Matrix and Soluble Factor Pathways to Lineage Specification

Sunday, February 28, 2016 489-Pos Board B269 Matrix and Soluble Factor Pathways to Lineage Specification Irena L. Ivanovska, Joe Swift, Kyle Spinler, Dave Dingal, Dennis E. Discher. University of Pennsylvania, Philadelphia, PA, USA. Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSC- derived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared sub- population of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove laminA dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive. 490-Pos Board B270 Tension-Regulated Actin Severing Revealed by Surface-Free SingleMolecule Force Spectroscopy Yan Jiang1,2, Theodore C. Feldman3, Hyeran Kang4, Enrique M. De La Cruz5, Wesley P. Wong1,2. 1 BCMP, Harvard Medical School, Boston, MA, USA, 2Pcmm, Boston Children’s Hospital, Boston, MA, USA, 3Applied Physics, Harvard University, Cambridge, MA, USA, 4NanoScience Technology Center, University of Central Florida, Orlando, FL, USA, 5Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. Actin-severing proteins, such as gelsolin, are key regulators of actin filament turnover. Studies suggest that the severing process is regulated by mechanical forces in actin filaments. However, the specific types and levels of force involved are unknown because the existing methods for applying force on actin filaments require surface attachment. These attachments introduce a combination of tension, compression, torsion and shear. We apply a surface-free force spectroscopy method that uses a high-speed cross-slot hydrodynamic trap to generate pure tension in actin filaments. Buffer containing actin filaments or severing proteins flows in from two opposite directions and exits via the two orthogonal outlets to create an elongational flow field with a stagnation point in the center. As a result, filaments near the stagnation point are stretched by the viscous drag from the flow. In addition, the pressure in one of the outlet reservoirs is electronically controlled with a high-speed feedback algorithm to stabilize the actin filament at the stagnation point. This allows us to measure the severing activity of actin filaments under pure tension. We found that the severing rate by gelsolin was independent of tension up to 10pN. In comparison, a previous magnetic tweezers experiment that applies a mixture of tension and bending forces shows a positive correlation between severing rate and pulling force for gelsolin over the range of 0.1 to 4 pN. Our result suggests that changes at actin monomer interfaces due to bending rather than tension enhances the severing rate by gelsolin. By enabling us to applying pure tension rather than a mixture of forces, our method provides a powerful tool for understand and differentiating the driving forces of structural changes in filamentous biomolecules. 491-Pos Board B271 Acoustic Tweezing Cytometry (ATC) on Dissociated Human Embryonic Stem Cells (HESCS) Xiaowei Hong1, Xufeng Xue2, Tugba Topal1, Jianping Fu2,3, Cheri X. Deng1. 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 2Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 3Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. Human embryonic stem cells (hESCs) have wide potential use in regenerative medicine and cell therapies. However, large-scale cell expansion is limited as hESCs undergo differentiation and apoptosis after single-cell dissociation. Currently, Rho-associated kinase (ROCK) inhibitors are used in hESC culture to maintain cell survival, yet with the risk of long-term negative effects on

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hESCs. In previous work, we have shown acoustic tweezing cytometry (ATC), an ultrasound-based subcellular mechanical stimulation technique, can significantly improve survival rate of dissociated hESCs while maintaining their pluripotency without the presence of ROCK inhibitor Y-27632. Herein, we further investigated the impact of ATC on dissociated hESCs and the mechanisms underlying the enhancement of hESC survival by ATC. To determine optimal ATC treatment conditions, we exposed hESCs to ATC treatments with different acoustic pressure levels (i.e., 0.05 - 0.25 MPa) and exposure frequencies, and assayed their effects on hESC survival. To explore the role of Ecadherin and integrin mediated signaling pathways in hESC survival, we used microbubbles targeted to E-cadherin and integrin on the cells in ATC application, and evaluated the effects on hESCs in terms of survival rate, cell area, colony size, and stemness. In addition, we monitored cell intracellular cytoskeleton contractility using a fluorescence-labeled micropost system in the initial cell spreading stage, and compared the contractility changes over time under various ATC stimulation conditions. In summary, results from this study will help us to develop an effective treatment scheme using ATC to enhance dissociated hESC survival and reveal the insights about mechanotransductive mechanisms involving E-cadherin and integrin-mediated adhesion signaling for hESC survival and pluripotency maintenance. 492-Pos Board B272 Electrostatic and Allosteric Response of Myosin as a Mechanosensor Jun Ohnuki, Takato Sato, Mitsunori Takano. Department of Pure & Applied Physics, Waseda University, Tokyo, Japan. Allostery is fundamental to the functions of proteins, where a locally-applied input travels within the molecule to a distant region. While the allostery due to chemical input such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the allostery due to mechanical input. One of the key proteins involved in mechanobiology is myosin. Myosin not only works as a motor, converting the chemical energy of the ATP hydrolysis to mechanical force, but also functions as a mechanosensor, changing the affinity for the ATP hydrolysis products and for the actin filament in response to the external force (load) that is applied to the so-called lever arm region. Since both the ATP-binding and the actin-binding regions are distant from the lever arm region, myosin is thought to be equipped with the allostery due to mechanical input. However, the underlying physical mechanism is unclear. By molecular dynamics simulation in combination with the replica exchange umbrella sampling, we investigated the response of myosin to the mechanical input applied to the lever arm. We found that the positional change of the basement of the lever arm (called converter region) induces significant electrostatic potential changes in the ATP-binding and the actin-binding regions. The mechanically-induced electrostatic responses we found are likely to be involved in the force generating function of the actin-myosin motor. We further show that allosteric response is caused by the large-scale rearrangement of the electrostatic bond network. 493-Pos Board B273 Screening Cell Mechanotype by Parallel Microfiltration Navjot Kaur Gill. Molecular, Cellular and Integrative Physiology MCIP, University of California, Los Angeles UCLA, Los Angeles, CA, USA. Cell mechanical phenotype or ‘mechanotype’ is emerging as a valuable labelfree biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRasV12transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application. 494-Pos Board B274 The Mechanism of Stress Granule Formation Induced by Intracellular Local Thermogenesis Beini Shi1, Kohki Okabe1,2, Takashi Funatsu1. 1 Dept Pharmac Sci, Univ Tokyo, Tokyo, Japan, 2JST, Presto, Tokyo, Japan. Under stress, eukaryotic cells assemble and form stress granule (SG), responsible for translation regulation. SG can dynamically spontaneously assemble