3D Simulations of Morphogen Transport in an Early Fish Embryo

Sunday, February 28, 2016 using a recently published and experimentally supported model of CFTR, we have carried out all-atom Molecular Dynamics (MD) in an attempt to further characterize the dynamics of this recalcitrant protein. First, we refined the ˚ model utilizing MD flexible fitting (MDFF) and structural data from a 9-A cryo-EM map of CFTR. Next, roughly 1 microsecond of equilibrium MD simulations of the model within a membrane was used to probe three physically relevant states: the apo state, the proposed semi-apo state (1 ATP bound), and the bound state (2 ATP bound). Results show both experimentally demonstrated protein interactions as well as new, untested ones. For instance, an ATPdependent interaction between the X-loop and Q-loop motifs was observed. A mechanistic picture of how ATP binding leads to channel activity will be reported. Specifically, structural metrics of protein motion are defined and used to explain the formation of a pore within CFTR in simulations when ATP is bound. 710-Pos Board B490 In Silico Characterization of P-Glycoprotein Substrate Entrance Pathways Laura Domicevica, Teresa Paramo, Philip C. Biggin. Biochemistry, Oxford University, Oxford, United Kingdom. Due to the wide variety of substrates it extrudes, P-glycoprotein is one of the main causes of multidrug resistance. Although the chemical structure of a ligand is likely to dictate interactions within the binding site, it is equally important to understand how access to the binding site itself is controlled. Access to the binding site is via an aqueous cavity formed in the main by transmembrane helices (TMHs). These TMHs also have helical structure that extends into the cytoplasm, but the hydrophobic vacuum cleaner model suggests that access to the aqueous cavity is via gates between the membranous regions of TMHs 4 and 6 or between TMHs 10 and 12. Previously, we observed that the intra-membrane sections of these gates tend to close while cytoplasmic regions remain open. We have explored this further via the use of steered MD with substrates initially positioned in the cytoplasmic leaflet of the bilayer. Rather unexpectedly, the simulations show that entrance through the intra-membrane gate requires a significantly larger amount of work compared to the cytoplasmic entrance and with a preference for the cavity between TMH 4 and 6 compared to that between TMH 10 and 12. Once the compound has reached the aqueous cavity, it can freely diffuse towards its binding site. The binding mode was independently assessed through the use of consensus docking and appears to be similar to the so-called ‘‘Rsite’’. The docked conformations were then used to calculate possible unbinding pathways of amitriptyline with the Protein Energy Landscape Exploration (PELE) method. Taken together, these combined approaches suggest that a likely access route to the binding site for many compounds is through a cytoplasmic gate formed by TMHs 4 and 6.

Cellular Signaling and Metabolic Networks 711-Pos Board B491 3D Simulations of Morphogen Transport in an Early Fish Embryo Ines Reinartz1, Claude Sinner1, Eliana Stanganello2, Benjamin Mattes2, Steffen Scholpp2, Alexander Schug1. 1 SCC, KIT, Eggenstein-Leopoldshafen, Germany, 2ITG, KIT, EggensteinLeopoldshafen, Germany. During the early stages of embryonic development, various means of cell communication orchestrate tissue development within the highly dynamic environment. Signalling gradients of morphogens determine cell fates, tissue generation, and in the long run organs of the final organism. In our research we focus on the distribution and effects of Wnt8a, a morphogen involved in the development and differentiation of the brain. In a previous work we were able to show experimentally that this protein is distributed by a novel shortrange propagation mechanism by means of specialized filopodia[1]. Here we present our results on extending these simulations of a simple flat tissue towards a more accurate description of the embryo in a 3D environment. The simulation models the tissue expansion on a 3D spherical surface and morphogen distribution via filopodia formation. It integrates length and angle distributions as well as growth frequencies of the filopodia, cell migration, and a slight ligand decay consistently with experimental measurements. Additionally it extends our previous model by explicitly considering the movement of the Wnt8a source. [1] Filopodia-based Wnt transport during vertebrate tissue patterning. Stanganello, E., Hagemann, A. I. H., Mattes, B., Sinner, C., Meyen, D., Weber, S., Schug, A., Raz, E. and Scholpp, S., Nature Communications 6, 2015

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712-Pos Board B492 Transplantation of Mesenchymal Stem Cells Enhances Angiogenesis after Ischemic Limb Injury in Mice Hua Zhu1, Nanzi Xie2, Timothy Adesanya1, Ahmet Kilic1, Tao Tan1, Xiaoyun Xie3. 1 The Ohio State University, Columbus, OH, USA, 2Tongji Hospital, Tongji University, Shanghai, China, 3Tongji Hospital, Tongji University, Columbus, China. Mesenchymal stem cell-based therapy has emerged as a promising approach for the treatment of peripheral arterial disease. This study was designed to examine the potential effects of human placenta-derived mesenchymal stem cells (PMSCs) on treatment of mouse hindlimb ischemia. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. An in vivo surgical ligation-induced mouse limb ischemia model was generated with fluorescent dye (CM-DiI) labelled PMSCs delivered via intramuscular injection. Our data show that PMSCs treatment significantly enhanced microvessel density, improved blood perfusion and diminished pathologies in ischemic mouse hindlimbs as compared to those in the control group. Further immunostaining studies suggested that injected PMSCs can incorporate into the vasculature and differentiate into endothelial and smooth muscle cells to enhance angiogenesis in ischemic hind limbs. This may in part explain the beneficial effects of PMSCs treatment. Taken together, we found that PMSCs treatment might be an effective treatment modality for treatment of ischemia-induced injury to mouse hind limbs by enhancement of angiogenesis. 713-Pos Board B493 Quantification of the Nutrient Dependence of Factors Controlling Start in Budding Yeast by 2-Photon Scanning Number and Brightness Savanna B. Dorsey1, Sylvain Tollis2, Jing Cheng2, Linnea Olofsson2, Mike Tyers2, Catherine A. Royer1. 1 Rensselaer Polytechnic Institute, Troy, NY, USA, 2Universite´ de Montre´al, Montreal, QC, Canada. Cell size homeostasis is a poorly understood universal property of all organisms which results from the coordination between cell growth and division. In budding yeast, cell size is modulated by nutrients. On poor nutrients cells commit to division (pass Start) at a smaller size. While many of the protein factors underlying the growth and division networks have been identified, there exists little quantitative data defining how much, when and where these proteins are present and interact at a single cell level. Even the key Start trigger, the G1 cyclin Cln3, which activates the Cdc28 kinase to phosphorylate and inactivate the Start transcription repressor Whi5, is notoriously difficult to detect and has never been observed on the single cell level. We used two-photon laser scanning Number and Brightness experiments to localize and quantify Cln3GFPmut3 and Whi5-GFPmut3 fusions expressed from their natural loci at the single cell level in live budding yeast cells grown on rich (glucose) and poor (ethanol) nutrients. We find homogeneous Cln3 concentrations (~3-4 nM) with no clear localization pattern or dependence on cell size or nutrients. This finding is in contrast to models suggesting changes in Cln3 concentration as a key factor in cell size control. In G1 phase cells we find Whi5 at ~150 nM in the nucleus and ~10-30-fold lower in the cytoplasm. In both G1 and postStart cells we find 3-fold higher concentrations of Whi5 in the cytoplasm on ethanol, 15 þ/- 3 nM, compared to glucose, 5-6 þ/- 1 nM. Higher cytosolic Whi5 on ethanol is consistent with increased inactivation and export of this repressor to trigger earlier initiation of Start. 714-Pos Board B494 Towards Predictive Modeling of Information Processing in Microbial Ecosystems with Quorum Sensing Interactions Tahir Yusufaly, James Boedicker. University of Southern California, Los Angeles, CA, USA. Bacteria communicate using external chemical signals in a process known as quorum sensing. However, the efficiency of this communication is reduced by both limitations on the rate of diffusion over long distances and potential interference from neighboring strains. Therefore, having a framework to quantitatively predict how spatial structure and biodiversity shape information processing in bacterial colonies is important, both for understanding the evolutionary dynamics of natural microbial ecosystems, and for the rational design of synthetic ecosystems with desired computational properties. As a first step towards these goals, we implement a reaction-diffusion model to study the dynamics of a LuxI/LuxR quorum sensing circuit in a growing bacterial population. The spatiotemporal concentration profile of acylhomoserine lactone (AHL) signaling molecules is analyzed, and used to define a measure of physical and functional signaling network connectivity. From