus Session: Novel Experimental Techniques for Probing Cellular Mechanics |
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Sponsoring Units: DBIO Chair: Cristian Staii, Tufts Room: Hilton Baltimore Holiday Ballroom 1 |
Friday, March 22, 2013 8:00AM - 8:36AM |
Y44.00001: Mechanosensitivity in axon growth and guidance Invited Speaker: Jeff Urbach In the developing nervous system, axons respond to a diverse array of cues to generate the intricate connection network required for proper function. The growth cone, a highly motile structure at the tip of a growing axon, integrates information about the local environment and modulates outgrowth and guidance, but little is known about effects of external mechanical cues and internal mechanical forces on growth cone behavior. We have investigated axon outgrowth and force generation on soft elastic substrates for dorsal root ganglion (DRG) neurons (from the peripheral nervous system) and hippocampal neurons (from the central) to see how the mechanics of the microenvironment affect different populations. We find that force generation and stiffness-dependent outgrowth are strongly dependent on cell type. We also observe very different internal dynamics and substrate coupling in the two populations, suggesting that the difference in force generation is due to stronger adhesions and therefore stronger substrate engagement in the peripheral nervous system neurons. We will discuss the biological origins of these differences, and recent analyses of the dynamic aspects of growth cone force generation and the implications for the role of mechanosensitivity in axon guidance. [Preview Abstract] |
Friday, March 22, 2013 8:36AM - 8:48AM |
Y44.00002: Electrophysiology of Axonal Constrictions Christopher Johnson, Peter Jung, Anthony Brown Axons of myelinated neurons are constricted at the nodes of Ranvier, where they are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions are generated by local regulation of the kinetics of neurofilaments the most important cytoskeletal elements of the axon. In this paper we discuss how this shape affects the electrophysiological function of the neuron. Specifically, although the nodes are short (about $1\mu m$) in comparison to the distance between nodes (hundreds of $\mu m$) they have a substantial influence on the conduction velocity of neurons. We show through computational modeling that nodal constrictions (all other features such as numbers of ion channels left constant) reduce the required fiber diameter for a given target conduction velocity by up to 50\% in comparison to an unconstricted axon. We further show that the predicted optimal fiber morphologies closely match reported fiber morphologies. [Preview Abstract] |
Friday, March 22, 2013 8:48AM - 9:00AM |
Y44.00003: Role of biomechanical cues on neuronal growth on asymmetric textured surfaces Cristian Staii, Elise Spedden, Timothy Atherton, Koray Sekeroglu, Melik Demirel Axonal growth and the formation of synaptic connections are key steps in the development of the nervous system. Here we present experimental and theoretical results on axonal growth on unidirectional nanotextured surface, and demonstrate that these surface can bias axonal growth. We also perform a systematic investigation of neuronal processes on these surfaces and quantify the role that biomechanical surface cues play in neuronal growth. We show that these surfaces provide a model growth substrates, which allow us to perform systematic studies of the interplay between mechanical, biochemical and topographical cues that contribute to neuronal growth. [Preview Abstract] |
Friday, March 22, 2013 9:00AM - 9:12AM |
Y44.00004: The formation of axonal caliber and nodes of Ranvier Yinyun Li, Peter Jung, Anthony Brown A remarkable feature of myelinated neurons is that their axons are constricted at the nodes of Ranvier. These are the locations where axons are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions emerge during development and have been observed to reduce axonal cross sectional area by factors of more than 10. Combining fluorescent imaging methods with computational modeling, we describe how the nervous system regulates the local caliber of its axons through the regulation of the transport kinetics of its most important cytoskeletal elements, the neurofilaments, matching axon caliber and shape to its physiologic function. [Preview Abstract] |
Friday, March 22, 2013 9:12AM - 9:24AM |
Y44.00005: Studying neuronal biomechanics and its role in CNS development Kristian Franze, Hanno Svoboda, Luciano da F. Costa, Jochen Guck, Christine Holt During the development of the nervous system, neurons migrate and grow over great distances. Currently, our understanding of nervous tissue development is, in large part, based on studies of biochemical signaling. Despite the fact that forces are involved in any kind of cell motion, mechanical aspects have so far rarely been considered. Here we used deformable cell culture substrates, traction force microscopy and calcium imaging to investigate how neurons probe and respond to their mechanical environment. While the growth rate of retinal ganglion cell axons was increased on stiffer substrates, their tendency to grow in bundles, which they show \textit{in vivo}, was significantly enhanced on more compliant substrates. Moreover, if grown on substrates incorporating linear stiffness gradients, neuronal axons were repelled by stiff substrates. Mechanosensing involved the application of forces driven by the interaction of actin and myosin II, and the activation of stretch-activated ion channels leading to calcium influxes into the cells. Applying a modified atomic force microscopy technique\textit{ in vivo}, we found mechanical gradients in developing brain tissue along which neurons grow. The application of chondroitin sulfate, which is a major extracellular matrix component in the developing brain, changed tissue mechanics and disrupted axonal pathfinding. Hence, our data suggest that neuronal growth is not only guided by chemical signals -- as it is currently assumed -- but also by the nervous tissue's mechanical properties. [Preview Abstract] |
Friday, March 22, 2013 9:24AM - 9:36AM |
Y44.00006: Contact nanomechanical measurements with the AFM Nicholas Geisse The atomic force microscope (AFM) has found broad use in the biological sciences largely due to its ability to make measurements on unfixed and unstained samples under liquid. In addition to imaging at multiple spatial scales ranging from micro- to nanometer, AFMs are commonly used as nanomechanical probes. This is pertinent for cell biology, as it has been demonstrated that the geometrical and mechanical properties of the extracellular microenvironment are important in such processes as cancer, cardiovascular disease, muscular dystrophy, and even the control of cell life and death. Indeed, the ability to control and quantify these external geometrical and mechanical parameters arises as a key issue in the field. Because AFM can quantitatively measure the mechanical properties of various biological samples, novel insights to cell function and to cell-substrate interactions are now possible. As the application of AFM to these types of problems is widened, it is important to understand the performance envelope of the technique and its associated data analyses. This talk will discuss the important issues that must be considered when mechanical models are applied to real-world data. Examples of the effect of different model assumptions on our understanding of the measured material properties will be shown. Furthermore, specific examples of the importance of mechanical stimuli and the micromechanical environment to the structure and function of biological materials will be presented. [Preview Abstract] |
Friday, March 22, 2013 9:36AM - 10:12AM |
Y44.00007: Quantitative nano-mechanics of biological cells with AFM Invited Speaker: Igor Sokolov The importance of study of living cells is hard to overestimate. Cell mechanics is a relatively young, yet not a well-developed area. Besides just a fundamental interest, large practical need has emerged to measure cell mechanics quantitatively. Recent studies revealed a significant correlation between stiffness of biological cells and various human diseases, such as cancer, malaria, arthritis, and even aging. However, really quantitative studies of mechanics of biological cells are virtually absent. It is not even clear if the cell, being a complex and heterogeneous object, can be described by the elastic modulus at all. Atomic force microscopy (AFM) is a natural instrument to study properties of cells in their native environments. Here we will demonstrate that quantitative measurements of elastic modulus of cells with AFM are possible. Specifically, we will show that the ``cell body'' (cell without ``brush'' surface layer, a non-elastic layer surrounding cells) typically demonstrates the response of a homogeneous elastic medium up to the deformation of 10-20{\%}, but if and only if a) the cellular brush layer is taken into account, b) rather dull AFM probes are used. This will be justified with the help of the strong condition of elastic behavior of material: the elastic modulus is shown to be independent on the indentation depth. We will also demonstrate that an attempt either to ignore the brush layer or to use sharp AFM probes will result in the violation of the strong condition, which implies impossibility to use the concept of the elastic modulus to describe cell mechanics in such experiments. Examples of quantitative measurements of the Young's modulus of the cell body and the cell brush parameters will be given for various cells. [Preview Abstract] |
Friday, March 22, 2013 10:12AM - 10:24AM |
Y44.00008: Tracking Cytoskeletal Dynamics in Living Neurons via Combined Atomic Force and Fluorescence Microscopy Elise Spedden, David Kaplan, Cristian Staii Living cells are active mechanical structures which evolve within and in response to their local microenvironments. Various cell types possess different mechanical properties and respond uniquely to growth, environmental changes, and the application of chemical stimuli. Here we present a powerful approach which combines high resolution Atomic Force Microscopy with Fluorescence Microscopy to systematically obtain real-time micrometer and sub-micrometer resolution elasticity maps for live neuronal cells cultured on glass substrates. Through this approach we measure the topography, the elastic properties, and the dynamics of neuronal cells, and identify changes in cytoskeletal components during axonal growth, chemical modification, and changes in ambient temperature. We will also show high resolution elasticity measurements of the cell body and of axons/dendrites during growth, as well as identification of cytoskeletal components during cell growth and environmental changes. [Preview Abstract] |
Friday, March 22, 2013 10:24AM - 10:36AM |
Y44.00009: Atomic Force Microscopy Based Cell Shape Index Usienemfon Adia-Nimuwa, Volkan Mujdat Tiryaki, Steven Hartz, Kan Xie, Virginia Ayres Stellation is a measure of cell physiology and pathology for several cell groups including neural, liver and pancreatic cells. In the present work, we compare the results of a conventional two-dimensional shape index study of both atomic force microscopy (AFM) and fluorescent microscopy images with the results obtained using a new three-dimensional AFM-based shape index similar to sphericity index [1]. The stellation of astrocytes is investigated on nanofibrillar scaffolds composed of electrospun polyamide nanofibers that has demonstrated promise for central nervous system (CNS) repair. Recent work by our group has given us the ability to clearly segment the cells from nanofibrillar scaffolds in AFM images [2]. The clear-featured AFM images indicated that the astrocyte processes were longer than previously identified at 24h. It was furthermore shown that cell spreading could vary significantly as a function of environmental parameters, and that AFM images could record these variations [3]. The new three-dimensional AFM-based shape index incorporates the new information: longer stellate processes and cell spreading. [1] AWl. Jay, Biophys. J.:15, 205 (1975) [2] VM Tiryaki, et al, Scanning:34, 316 (2012) [3] VM Tiryaki, et al, Int. J. Nanomed.:07, 3891 (2012) [Preview Abstract] |
Friday, March 22, 2013 10:36AM - 10:48AM |
Y44.00010: Response of Quiescent Cerebral Cortical Astrocytes to Nanofibrillar Scaffold Properties Virginia Ayres, Volkan Mujdat Tiryaki, Kan Xie, Ijaz Ahmed, David I. Shreiber We present results of an investigation to examine the hypothesis that the extracellular environment can trigger specific signaling cascades with morphological consequences [1]. Differences in the morphological responses of quiescent cerebral cortical astrocytes cultured on the nanofibrillar matrices versus poly-L-lysine functionalized glass and Aclar, and unfunctionalized Aclar surfaces were demonstrated using atomic force microscopy (AFM) and phalloidin staining of F-actin. The differences and similarities of the morphological responses were consistent with differences and similarities of the surface polarity and surface roughness of the four surfaces investigated in this work, characterized using contact angle and AFM measurements. The three-dimensional capability of AFM was also used to identify differences in cell spreading. An initial quantitative immunolabeling study further identified significant differences in the activation of the Rho GTPases: Cdc42, Rac1, and RhoA, which are upstream regulators of the observed morphological responses: filopodia, lamellipodia, and stress fiber formation. The results support the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family with demonstrable morphological consequences for cerebral cortical astrocytes. [1] VM Tiryaki et al, Int. J. Nanomed.: 07, 3891 (2012) [Preview Abstract] |
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