Sunday, February 26, 2012 177a

879-Pos Board B665Mechanosensitive Vinculin Signaling Regulates Stem Cell FateAndrew W. Holle, Xinyi Tang, Damini Tandon, Alex Fuhrmann,Juan Carlos Del Alamo, Adam J. Engler.UC San Diego, La Jolla, CA, USA.Human mesenchymal stem cell (hMSC) proliferation, migration, and differen-tiation have all been linked to extracellular matrix stiffness, but despite seriousscientific inquiry, a consensus on the signaling pathways that are necessary andsufficient for this mechanosensitive ability has yet to be reached. An analysis ofkinase binding site accessibility revealed MAPK1 to be both prevalent and in-accessible in many focal adhesion/mechanosensing candidate proteins. Onebinding partner for MAPK1, vinculin, is activated upon binding to talin ina force-sensitive manner. A Cysteine Shotgun/Western Blot was used to con-firm the unfolding of talin in response to changes in substrate stiffness. RNAinterference was used to knock down vinculin, resulting in an 80% decreasein stiffness-induced MyoD, a muscle transcription factor; control culturesthat induce Runx2 expression, an osteoblast transcription factor, were insensi-tive to vinculin knockdown. Vinculin knockdown was not observed to interferewith focal adhesion assembly, alter adhesive properties, or diminish cell trac-tion force generation, indicating that its deletion only adversely affectedMAPK1 signaling. In addition, vinculin domains were also selectively deletedand added back into knocked-down cells to assess which domains of the proteinwere sufficient for stiffness-induced differentiation. Together, these data pro-vide some of the first in situ evidence that force-sensitive focal adhesion pro-teins can activate stem cell differentiation signals.

880-Pos Board B666Measuring Cell Mechanics by Optical Alignment DeformationSpectroscopyKevin B. Roth, Keith B. Neeves, David W.M. Marr.Colorado School of Mines, Golden, CO, USA.Cell mechanical properties are a useful measure of phenotype that can be quan-tified by cell deformability. There is a lack of high-throughput methods to inves-tigate the mechanical properties of large populations of individual cells. Toaddress this need, we developed optical alignment deformation spectroscopy(OADS), a technique where hydrodynamic interactions between individual cellsare used to create deformation. InOADS, a linear optical trap is used to align twoincoming cells in a microfluidic cross-flow geometry, allowing hydrodynamicforces to induce a collision between cells at the stagnation point (see figure). Af-ter the interaction, the cells leave the stagnation point and a new pair of cells en-ters the trap. A convenient model cell to characterize OADS is the humanerythrocyte because of its well-known mechanical properties. We fit deforma-tion data of erythrocytes to a linear viscoelastic constitutive model (Voigt).This model incorporates a spring and dashpot in parallel, for the elastic (k)and viscous (h) parameters of the cell, respectively. Our measured values of

k = 14.5 mN/m andh = 4.9 mN*s/mcompare favorablywith literaturevalues. Our resultsshow OADS has po-tential as an accuratehigh-throughput in-dividual cell me-chanical cytometer.

881-Pos Board B667Viscoelastic Response of Red Blood Cells in Linear Optical TrapsTobias Sawetzki, David W.M. Marr.Colorado School of Mines, Golden, CO, USA.The mechanical properties of biological cells can be used as a marker for in-dividual cell’s health, information not accessible by bulk measurements. Forexample malaria parasites are known to significantly stiffen the red bloodcell (RBC) membrane, allowing identification of single infected cells basedon their deformability. We have developed a non-contact method employingnon-invasive optical forces, efficiently elongating RBCs within microfluidicchannels to determine cell elastic properties. In this, the anisotropic beam ofa single laser diode bar is used to create a line-shaped optical trap, capturingand elongating specimens along the laser axis. We perform static measure-ments on single RBCs to demonstrate the utility of this method. Simulationsemploying ray-tracing methods illustrate how the refraction of the asymmetriclaser profile inherently creates antipodal stretching forces. Applying themembrane theory of thin shells enables us to determine the expected deforma-tion based on our simulations and compare results to measured data.

As the behavior of a viscoelastic material, such as the RBC’s membrane,strongly depends on the timescales of applied forces, we perform frequency-dependent measurements with modulated external stimulus to determine theRBC’s complex elastic moduli, properties not accessible by comparable tech-niques. Laser intensity, and therefore the forces responsible for cell deforma-tion, is modulated at frequencies varying over three orders of magnitude.Cell response is recorded using a high-speed camera system, allowing us to cor-relate the applied external load to the phase-shifted viscoelastic behavior of theRBC and to obtain the frequency-dependent dissipation of energy during onecycle of oscillation.Employing this new technique, cells can be deformed while streaming alongthe line-shaped trap in flowing environment, allowing application in high-throughput systems. Use of comparably simple optics and inexpensive lasersources facilitates potential implementation in small platforms and hand-helddevices.

882-Pos Board B668Modulation of Integrin-Extracellular Matrix Adhesions by RhoA and SrcHarini Sreenivasappa1,2, Soon-Mi Lim1, Jerome P. Trzeciakowski1,Gonzalo Rivera2, Michael Davidson3, Andreea Trache1,2.1Texas A&M Health Science Center, College Station, TX, USA,2Texas A&M University, College Station, TX, USA,3Florida State University - National High Magnetic Field Laboratory,Tallahassee, FL, USA.RhoA and Src are considered potential modulators of the mechanical signalingthat promotes actin polymerization and focal adhesion formation. Here, we per-formed adhesion force spectroscopy measurements on vascular smooth musclecells (VSMC) expressing RhoA or Src (wild type, constitutively active or dom-inant negative mutants) using an atomic force microscope (AFM) tip function-alized with fibronectin. Integrin alpha5beta1-fibronectin adhesion force, localcell stiffness at the point of contact, and adhesion probability were determined.The results show that the mechanical properties of the VSMC as well asintegrin-fibronectin adhesion force are modulated by RhoA and Src. Inhibitionof RhoA activity induces low cytoskeletal tension and decreased integrin-fibronectin adhesion force. Also, VSMC expressing RhoA or Src dominant neg-ative constructs were found to be the softest. Conversely, RhoA and Src acti-vation increased alpha5beta1 integrin-fibronectin adhesion force and cellstiffness. To independently verify the effect of RhoA and Src on cytoskeletalchanges, fluorescence imaging experiments were performed to quantify actinfibers (confocal imaging of cells co-expressing RhoA-GFP and actin-mRFP)or protein phosphorylation at focal adhesions (total internal reflection fluores-cence imaging of cells co-expressing Src-mCherry and dSH2-YFP), respec-tively. Consistent with the AFM measurements, we observed an increase inactin fibers, and in protein phosphorylation at focal adhesion sites in cells ex-pressing constitutively active RhoA and Src, respectively. In addition, thesetwo proteins were maximally decreased by expression of dominant negativeRhoA and Src. Taken together, these results show that RhoA and Src are criticalmodulators of the mechanical signaling cross-talk between the cell and the ex-tracellular matrix.

883-Pos Board B669Measuring and Exploiting the Perturbative Effects of Surfaces onBiomoleculesEric A. Josephs, Jingru Shao, Gary Abel, Tao Ye.University of California, Merced, Merced, CA, USA.When proteins or nucleic acids are adsorbed onto a surface, their structure andkinetics may be perturbed relative to those under physiological conditions.Understanding these effects, which may be subtle or weak, are essential for ac-curate measurement of single-molecule behavior and important to the operationof essential biotechnologies such as microarrays. Using in-situ electrochemicalatomic force microscopy on a model system often used in biosensors – DNA co-valently tethered to a gold electrode that has been passivated by a self-assembledmonolayer of 6-mercaptohexanol– we examine the effects of both static andtime-varying surface charge onDNAconformation and find an extreme sensitiv-ity to its nanoscale chemical environment. Herewe present, within the context ofsingle-molecule biophysical measurements, methods to determine the interac-tion energy between the molecule and surface using a sensitive variation of tra-ditional force spectroscopy, as well as techniques to reduce the heterogeneity inthe chemical environment across the surface by direct placement of the biomol-ecules on the surface or through specific exploitation of defectswithin themono-layer. Furthermore, we have found that by controlling the surface potential wecan dynamically modulate the activity and kinetics of surface-bound biomole-cules, which may permit the development of new devices or measurement tech-niques that control the activity of single molecules in time and space.