Bayesian Parameter Estimation and Model Selection for Biophysical ...

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Monday, February 29, 2016

production. Estradiol (E2) is the major sex hormone signal to skeletal muscle in females, and strength loss is rescued by E2 treatment in ovariectomized (OVX) mice. We previously showed that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in single fibers isolated from OVX mice. To investigate E2 mechanisms on aged skeletal muscle, single fibers were isolated from young (2 months) or old (28 months) male and female mice, and were incubated with mantATP. Old female mice receiving 60-day E2 treatment were also studied. We measured the decay of mantATP fluorescence in an ATP-chase experiment to characterize the slow nucleotide turnover, called the super-relaxed state (SRX), detected in approximately one-third of the myosin heads. The SRX turnover was faster in aged female fibers compared to young female fibers. In contrast, ATP turnover in old male fibers was not significantly different than in young males, and even trended toward slower rather than faster turnover with aging. We conclude that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in aging female mice. Ageand hormone-related functional deficits may be targetable at the level of myosin and other contractile protein structure/function for strategies to offset muscle weakness and metabolic changes that occur with age. This work was supported by NIH grants to DDT (R37 AG026160 and R01 AR032961), to DL (R01 AG31743), to BAC (R00 HL122397), and to KJP and BCC (T32 AR7612). 1492-Pos Board B469 Destabilizing the Super Relaxed State of Skeletal Muscle Myosin to Treat Obesity and Type 2 Diabetes Leonardo Nogara1, Nariman Naber2, Marcella Canton3, Carlo Reggiani3, Edward Pate4, Roger Cooke2. 1 Dept. of Biomedical Sciences, Univ of Padua, Padua, Italy, 2Biochem/ Biophys, Univ California, San Francisco, CA, USA, 3Dept. of Biomedical Sciences, Univ Padua, Padua, Italy, 4Voiland school Bioengineering, Washington State Univ, Pullman, WA, USA. In resting skeletal muscle, myosin is found in 2 states, the super-relaxed state (SRX) and the disordered relaxed state (DRX). In the SRX the ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament; in the DRX the myosin heads are not bound to the core of the thick filament and have an ATPase rate that is an order of magnitude greater. We made a series of single cysteine mutants of the myosin regulatory light chain, placed fluorescent probes on them, and exchanged them into skinned rabbit fast skeletal muscle fibers. We found fluorescent probes that reported on the relative populations of the SRX and DRX, and used them to carry out a high throughput screen. The screen identified one compound, piperine, which destabilized the SRX as measured by probe fluorescence. The kd for inhibition was ~3 mM with a maximum inhibition of ~50% at high concentations. The compound was also shown to destabilize the SRX using single nucleotide turnover measurements. The effect was only seen in fast twitch fibers. Piperine was shown to increase the ATPase activity of relaxed fibers by 40%. It had no effect on the mechanics of either active or resting muscle fibers. Previous work has shown that piperine can attenuate both obesity and type 2 diabetes in rodent models of these conditions. We propose that these effects are due to the up-regulation of resting muscle metabolism. The results described here suggest that piperine would provide a useful lead compound for the development of therapies to treat obesity and type 2 diabetes in humans.

Cell Mechanics, Mechanosensing, and Motility II 1493-Pos Board B470 Space and Time in Leukocyte Migration Donald M. Guu1, Thomas Quast2, Luis Alvarez1, U. Benjamin Kaupp1, Waldemar Kolanus2. 1 Molecular Sensory Systems, Research Center CAESAR Bonn, Germany, Bonn, Germany, 2LIMES, University of Bonn, Bonn, Germany. Dendritic cells (DCs) are an essential component of the mammalian immune system because they link the innate and the adaptive immune responses. They are sentinels that patrol and scan the peripheral tissue where they capture pathogen-derived antigens at the site of infection. After this, they migrate to the secondary lymphoid organs, where they prime naive T lymphocytes by presenting the captured antigens. For migration towards the lymphoid organs, DCs make use of chemoattractant cues. They detect the distribution of the chemoattractant in the tissue and extend cellular protrusions (lamellipodia) to migrate up the chemical gradient in a process called chemotaxis. Although several of the molecular components for DC chemotaxis have been identified, little is

known about the mechanisms used by DCs to sense gradients. We used a microfluidic chemotaxis chamber and optical microscopy to elucidate the gradient sensing mechanism of DCs. The microfluidic chemotaxis assay allows us to control with high precision the properties of the gradient in space and time. We will present several novel features of DC migration in response to different spatio-temporal stimulation patterns. 1494-Pos Board B471 Bayesian Parameter Estimation and Model Selection for Biophysical Models of Leukocyte Rolling Mats L. Moskopp1, Andreas Deussen1, Triantafyllos Chavakis2, Peter Dieterich1. 1 Institut fu¨r Physiologie, Medizinische Fakulta¨t Carl Gustav Carus, TU Dresden, Dresden, Germany, 2Klinische Pathobiochemie, Medizinische Fakulta¨t Carl Gustav Carus, TU Dresden, Dresden, Germany. Leukocytes leave the bloodstream and enter into tissue by undergoing the leukocyte adhesion cascade. The endothelium, which covers the inner vessel surface, regulates this process by expressing different adhesion molecules or chemoattractants. This study focuses on the initial interaction between leukocytes and endothelium - the so called selectin dependent leukocyte rolling. Bindings between selectins on the endothelium and their counter ligands on leukocytes are able to slow down the cell to a small fraction of the velocity of the convective blood stream. Interestingly, the rolling process shows a characteristic stop-and-go pattern. We hypothesize that this pattern is caused by binding, unbinding and stretching behavior of microvilli and attached selectin-bindings. In addition, we claim that the analysis of the cell paths allows extracting biophysical parameters of the selectin-cell-interaction. Therefore, we have applied an in vitro flow chamber assay to measure monocytic like THP-1 cell rolling on isolated E-selectin under defined flow conditions. The positions of cells are obtained from sequences of phase contrast images applying self-written image processing software. In addition, we constructed different mathematical models of rolling leukocytes differing in the (visco-) elastic behavior of the simulated selectin-bonds. Bayesian data analysis is applied to estimate the parameters of the mathematical models. Bayesian model selection shows that a viscoelastic model gives the best description of the observed data. Thus, we could identify single molecule events causing the typical stop-and-go pattern of leukocyte rolling. As a byproduct the algorithm permits to directly estimate biomechanical parameters of the nano-scale from microscopic analysis of whole cells. 1495-Pos Board B472 Forward and Inverse Approaches to Characterizing Cellular Traction Forces Ankur H. Kulkarni1, Prasenjit Ghosh1, Nagaraj Balasubramanian2, Namrata Gundiah1. 1 Indian Institute of Science, Bangalore, India, 2Indian Institute of Science Education and Research, Pune, India. Adherent cells exert tractional stresses on substrates through transmission of intracellular contractile forces. Traction forces influence processes including cell migration, crucial in cancer metastasis and wound healing. Quantification of these forces is hence essential to our understanding of cell-ECM interactions in normal and pathological conditions. In this study, we adapted the finite element (FE) method to quantify cellular tractions and used strain energy, stress and reaction forces as measures to characterize cell-substrate interactions using fibroblasts transfected with GFP-paxillin cultured on polyacrylamide substrates. FE analysis showed rapid convergence in strain energy and net reaction force with mesh refinement; stresses however converged slowly. We compared these results with Fourier Transform Traction Cytometry (FTTC) and regularised FTTC (Reg-FTTC) method which overcomes problems in inversion occurring due to noise. Mesh convergence for high stress nodes in FE was comparable to Reg-FTTC; FTTC stresses had poor convergence in comparison. Strain energy from FE method was lower than that obtained from both Fourier methods. Further, maximum stress was highest for FTTC, followed by FE and Reg-FTTC methods. Overall, the FE method performed significantly better than FTTC based on these metrics. The Reg-FTTC provides an improvement over slow stress convergence in FE but depends crucially on choice of regularization parameter which necessitates optimization for individual cell types. We used reaction force output, unique to FE, to quantify localized adhesion forces acting on discrete regions of cell periphery that may provide insights into spatial organization of cell-ECM interactions. Together, we hope these studies will help in creation of robust tools to characterize the mechanical interplay between cell and microenvironment which will further our understanding of disease progression and cell behaviour.