conductances, raising the possibility that the proteins may interact to form channels with a range of biophysical properties. Both dominant and recessive mutations in Tmc1 cause deafness in mice and humans. To further charac- terize the contributions of TMC1 to hair cell function we examined hair cells of mice that carried a dominant point mutation in Tmc1 known as Beethoven (Bth). The Bth mutation is methionine to lysine substitution at amino acid position 412. We find that the Bth point mutation is not a dominant- negative, or loss-of-function mutation, but alters several biophysical proper- ties of hair cell transduction. Relative to cells that express wild-type Tmc1, cells that express the Bth mutation have smaller single-channel conductance, larger calcium block, reduced calcium permeability and slower adaptation. These data provide strong evidence that implicate TMC1 as a component of the hair cell mechanotransduction channel. Platform: Cell Mechanics and Motility III 2144-Plat The Predominant Role of Tension in the Nanoscale Mechanical Behavior of Cells Visualized by a New Imaging Platform Nicola Mandriota, Ozgur Sahin. Columbia University, New York, NY, USA. Mechanical properties of cells are crucial regulators of a plethora of physio- logical processes. Yet, we still lack the spatiotemporal resolution to probe them at the nanoscale, where they are most relevant for cell physiology. Here we present a new AFM-based platform compatible with a wide variety of cells - including human - producing cell mechanical maps that resolve fea- tures separated by 18 nm, thus approaching biological molecules length scales. Our mechanical maps are high in contrast and rich in nanometer- scale features, so we perform simultaneous fluorescence microscopy to identify molecular counterparts. Although cytoskeletal elements correlate with stiffness, mechanical contrast changes over time or in response to drug. This suggests that tension - rather than elastic modulus - dominates the mechanical response at the nanoscale, with the unique exception of regions corre- sponding to exceptionally- rigid focal adhesion. We finally synthesized our findings showing how ten- sion levels can determine and predict cell shape at different length scales. 2145-Plat Investigating Focal Adhesion Mechanics using Nanopatterned Molecular Tension Fluorescence Microscopy (MTFM) Yang Liu 1 , Rebecca Medda 2,3 , Elisabetta Ada Cavalcanti-Adam 2,3 , Khalid Salaita 1 . 1 Department of Chemistry, Emory University, Atlanta, GA, USA, 2 Department of Biophysical Chemistry, Ruprecht-Karls-University, Heidelberg, Germany, 3 Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Stuttgart, Germany. The mechanical interaction between integrins and the ECM regulates pro- cesses such as adhesion and motility, as well as the remodeling of the ECM. Despite a large body of literature describing mechanoregulatory processes of integrins, the precise relationship between integrin-mediated forces and recep- tor lateral clustering are still poorly understood. Moreover, the fundamental structure responsible for force transduction remains unknown, and may occur at the scale of individual integrin receptors or within micron-scale focal adhesions. To address these questions, we generated hexagonal arrays of force-sensing nanoparticles and used these to visualize the mechanical forces exerted by in- tegrins in living cells. Recently developed gold nanoparticle molecular tension fluorescence microscopy (MTFM) probes were combined with block copol- ymer micellar nanolithography to generate tension sensing particle arrays with precisely defined inter-particle spacings at the nanometer scale. Fluores- cently labeled cyclicRGD probes were functionalized and efficiently quenched on the surface-anchored 9 nm gold nanoparticles. This probe features an entropic spring, polyethylene glycol (PEG) polymer, enabling pN tension mea- surements. Under cellular tension, the RGD-termini of the sensors extend away from the nanoparticle surface and fluorescence increases 10-15 fold. By using the worm-like chain model, the tension magnitude across the entire cell mem- brane can be calculated and mapped. In nanopatterned experiments, we fabri- cated gold nanoparticle arrays with 50 nm and 100 nm interparticle distance. Our results suggest that the average tension per integrin was highly dependent on RGD spacing, and therefore on integrin lateral clustering. When cultured on substrates with 50 nm spacing, fibroblast cells generated a tension of approxi- mately 4 pN per integrin, whereas the 100 nm spacing leads to forces of 1-2 pN. This indicates that when integrin clustering is hindered, high tension forces can not be generated in part due to the instability of focal complexes. 2146-Plat Probing the Mechanical Coupling of the Cell Membrane to the Nucleus with Vertical Nanopillar Arrays Lindsey Hanson 1 , Wenting Zhao 2 , Ziliang Lin 3 , Yi Cui 2,4 , Bianxiao Cui 1 . 1 Chemistry, Stanford University, Stanford, CA, USA, 2 Materials Science and Engineering, Stanford University, Stanford, CA, USA, 3 Applied Physics, Stanford University, Stanford, CA, USA, 4 Stanford Institute for Materials and Energy Sciences, SLAC National Accelerator Facility, Menlo Park, CA, USA. The structure of the nuclear envelope is crucial to many cell processes, from signal transduction to migration and motility, yet few studies have been carried out about the mechanical properties of the nuclear envelope. Previous studies rely on invasive techniques like micropipette aspiration that require isolation of the nucleus from the cell and neglect the participation of the cytoskeleton and extracellular membrane. In order to fully understand this process, we need a technique that can measure the response of the nucleus in intact cells as they function normally. For this purpose, we fabricated arrays of vertical nanopillars with various diameters, heights, and spacing to observe the extent of both plasma membrane deformation and nuclear deformation in response to non-invasive mechanical perturbation by vertical nanopillars. We found that while the plasma membrane is very flexible and conforms to the shape of the nanopillars, the nuclear membrane is much more rigid and deforms to a lesser degree. All three of those geometric parameters determine the stress relayed to the nucleus by the cell membrane and cytoskeleton. We investigated the con- tributions of different components of the cytoskeleton and found that while actin pulls the nucleus toward the cell membrane, intermediate filaments resist nuclear deformation. Nanostructures provide a new and exciting platform for studying cell and nuclear mechanics in length scales and contexts that are inac- cessible to traditional techniques. 2147-Plat Outward Microtubule-Mediated Pushing Forces Dictate Mitosis Hedde van Hoorn, Martin de Valois, Claude Backendorf, Thomas Schmidt. Leiden University, Leiden, Netherlands. Cellular force generation is essential for tissue morphogenesis and cell prolif- eration. The balance of forces is in particular needed for successful chromo- some condensation, separation and ultimately cell division. Although cellular force exertion has been studied extensively during interphase, its characteriza- tion during mitosis is still lacking. Recently, a fascinating study already showed the importance of the mechanical outside-in coupling during mitosis [1]. Here, we directly measured the inside-out force exerted during mitosis with GFP- labeled H2B to follow the cell cycle and GFP-labeled tubulin to examine the role of microtubules. Using micropillars of different heights, we quantified force exertion of dividing HeLa cells. Before prophase, we observed cell polarization where in the direc- tion of polarization pulling forces were exerted. Polarization corresponded to the direction of division, as was expected from stretch-induced directionality of division [1]. Surprisingly, we consistently found radial outward forces dur- ing the subsequent phases in mitosis. This pushing force increased greatly in prophase, slowed in increase in metaphase and reached its peak at the start of anaphase. These displacement-controlled forces increase 5-fold over these phases. During anaphase and telophase, the forces decreased rapidly until the two daughter cells spread into their normal interphase conformation. Experi- ments with GFP-labeled tubulin demonstrated that pushing forces were present where microtubules were present, emanating from the centrosomes. From these observations, we developed a model for the mechanical balancing act through which pushing forces mediated by microtubules ensure proper cell division. Reference 1. Fink et al. (2011), Nature Cell Biology. 2148-Plat Analysis and Modeling of Dendritic Spine Morphogenesis Olena Marchenko 1 , Charles W. Wolgemuth 2 , Leslie M. Loew 1 . 1 CCAM, Uconn Health Center, Farmington, CT, USA, 2 Physics and Molecular and Cellular Biology, University of Arizona, Tucson, AZ, USA. Dendritic spines receive the majority of excitatory inputs in the central nervous system, and they are key structures in the regulation of neural activity. Spine 424a Tuesday, February 18, 2014