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Microrheology of Astrocytes and Gliomas and Contribution of Intermediate Filaments to their Mechanics
Tuesday, February 14, 2017 each ligand (for a total of 200 AcrB-ligand structures) were then subjected to MD ms- long simulations and analyzed with respect to descriptors of their structure, dynamics, and thermodynamics of binding. The outcome of this study, unprecedented for its extensiveness, will provide key information to medicinal chemists for the design of new compounds with improved efficiency. 1650-Plat Voltage-Gated Calcium Channels as Detecting Tools of Psychoactive Drugs Iwona Ruchala, Alan Harris, Louis J. De Felice, Jose M. Eltit. Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA. The dopamine, serotonin, and norepinephrine transporters are important determinants of monoaminergic neurotransmission. The pharmacological manipulation of these systems contributes not only to drug addiction, but also to the treatment of many mental diseases. The development of novel tools to expedite the identification of drug-effect profiles on monoamine transporters can benefit efforts to discover new psychotherapeutics. Monoamine transporters are natively expressed in the plasma membranes of presynaptic neurons, where they mediate the uptake of monoamine neurotransmitters. When expressed in heterologous expression systems, the uptake of substrates is associated with an inward current that depolarizes the cell membrane. In addition, voltage-gated Ca2þ channels modulate Ca2þ permeability as a function of the membrane potential. We took advantage of these two elemental principles to develop a new assay to detect and characterize ligands of monoamine transporters. Using the Flp-In T-REx expression system, we established permanent cell lines expressing the human dopamine or serotonin transporters. We studied the ability of these cells to produce Ca2þ signals in response to monoamine transporters’ substrates after transient transfection with different voltage-gated Ca2þ channels. In addition we studied the dynamic range and sensitivity of two genetically encoded Ca2þ sensors. Our results show that the co-expression of CaV1.2 and GCamp6s in monoamine transporterexpressing cells generates significant Ca2þ signals in response to monoamine transporter substrates. Moreover monoamine transporter blockers can inhibit these Ca2þ signals. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2þ channel, a monoamine transporter and a genetically encoded Ca2þ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Supported by R01 DA033930 and R01 AR067738. 1651-Plat Homology Modeling and Ligand Optimization for the Human Amino Acid Exchanger Lat-1 (SLC7A5) Claire Colas1, Avner Schlessinger2. 1 Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Pharmacological sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. LAT-1 (SLC7A5) is a sodium-independent exchanger mainly located in the blood brain barrier (BBB) where it mediates the transport of large neutral amino acids, thyroid hormones and prescription drugs. LAT-1 is also upregulated in a variety of cancer types (e.g., prostate cancer), to provide amino acids that are used as nutrients and signaling molecules for growth. Thus, LAT-1 is an emerging drug target for substrates that can serve as prodrugs with optimal BBB or tumor permeability, or inhibitors that starve cancer cells. Here, we use computational modeling using homology modeling and ligand docking, followed by experimental testing using synthetic chemistry and cellular uptake measurements, to develop homology models for LAT-1 and identify novel chemical tools for this transporter. We refine a homology model of LAT-1 based on the arginine/agmatine transporter AdiC from E. Coli as modeling template and conduct SAR studies on amino acid derivatives to design compounds with optimal Km/Vmax balance. Particularly, initial LAT-1 models and binding evaluation of putative compounds guided the synthesis of distinct compounds, including tyrosine and phenylalanine derivatives, carboxylic acid bioisosteres, and other unique scaffolds. These compounds were tested experimentally by your collaborators using cis-inhibition and trans-stimulation assays. For example, we identified hydroxamic acids derivatives as LAT-1 substrates for the first time. Furthermore, iterative evaluation of these compounds by computational methods and experimental testing permitted to progressively refine our LAT-1 models, reaveling previously unknown druggable pockets on its surface. Our results improve our understanding of how inhibition and transport specificity is determined in LAT-1, as well as provide novel chemical probes to further characterize the role of this transporter in cancer and neurological disorders.
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Platform: Cell Mechanics, Mechanosensing, and Motility II 1652-Plat Mechanical Response of Nesprin-2G to Cytoskeletal Forces Regulates Linc Complex-Dependent Mechanotransduction Hengameh Shams1, Gant Luxton2, Mohammad R.K. Mofrad1. 1 Bioengineering, University of California, Berkeley, Berkeley, CA, USA, 2 Genetics, Cell Biology, and Development, University OF Minnesota, Minneapolis, CA, USA. Nuclear geometry and positioning are affected by cytoskeletal forces, which plays an important role in cellular mechanotrasnduction. The LINC (LInkers of the Nucleoskeleton and Cytoskeleton) complex has been shown to bear mechanical load and mediate force transmission from the cytoskeleton into the nucleus, however, molecular mechanisms of this phenomenon are not yet understood. Giant nesprin-2 (nesprin-2G) forms a direct linkage between the actin cytoskeleton and SUN proteins inside the perinuclear space. These linkers are shown to be under constant tension especially in the apical and equatorial planes of the nucleus and have 56 spectrin repeats along their rod domain. A functional construct of nespring-2G containing only four spectrin repeats, referred to as mini-nesprin-2G, was shown to rescue the nuclear movement defects observed in nesprin-2Gdepleted fibroblasts. Since it is intrinsically difficult to measure forces and track residue-level interactions across individual proteins using current experimental techniques, we used molecular dynamics simulations to study mechanical response of nesprin molecules to cytoskeletal forces. Mininesprins are excellent candidates for this purpose as their small size allows for long simulation periods necessary for capturing important molecular events. Our results provides a detailed understanding of how mechanical modes of mini-nesprin-2G are excited and contribute to its response to various cytoskeletal forces around the nucleus. Also, we simulated forceinduced conformational changes of the Actin Binding Domain (ABD) of mini-nesprin-2G, which are most likely induced by the axial tension along the actin bundles at the apical plane of the nucleus. Furthermore, we examined the effect of ABD conformation on its actin binding affinity by comparing the strength of interactions between distinct conformations of the ABD domain of mini-nesprin-2G and actin. Lastly, we used force distribution analysis to identify force-sensitive residues along the mini-nesprin2G structure and tested the effect of mutating them to alanine on transferring forces to the nucleus. 1653-Plat Nuclear Constriction Segregates Mobile Nuclear Proteins Away from Chromatin Charlotte R. Pfeifer, Jerome Irianto, Rachel R. Bennett, Yuntao Xia, Irena L. Ivanovska, Andrea J. Liu, Roger A. Greenberg, Dennis E. Discher. University of Pennsylvania, Philadelphia, PA, USA. As a cancer cell squeezes through adjacent tissue, penetrates a basement membrane, or enters the smallest blood capillaries, its nucleus can be highly constricted, but any effects on chromatin density and other nuclear factors are poorly understood. Here, in cancer cell migration through rigid micropores and also in passive pulling into micropipettes, local compaction of chromatin is observed coincident in space and time with depletion of mobile factors. Hetero/eu-chromatin has been previously estimated from molecular mobility measurements to occupy a volume fraction f roughly two-thirds of the nucleus, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g. 53BP1) or nucleases (e.g. Cas9, FokI), are seen to exhibit a much reduced density within the constriction, approaching 0%. Such loss of mobile nuclear factors—compounded by the occasional rupture of the nuclear envelope—has important functional consequences for the cell. Constricted migration indeed delays DNA cleavage by a FokI-lacR fusion of a target locus integrated into chromosome 1. 1654-Plat Microrheology of Astrocytes and Gliomas and Contribution of Intermediate Filaments to their Mechanics Alibert Charlotte1, Atef Asnacios2, Bruno Goud1, Jean-Baptiste Manneville1. 1 Institut Curie, Paris, France, 2Matie`re et Syste`mes complexes, Paris, France. Several studies show that cancerous tissues are stiffer than normal tissues. However, at the scale of the individual cell, tumor cells appear to be softer
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than normal cells. Here, we use two different microrheology techniques allowing us to probe the mechanical properties of cells at two different scales: the scale of the whole cell using a single cell uniaxial rheometer, and the intracellular scale using a combination of micropatterning and optical tweezers. These two techniques enable us to compare and correlate the internal visco-elastic properties with the mechanics of the entire cell. We focus our study on the mechanics of astrocytes, the major glial cell type in the brain, and gliomas, brain tumors derived from astrocytes. We first discriminate astrocytes and gliomas of different grades based on their mechanical properties. We show that rat primary astrocytes are stiffer than glioma cells from both human and rat grade III and grade IV cell lines, and that mechanical differences exist between glioma cells grades. Next, since intermediate filaments (IFs) have been involved in the migration of glial cells and since the expression of IF proteins is modified in gliomas, we evaluate the contribution of IFs to the mechanics of gliomas and astrocytes. We show that the level of IF proteins correlate with the mechanical properties of the cells and that downregulation of IF proteins lowers cell rigidity. Finally, using our intracellular technique, we are able to measure the force-deflection curve and the bending rigidity of IF bundles in cellulo. 1655-Plat Modelling the Flow of Dynamically Cross-Linked Biopolymer Networks Arjan E. Boerma1, Patrick R. Onck1, Erik van der Giessen1, Stefanos Papanikolaou2. 1 Micromechanics of Materials, University of Groningen, Groningen, Netherlands, 2Hopkins Extreme Materials Institute, Johns Hopkins University, Baltimore, MD, USA. Networks of semi-flexible actin filaments in the cell cytoskeleton play a key role in biological functions such as cell motility and force transduction. Connections between actin filaments are formed and broken by crosslinker molecules that thermally diffuse through the cell. The dynamic character of these cross-links can be critical for the mechanical behavior of the network. We present a coupled finite-element/Monte Carlo approach to model cross-linked filaments, and show that thermal diffusion of crosslinks causes a transition from elastic to flowing behavior in response to mechanical loads. 1656-Plat Dictyostelium Mechanics Accurately Identifies New Targetable Drug Space for Pancreatic Cancer Delineated by Myosin IIS, Filamins, and Alpha-Actinins, Collectively Comprimising the Mechanobiome Alexandra Surcel1, Eric Schiffhauer1, Dustin Thomas1, Qingfeng Zhu2, Robert Anders2, Douglas Robinson1. 1 Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Pancreatic ductal adenocarcinoma (PDAC) is largely incurable, with a 5-year survival rate of ~6% and a median survival of <6 months. With 37,000 people dying annually in the US from pancreatic cancer, it is predicted to become the 2nd leading cause of cancer deaths. PDAC cells show extremely elevated rates of de novo and acquired resistance to traditional chemotherapies. Uncovering new drug spaces that target metastatic hepatic disease, the primary mortality factor for patients, is essential. The ability of metastasizing cells to navigate through different microenvironments requires them to have altered mechanical profiles characterized by the cytoskeletal proteins that define the structural elements of the mechanobiome – the collection of proteins that sense and respond to mechanical stimuli. To identify new targetable drug spaces, we assessed the PDAC mechanobiome. We used our understanding of Dictyostelium mechanics and in silico analysis to identify the players and to accurately predict the mechanoresponsiveness of specific isoforms of the actin crosslinkers alpha-actinin and filamin. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation – myosin IIA, IIC, alpha-actin-4, and filamin B – show increased expression in cancerous ductal epithelial over normal tissue, while nonmechanosensory paralogs show decreased expression. We have determined the contributions of each of isoform on PDAC mechanics across short and long-time scales and have assessed the impact of knockdowns and overexpression PDAC cell mechanics, migration, and invasion both 2D and 3D. Additionally, we have previously shown that targeting of myosin IIC by the small molecule mechanical modulator 4-HAP affects PDAC mechanics. Mice treated with 4-HAP show a reduction in metastasis, suggesting that direct targeting of the mechanobiome can be developed into new treatments.
1657-Plat Myofibril Organization in Cardiomyocytes is Guided by the Maximal Tensile Stress Direction Hongyan Yuan, Bahador Marzban. Mechanical Engineering, University of Rhode Island, Kingston, RI, USA. The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a remarkable correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we revealed that the different myofibril organizations in mature versus immature cardiomyocytes are due to the different levels of force-dependent remodeling of focal adhesions. Further we found that the myofibril organizations in microtissues are regulated by a combination of multiple factors including cell/tissue shape, cell-substrate adhesions, and cell-cell adhesions. These findings can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart. 1658-Plat Microtubule Bundles in the Adult Cardiomyocyte Patrick Robison, Alexey I. Bogush, Benjamin L. Prosser. Physiology, University of Pennsylvania, Philadelphia, PA, USA. The role of Microtubules (MTs) in mechanosignalling and mechanical properties has seen a recent surge of interest among the cardiac and skeletal muscle community. We recently reported that MTs buckle and bear load during the cardiac cycle. This has a significant effect on contractile mechanics, suggesting that myocyte function is tunable by any regulatory pathway that alters the mechanical behavior of MTs. Other work ongoing in our lab indicates that a number of Microtubule Associated Proteins (MAPs) are robustly upregulated in human heart disease. Multi-MT assemblies or bundles associated with these and other MAPs have been observed and studied extensively in other contexts, including unicellular motile structures and neuronal axons, but have not been examined in cardiac myocytes. The number of MTs involved, their geometry and the relative abundance of such structures compared to isolated MTs have significant implications for the mechanical properties of a myocyte. Using targeted overexpression of several MAPs in adult rat cardiomyocytes, we examine the structure, magnitude and prevalence of MT bundles by electron microscopy. We find that single MTs are the most prevalent MT elements in the unmanipulated myocyte, accompanied by comparatively rare and small groupings of 2-3 MTs with wide spacing. MTs are preferentially located at the interface between myofibril and mitochondria. Manipulation of MT binding partners can modestly increase the size and likelihood of MT bundles, and also dramatically reduces the inter-tubule spacing to be in-line with canonical bundle spacing. When we further characterized the functional consequences of MT bundling on cardiac contractility, we were surprised to find that the promotion of bundling, at least with certain MAPs, does not appear to impair contraction by reinforcing compliant microtubules, but rather uncouples the MTs from the process of contraction-associated buckling.
Platform: Protein-Small Molecule Interactions 1659-Plat Combined in Vitro and in Silico Approaches to the Assessment of Stimulant Properties of Novel Psychoactive Substances Michelle A. Sahai1, Colin Davidson2, George Khelashvili3, Vincenzo Barrese4, Neelakshi Dutta1, Harel Weinstein3, Jolanta Opacka-Juffry1. 1 Department of Life Sciences, University of Roehampton, London, United Kingdom, 2Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, United Kingdom, 3Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA, 4St George’s University of London, London, United Kingdom. Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fastcyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a
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Platform: Cell Mechanics, Mechanosensing, and Motility II 1652-Plat Mechanical Response of Nesprin-2G to Cytoskeletal Forces Regulates Linc Complex-Dependent Mechanotransduction Hengameh Shams1, Gant Luxton2, Mohammad R.K. Mofrad1. 1 Bioengineering, University of California, Berkeley, Berkeley, CA, USA, 2 Genetics, Cell Biology, and Development, University OF Minnesota, Minneapolis, CA, USA. Nuclear geometry and positioning are affected by cytoskeletal forces, which plays an important role in cellular mechanotrasnduction. The LINC (LInkers of the Nucleoskeleton and Cytoskeleton) complex has been shown to bear mechanical load and mediate force transmission from the cytoskeleton into the nucleus, however, molecular mechanisms of this phenomenon are not yet understood. Giant nesprin-2 (nesprin-2G) forms a direct linkage between the actin cytoskeleton and SUN proteins inside the perinuclear space. These linkers are shown to be under constant tension especially in the apical and equatorial planes of the nucleus and have 56 spectrin repeats along their rod domain. A functional construct of nespring-2G containing only four spectrin repeats, referred to as mini-nesprin-2G, was shown to rescue the nuclear movement defects observed in nesprin-2Gdepleted fibroblasts. Since it is intrinsically difficult to measure forces and track residue-level interactions across individual proteins using current experimental techniques, we used molecular dynamics simulations to study mechanical response of nesprin molecules to cytoskeletal forces. Mininesprins are excellent candidates for this purpose as their small size allows for long simulation periods necessary for capturing important molecular events. Our results provides a detailed understanding of how mechanical modes of mini-nesprin-2G are excited and contribute to its response to various cytoskeletal forces around the nucleus. Also, we simulated forceinduced conformational changes of the Actin Binding Domain (ABD) of mini-nesprin-2G, which are most likely induced by the axial tension along the actin bundles at the apical plane of the nucleus. Furthermore, we examined the effect of ABD conformation on its actin binding affinity by comparing the strength of interactions between distinct conformations of the ABD domain of mini-nesprin-2G and actin. Lastly, we used force distribution analysis to identify force-sensitive residues along the mini-nesprin2G structure and tested the effect of mutating them to alanine on transferring forces to the nucleus. 1653-Plat Nuclear Constriction Segregates Mobile Nuclear Proteins Away from Chromatin Charlotte R. Pfeifer, Jerome Irianto, Rachel R. Bennett, Yuntao Xia, Irena L. Ivanovska, Andrea J. Liu, Roger A. Greenberg, Dennis E. Discher. University of Pennsylvania, Philadelphia, PA, USA. As a cancer cell squeezes through adjacent tissue, penetrates a basement membrane, or enters the smallest blood capillaries, its nucleus can be highly constricted, but any effects on chromatin density and other nuclear factors are poorly understood. Here, in cancer cell migration through rigid micropores and also in passive pulling into micropipettes, local compaction of chromatin is observed coincident in space and time with depletion of mobile factors. Hetero/eu-chromatin has been previously estimated from molecular mobility measurements to occupy a volume fraction f roughly two-thirds of the nucleus, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g. 53BP1) or nucleases (e.g. Cas9, FokI), are seen to exhibit a much reduced density within the constriction, approaching 0%. Such loss of mobile nuclear factors—compounded by the occasional rupture of the nuclear envelope—has important functional consequences for the cell. Constricted migration indeed delays DNA cleavage by a FokI-lacR fusion of a target locus integrated into chromosome 1. 1654-Plat Microrheology of Astrocytes and Gliomas and Contribution of Intermediate Filaments to their Mechanics Alibert Charlotte1, Atef Asnacios2, Bruno Goud1, Jean-Baptiste Manneville1. 1 Institut Curie, Paris, France, 2Matie`re et Syste`mes complexes, Paris, France. Several studies show that cancerous tissues are stiffer than normal tissues. However, at the scale of the individual cell, tumor cells appear to be softer
338a
Tuesday, February 14, 2017
than normal cells. Here, we use two different microrheology techniques allowing us to probe the mechanical properties of cells at two different scales: the scale of the whole cell using a single cell uniaxial rheometer, and the intracellular scale using a combination of micropatterning and optical tweezers. These two techniques enable us to compare and correlate the internal visco-elastic properties with the mechanics of the entire cell. We focus our study on the mechanics of astrocytes, the major glial cell type in the brain, and gliomas, brain tumors derived from astrocytes. We first discriminate astrocytes and gliomas of different grades based on their mechanical properties. We show that rat primary astrocytes are stiffer than glioma cells from both human and rat grade III and grade IV cell lines, and that mechanical differences exist between glioma cells grades. Next, since intermediate filaments (IFs) have been involved in the migration of glial cells and since the expression of IF proteins is modified in gliomas, we evaluate the contribution of IFs to the mechanics of gliomas and astrocytes. We show that the level of IF proteins correlate with the mechanical properties of the cells and that downregulation of IF proteins lowers cell rigidity. Finally, using our intracellular technique, we are able to measure the force-deflection curve and the bending rigidity of IF bundles in cellulo. 1655-Plat Modelling the Flow of Dynamically Cross-Linked Biopolymer Networks Arjan E. Boerma1, Patrick R. Onck1, Erik van der Giessen1, Stefanos Papanikolaou2. 1 Micromechanics of Materials, University of Groningen, Groningen, Netherlands, 2Hopkins Extreme Materials Institute, Johns Hopkins University, Baltimore, MD, USA. Networks of semi-flexible actin filaments in the cell cytoskeleton play a key role in biological functions such as cell motility and force transduction. Connections between actin filaments are formed and broken by crosslinker molecules that thermally diffuse through the cell. The dynamic character of these cross-links can be critical for the mechanical behavior of the network. We present a coupled finite-element/Monte Carlo approach to model cross-linked filaments, and show that thermal diffusion of crosslinks causes a transition from elastic to flowing behavior in response to mechanical loads. 1656-Plat Dictyostelium Mechanics Accurately Identifies New Targetable Drug Space for Pancreatic Cancer Delineated by Myosin IIS, Filamins, and Alpha-Actinins, Collectively Comprimising the Mechanobiome Alexandra Surcel1, Eric Schiffhauer1, Dustin Thomas1, Qingfeng Zhu2, Robert Anders2, Douglas Robinson1. 1 Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Pancreatic ductal adenocarcinoma (PDAC) is largely incurable, with a 5-year survival rate of ~6% and a median survival of <6 months. With 37,000 people dying annually in the US from pancreatic cancer, it is predicted to become the 2nd leading cause of cancer deaths. PDAC cells show extremely elevated rates of de novo and acquired resistance to traditional chemotherapies. Uncovering new drug spaces that target metastatic hepatic disease, the primary mortality factor for patients, is essential. The ability of metastasizing cells to navigate through different microenvironments requires them to have altered mechanical profiles characterized by the cytoskeletal proteins that define the structural elements of the mechanobiome – the collection of proteins that sense and respond to mechanical stimuli. To identify new targetable drug spaces, we assessed the PDAC mechanobiome. We used our understanding of Dictyostelium mechanics and in silico analysis to identify the players and to accurately predict the mechanoresponsiveness of specific isoforms of the actin crosslinkers alpha-actinin and filamin. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation – myosin IIA, IIC, alpha-actin-4, and filamin B – show increased expression in cancerous ductal epithelial over normal tissue, while nonmechanosensory paralogs show decreased expression. We have determined the contributions of each of isoform on PDAC mechanics across short and long-time scales and have assessed the impact of knockdowns and overexpression PDAC cell mechanics, migration, and invasion both 2D and 3D. Additionally, we have previously shown that targeting of myosin IIC by the small molecule mechanical modulator 4-HAP affects PDAC mechanics. Mice treated with 4-HAP show a reduction in metastasis, suggesting that direct targeting of the mechanobiome can be developed into new treatments.
1657-Plat Myofibril Organization in Cardiomyocytes is Guided by the Maximal Tensile Stress Direction Hongyan Yuan, Bahador Marzban. Mechanical Engineering, University of Rhode Island, Kingston, RI, USA. The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a remarkable correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we revealed that the different myofibril organizations in mature versus immature cardiomyocytes are due to the different levels of force-dependent remodeling of focal adhesions. Further we found that the myofibril organizations in microtissues are regulated by a combination of multiple factors including cell/tissue shape, cell-substrate adhesions, and cell-cell adhesions. These findings can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart. 1658-Plat Microtubule Bundles in the Adult Cardiomyocyte Patrick Robison, Alexey I. Bogush, Benjamin L. Prosser. Physiology, University of Pennsylvania, Philadelphia, PA, USA. The role of Microtubules (MTs) in mechanosignalling and mechanical properties has seen a recent surge of interest among the cardiac and skeletal muscle community. We recently reported that MTs buckle and bear load during the cardiac cycle. This has a significant effect on contractile mechanics, suggesting that myocyte function is tunable by any regulatory pathway that alters the mechanical behavior of MTs. Other work ongoing in our lab indicates that a number of Microtubule Associated Proteins (MAPs) are robustly upregulated in human heart disease. Multi-MT assemblies or bundles associated with these and other MAPs have been observed and studied extensively in other contexts, including unicellular motile structures and neuronal axons, but have not been examined in cardiac myocytes. The number of MTs involved, their geometry and the relative abundance of such structures compared to isolated MTs have significant implications for the mechanical properties of a myocyte. Using targeted overexpression of several MAPs in adult rat cardiomyocytes, we examine the structure, magnitude and prevalence of MT bundles by electron microscopy. We find that single MTs are the most prevalent MT elements in the unmanipulated myocyte, accompanied by comparatively rare and small groupings of 2-3 MTs with wide spacing. MTs are preferentially located at the interface between myofibril and mitochondria. Manipulation of MT binding partners can modestly increase the size and likelihood of MT bundles, and also dramatically reduces the inter-tubule spacing to be in-line with canonical bundle spacing. When we further characterized the functional consequences of MT bundling on cardiac contractility, we were surprised to find that the promotion of bundling, at least with certain MAPs, does not appear to impair contraction by reinforcing compliant microtubules, but rather uncouples the MTs from the process of contraction-associated buckling.
Platform: Protein-Small Molecule Interactions 1659-Plat Combined in Vitro and in Silico Approaches to the Assessment of Stimulant Properties of Novel Psychoactive Substances Michelle A. Sahai1, Colin Davidson2, George Khelashvili3, Vincenzo Barrese4, Neelakshi Dutta1, Harel Weinstein3, Jolanta Opacka-Juffry1. 1 Department of Life Sciences, University of Roehampton, London, United Kingdom, 2Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, United Kingdom, 3Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA, 4St George’s University of London, London, United Kingdom. Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fastcyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a