Compressive Stress Stalls Growth and Decreases Cytoplasmic Diffion

Tuesday, February 14, 2017 interaction of cell adhesion can provide advances in biomaterial for implants, potential drug treatments for improvement of cell adhesion of implants, and fundamental understanding of signaling pathways related to cell adhesion. We have developed a non-invasive real time method using the quartz crystal microbalance with dissipation monitoring (QCM-D) to quantitatively monitor such cellular processes using the dissipation factor DD. In this study, we have used this method to examine the adhesion of human epidermal keratinocytes to the QCM-D sensor surface coated with titanium, a common material for medical implant. The key results from this study have validated this method as an in-vitro approach for examining cell-implant interaction. 2137-Pos Board B457 The Role of Global and Local Mechanical Signals in Modulating Cell Spreading Magdalena Stolarska1, Aravind Rammohan2. 1 Mathematics, University of St. Thomas, St. Paul, MN, USA, 2Mathematics, Corning, Inc., Corning, NY, USA. Cell spreading on a flat surface is controlled by the interaction of cell adhesion, actin-based cellular deformations, and intracellular stresses. It has been shown that many cell types obtain larger spread areas on stiffer substrates [1]. We present a 2D model and finite element simulations of a spreading cell interacting with a deformable substrate through focal adhesion complexes. Focal adhesion complexes are modeled by collections of linear springs that depend on local concentrations of a ligand-activated bound integrin and can form and break dynamically in a stress and strain dependent manner. The cell is treated as a hypoelastic material that undergoes active deformations that represent cell spreading. By considering various formulations for the active deformation, which is characterized by an active rate of deformation tensor field, we use this model to better understand whether the mechanism of cell spreading is localized or governed by a global signal. Specifically, we consider the ability of a cell to integrate local signals and define an active rate of deformation that depends on the cell-level integration of signals arising from bound integrin concentrations, which enhance cell spreading, and from intracellular tension, which inhibit cell spreading. We also consider a spatially inhomogeneous active rate of deformation field that depends on local values of bound integrin concentrations and intracellular tension. We find that cell spread areas are governed by a combination of global signal integration, which accounts for longrange communication within a cell, and local integrin binding and tensile stress. It is this combination of signaling mechanisms that allows us to obtain experimentally observed cell spread area dependence on substrate stiffness. [1] Yeung, T. et al. ‘‘Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.’’ Cell Motility and the Cytoskeleton 60 (2005): 24-34. 2138-Pos Board B458 Force Localization and Cell Shape in Epithelial Monolayers Erik Schaumann1, Margaret Gardel2. 1 Department of Chemistry, University of Chicago, Chicago, IL, USA, 2 Institute for Biophysical Dynamics, James Franck Institute, Department of Physics, University of Chicago, Chicago, IL, USA. Epithelial monolayers are a classic system of active matter, in which the component particles (cells) endogenously generate forces and change their own shapes without external stimuli. The mechanics of single cells and circular colonies of keratinocytes can be successfully described using a continuum model, however, it is not known how general this model is. Using micropatterning to constrain colonies of MDCK cells to reproducible geometries, the relationship between the shapes of individual cells within the colonies and the mechanical output of otherwise similar colonies can be assessed. We find that wild-type cells are motile with respect to the colony borders and distribute traction forces to localized hot spots that move over time, thus breaking from the continuum model. ZO-1/ZO-2 knockdown cells, which display elevated contractility and straighter cell-cell borders, are employed to examine the impact of these parameters on the magnitude and localization of traction forces. 2139-Pos Board B459 Real-Time Identification of Cell Mechanical Properties Alice Bartolozzi1, Alessandro Soloperto2, Gemma Palazzolo2, Michele Basso1, Francesco Difato2, Massimo Vassalli3. 1 Information Engineering (DINFO), University of Florence, Florence, Italy, 2 Neuroscience and Brain Technologies, Italian Institute of Technology, Genova, Italy, 3IBF, CNR National Research Council, Genova, Italy. In the last years, the exploitation of nanotechnology to the study of biological systems opened new avenues towards innovative clinical approaches based on single cell mechanical characterization. Nevertheless, due to the lack of throughput typical of high resolution tools, a complete translation of research findings to real life applications has not been accomplished yet. One of the as-

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pects hindering this process is associated to the analytical estimation of physical parameters belonging to single cells. The typical approach consists in fitting a mathematical model to the experimental signals or, in other words, in optimizing the parameters of a chosen functional. This strategy is fast and effective for deterministic models, slightly affected by environmental noise, but a paradigm shift is required when dealing with nanoscale systems, intrinsically described by a thermal-driven statistical distribution. The method proposed in this work consists in extracting meaningful mechanical parameters from experimental data based on solving the associated estimation problem. An approach to cell mechanics investigation based on Sequential Monte Carlo algorithms have been implemented. Starting from a generalized state space representation, the experiment and its uncertainties and noise have been modelled and the characterizing parameters have been identified through the exploitation of estimation algorithms. The analysis has been applied in a simulation environment, allowing to check the algorithm inference capability and to define the optimal experimental protocol. Single cell mechanical estimation experiments have been designed to match the selected procedure, implementing the estimation strategy in an embedded platform based on a single core dsPIC device. The effectiveness of the proposed approach has been tested on a cell line before and after treatment with cytoskeleton-disrupting drugs. Preliminary results show that modern filtering theory can be effectively applied to extract single cell mechanical parameters in real-time, also providing a valuable guide to identify the most efficient experimental design. 2140-Pos Board B460 The Expression and Degradation of SM22-Alpha/Transgelin are Regulated by Mechanical Tension in the Cytoskeleton Rong Liu, M. Moazzem Hossain, J.-P. Jin. Wayne State University, Detroit, MI, USA. SM22-alpha, also named transgelin, encoded by the gene TAGLN is a calponinrelated protein found in smooth muscle, fibroblast and cancer cells. SM22alpha was discovered three decades ago but its biological function remains unclear. In addition to an application as a differentiation marker for smooth muscle cells, SM22-alpha has been reported to regulate the structure and dynamics of actin cytoskeleton and cell motility in fibroblasts and cancer cells. Here we report a novel finding that the expression and degradation of SM22alpha/transgelin are both regulated by mechanical tension. Mass spectrometry detected that SM22-alpha was significantly decreased in mouse aortic rings after incubation under low mechanical tension. Using specific monoclonal antibodies developed against chicken gizzard SM22-alpha, we found high levels of SM22-alpha in human fetal lung fibroblast cells line MRC-5 and primary neonatal mouse skin fibroblasts. Similar to that of calponin 2, the level of SM22-alpha is positively dependent on the mechanical tension in the cytoskeleton as determined by the stiffness of culture substrate. Quantitative RT-PCR demonstrated a transcriptional regulation of TAGLN gene expression by mechanical tension in the cytoskeleton. The cellular localization of SM22-alpha overlaps with that of myosin IIA and blebbistatin inhibition of myosin motor decreased the expression of SM22-alpha. The level of SM22-alpha is decreased in skin fibroblasts isolated from calponin 2 knockout mice compared to that in calponin 2-positive wild type cells, suggesting their correlated functions. With the close phylogenetic relationship between TAGLN and the calponin genes, SM22-alpha is identified as a calponin-like cytoskeleton regulatory protein. These findings laid a groundwork for understanding the physiological function of SM22-alpha in mechanoregulation of cytoskeleton and cell motility and its relationship with calponins. 2141-Pos Board B461 Compressive Stress Stalls Growth and Decreases Cytoplasmic Diffion Morgan Delarue1, Greg Brittingham1, Oskar Hallatschek2, Liam Holt1. 1 NYU Langone Medical Center, New York, NY, USA, 2UC Berkeley / Stanley Hall, Berkeley, NY, USA. Any cell population growing in a limited space can generate mechanical compressive stresses. Tumors growing within tissues and microbes that are naturally confined by their environment both build up growth-induced pressure. While the effects of tensile mechanical stresses have been widely studied, much less is known about the effects of compressive mechanical stresses on cell physiology. We developed a microfluidic mechano-chemostat that enables a precise temporal control of mechanical and chemical conditions. We found that rate of cell growth is affected by compressive stress: Cell growth decreased exponentially with pressure. In order to investigate the molecular origin for such a growth decrease, we developed genetically encoded multimeric nanoparticles (GEMs) to assess the regulation of cellular crowding and the effects of a mechanical compressive stress on cell microrheology. GEMs are particles of 16nm or 35 nm in size naturally expressed by cells that enable direct particle tracking. We observe that the motion of GEMs is non-ergodic and subdiffusive,

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Tuesday, February 14, 2017

and that crowding is highly regulated in a cell by the regulation of ribosome biogenesis. Moreover, similar to overall growth-rate, GEM diffusion follows the same exponentially decreased with increasing compressive stress that growth rate. To conclude, we speculate that macromolecular diffusion becomes rate limiting for growth under compressive stress. Intermediate mechanical stresses could drive changes in crowding and diffusion to regulate cell physiology, from metabolism to signaling. 2142-Pos Board B462 Cellular Durotaxis from Differentially Persistent Motility Elizaveta A. Novikova1, Matthew Raab2, Dennis E. Discher3, Cornelis Storm4. 1 CEA, Institut de Biologie en de Technologies de Saclay, Gif-sur-Yvette, France, 2Institut Curie, Paris, France, 3University of Pennsylvania, Philadelphia, PA, USA, 4Eindhoven University of Technology, Eindhoven, Netherlands. Cells move differently on substrates with different elasticities. In particular, the persistence of their directionality is greater on substrates with a higher elastic modulus. We show that this behavior - without any further assumptions will result in a net transport of cells directed up a soft-to-stiff gradient. Using simple random walk models with controlled persistence and stochastic simulations, we characterize this propensity to move in terms of the durotactic index measured in experiments. A one-dimensional model captures the essential features of this motion and highlights the competition between diffusive spreading and linear, wavelike propagation. Since the directed motion is rooted in a nondirectional change in the behavior of individual cells, the motility is a kinesis rather than a taxis. Persistence-driven durokinesis is generic and may be of use in the design of instructive environments for cells and other motile, mechanosensitive objects. 2143-Pos Board B463 Characterization of the Frustrated Differentiation of Mesenchymal Stem Cells Induced by Normadic Migration Between Stiff and Soft Region of Gel Matrix Satoru Kidoaki1, Hiroyuki Ebata1, Rumi Sawada2, Kousuke Moriyama1, Thasaneeya Kuboki1, Yukie Tsuji1, Ken Kono2, Kazusa Tanaka2, Saori Sasaki1. 1 IMCE, Kyushu University, Fukuoka, Japan, 2National Institute of Health Sciences, Tokyo, Japan. Mesenchymal stem cells (MSCs) have been known to exhibit substrate stiffness-dependent differentiation, and history of the mechanical dose from culture environment to MSCs sensitively is found to alter its phenotype. A certain level of substrate stiffness and duration period on that determine the fate of MSCs. In relation to this, we have found before that microelasticallypatterned hydrogel with heterogeneous distribution of matrix stiffness allow MSCs to suppress fate determination into specific differentiation lineages, and contribute to keep the undifferentiated state. We call such mode of MSCs as ‘‘frustrated differentiation’’, which serves to construct culture substrate for MSCs to maintain their stemness in high-qualified state. The basis of this phenomenon is in the enforced oscillation of mechanical dose from environment to MSCs during the nomadic migration between stiff and soft region of gel matrix, which eliminate the history of experience on a certain level of stiffness. To design such heterogeneous microelastically-patterned gels, we have applied the photolithographic microelasticity patterning using photoculable gelatins. The emergence of frustrated mode of differentiation was previously confirmed by immunofluorescence and RT-PCR analysis for the expression markers, but more detailed and precise characterizations are of course required. In this study, to fully characterize the frustrated differentiation of MSCs, we investigated oscillation of the mechanical dose and mechanical signal input to MSCs employing the long-term traction force microscopy for MSCs culture on the microelastically-striped patterned gels. In addition, we performed cDNA microarray analysis for the MSCs culture in such mode of frustrated differentiation. As the result, MSCs in normadic movement between stiff and soft region of gel surface were confirmed to exhibit characteristic transcriptome and marked large fluctuation profile of traction forces compared with plain control gels. 2144-Pos Board B464 Cells as Strain-Cued Automata Brian Cox. Arachne Consulting, Sherman Oaks, CA, USA. Autonomous cells can form patterns by responding to local variations in the strain fields that arise from their individual or collective motions. Evidence of cells acting as strain-cued automata have been inferred from patterns observed in nature and from experiments conducted in vitro. In simulations, cells are assumed to pass information among themselves solely via mechanical

boundary conditions, i.e., the tractions and displacements present at their membranes. This assumption opens three mechanisms for pattern formation in large cell populations: wavelike behavior, kinematic feedback in motility that leads to sliding layered patterns, and directed migration during invasions. 1. Wavelike behavior among ameloblast cells during amelogenesis has been inferred from enamel microstructure, while strain waves in populations of epithelial cell shave been observed in vitro. We show that ‘‘determination fronts’’, where cells transition into a new state, can be governed by tsunami-like wavefronts propagating across a cell population, a depiction that is supported by data for ameloblasts. 2. A kinematic feedback mechanism, ‘‘enhanced shear motility’’, accounts successfully for the spontaneous formation of layered patterns during amelogenesis. 3. Directed migration is exemplified by a theory of invader cells that sense and respond to the strains they themselves create in the host population as they invade it: analysis shows that the strain fields contain positional information that could aid the formation of network structures, stabilizing the slender geometry of branches and helping govern the frequency of branch bifurcation and branch coalescence (the formation of closed networks). In all cases, morphological outcomes are governed by the ratio of the rates of two competing processes, one a migration velocity and the other a relaxation velocity related to the propagation of strain information. Relaxation velocities are approximately constant for different species and organs, whereas cell migration rates vary by three orders of magnitude. We conjecture that developmental processes use rapid cell migration to achieve certain outcomes, and slow migration to achieve others. We infer from analysis of host relaxation during network formation that a transition exists in the mechanical response of a host cell from animate to inanimate behavior when its strain changes at a rate that exceeds 10 4 - 10 3 s 1. The transition has previously been observed in experiments conducted in vitro. In initially homogeneous populations, outcomes are strongly influenced by the presence of double-curvature in the cell sheet: the evolving metric of the sheet triggers strain-cues among cells, which break symmetry. 2145-Pos Board B465 Mitochondrial Fluctuations as a Measure of Biomechanical Properties of Murine Cells Wenlong Xu1, Elaheh Alizadeh1, Jordan Castle2, Ashok Prasad1,3. 1 Chem. & Biol. Engr., Colorado State University, Fort Collins, CO, USA, 2 Dept. of Biology, Colorado State University, Fort Collins, CO, USA, 3 School of Biomed. Engr., Colorado State University, Fort Collins, CO, USA. Introduction: The active mechanical properties of the cellular cytoskeleton are important in a number of significant cellular processes from migration to cell division, and are altered in cancer. A non-invasive single-cell assay of these properties would be of significant value for understanding these cellular processes, would give insights into mechanical heterogeneity between cells, and would be of therapeutic importance in cancer. A promising technique is derived from passive particle microrheology, wherein fluorescent beads are inserted into the cytoplasm, their mean square displacement (MSD) measured, and mechanical parameters calculated using viscoelastic theory. The non-invasive version of this method uses mitochondrial fluctuations towards the same goal which is potentially a very useful assay due to its relative simplicity. Methods: We carried out a detailed study of mitochondrial fluctuations in the C3H-10T1/2 cell line, a murine embryonic mesenchymal cell line, as well as two osteosarcoma cell lines with low and high metastatic potentials, DUNN and DLM8. Cells were cultured under standard growth condition for 24 hr, and then stained with MitoTracker Green FM (ThermoFisher Scientific). Random cells were selected for imaging using spinning disk confocal microscopy at a high temporal resolution (100 ms per frame) for 100 s. We optimized a multi-step image processing protocol to identify mitochondria with high fidelity. We selected the mitochondria with life-time spanning the entire imaging period and with no interaction with other mitochondria and calculated the MSD. Results: The MSD of isolated mitochondria resembles that of a particle in a viscoelastic medium, in agreement with previous results. However comparison of MSD between controls and cells treated with drugs that disrupt the actin and microtubule network showed surprisingly small effects, while treatment with ATP synthesis inhibitors significantly decreased the MSD. Our results imply that mitochondrial fluctuations are primarily driven by active fluctuations of the cytoskeleton, and are relatively unaffected by perturbing the actin or microtubule network, at least in the cell lines in our study. Comparison of MSDs between less invasive and more invasive murine osteosarcoma cells showed significant differences at long time scales. Hence active fluctuations differ significantly between different cell types. We also observed significant heterogeneity among cells of the same type and even more heterogeneity in the mitochondria from a single cell. Our analysis suggests that mitochondrial fluctuations reflect the active contractile properties of the cytoskeleton.