- Email: [email protected]
Mechanical Induction of the Tumorigenic β-Catenin Pathway by Tumour Growth Pressure in vivo
622a
Wednesday, March 2, 2016
3071-Pos Board B448 Mechanical Induction of the Tumorigenic b-Catenin Pathway by Tumour Growth Pressure in vivo Emmanuel Farge1,2. 1 Institut Curie, Paris, France, 2INSERM, Paris, France. The tumour microenvironment may contribute to tumorigenesis due to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the NotchþApcþ/1638N mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, associated with increased Ret and beta-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by acoustic strain imaging and shear wave elastography. Exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of beta-catenin on Tyr654, which impairs its interaction with the E-cadherin in adherens junctions, and which was followed by beta-catenin nuclear translocation after 15 days. As a consequence, elevated expression of beta-catenintarget genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic beta-catenin pathway, conserved from early embryos betacatenin dependent mechanical induction of patterning-genes expression, suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity. Fernandez-Sanchez, Barbier et al, Nature 2015, Jul 2;523(7558):92-5. http:// dx.doi.org/10.1038/nature14329. Epub 2015 May 11. 3072-Pos Board B449 Segregation of Mobile Nuclear Proteins Away from Chromatin when the Nucleus is Constricted Charlotte R. Pfeifer, Jerome Irianto, Dennis E. Discher. Molecular & Cell Biophysics Lab and Physics & Astronomy Grad Group, University of Pennsylvania, Philadelphia, PA, USA. As a cancer cell or adult stem cell squeezes through adjacent tissue or penetrates a basement membrane, its nucleus can exhibit very large deformations, but the effects of such constricted migration on chromatin organization and nuclear factors is poorly understood. Here, in micropipette aspiration and cell migration through rigid micropores, local densification and extension of chromatin is visualized together with a surprising squeeze-out of mobile factors. Hetero/euchromatin has been estimated to occupy f ~ 70 5 10% of the nucleus, but based on relative intensity of DNA and GFP-histones in a human osteosarcoma cell line drawn into a narrow constriction, f can easily increase locally to f* ~ 85%. A fluorescent locus within Chromosome-1 is also seen to re-orient into the constriction, consistent with chromosomal alignment and tighter packing upon lateral squeezing when compared to the usual fractal globule state of chromosomes. In contrast, any mobile protein in the nucleus, including a ~dozen proteins that function as either DNA repair proteins (e.g. BRCA1), transcription factors (e.g. RelA), or Cas9 nuclease, is seen to exhibit a reduced density within the constriction. A local change in free volume from (1 - f) ~ 30% to (1 - f*) ~ 15% within the constriction is consistent with a ~2-fold decrease in the relative intensity for all of these mobile protein within the constriction. Such a large decrease in mobile nuclear factors away from the constriction where DNA concentration is highest has important implications for regulating DNA repair, transcription, and nuclease-breakage, especially when combined with an increased tendency for rupture of the lamina and loss of the nuclear factors. 3073-Pos Board B450 A Chemo-Mechanical Model for Extracellular Matrix and Nuclear Rigidity Regulated Size of Focal Adhesion Plaques Xuan Cao1, Yuan Lin2, Tristian P. Driscoll3, Janusz Franco-Barraza4, Edna Cukierman5, Robert L. Mauck3,6, Vivek Shenoy1,6. 1 Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Mechanical Engineering, University of Hong Kong, Hong Kong, Hong Kong, 3Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA, 4 Cancer Biology Program, University of PennsylvaniaFox Chase Cancer Center, Philadelphia, PA, USA, 5Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA, 6Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
In this work, a chemo-mechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e. focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced back to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, the effective stiffness of the FA-ECM complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer ECM and/or nucleus, as well as a stronger chemo-mechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a 1D fiber does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model provides a framework in which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size. 3074-Pos Board B451 Spindle Micro-Fluctuations of Length Reveal its Dynamics Over Cell Division Benjamin Mercat1, Xavier Pinson1, Jonathan Fouchard2, Hadrien Mary2, Sylvain Pastezeur1, Zahraa Alayan1, Yannick Gachet2, Sylvie Tournier2, He´le`ne Bouvrais1, Jacques Pe´cre´aux1. 1 Team CeDRE, Institut de Ge´ne´tique et De´veloppement de Rennes, Rennes, France, 2Laboratoire de biologie cellulaire et mole´culaire du controˆle de la prolife´ration, Toulouse, France. The mitotic spindle ensures correct segregation of sister chromatids and correct partitioning in daughter cells. It comprises dynamical microtubules (alternating polymerizing and depolymerizing), a variety of molecular motors and their regulators. Although spindle structure is well known, the link to its functions remains elusive, calling for including the dynamics of its components and their interactions. This question was mostly investigated by in silico or in vitro approaches. But a detailed characterizing of spindle mechanics, in physiological conditions, is missing. We propose an image processing based, non invasive, method combined to an heuristic model to measure mechanical parameters along time. We tracked fluorescently labeled spindle pole at high temporal and spatial resolution and measured the micro-fluctuations of spindle length, in vivo. We computed its power density spectrum using short time Fourier transform (sliding window) — a blueprint of spindle mechanics. Such a spectrum is then fitted with a Kelvin Voigt model with inertia (a spring, a damper, an inertial element in parallel) convolved with the window. We validated this method by recovering the mechanical parameters over time from simulated data. Then we measured them, in vivo, in two model organisms, nematode C. elegans onecell embryo and fission yeast S. pombe. In control C. elegans embryos, metaphase appeared dominated by damping element, consistent with the slow spindle elongation observed. At anaphase onset, all three parameters collapsed, before increasing about 50s later to reach a regime where damping and inertia dominated, suggesting a dramatic rearrangement of the spindle. Using a gene candidate approach, we could relate these mechanical behaviors to known structures at work, overlapping and kinetochore microtubules during metaphase and centralspindle at anaphase. With such a tool, we will propose a model of the spindle accounting for the dynamics of its component. 3075-Pos Board B452 Actomyosin Network Contractility Triggers a Stochastic Transformation into Highly Motile Amoeboid Cells Verena Ruprecht1, Stefan Wieser2, Andrew Callan-Jones3, Michael Smutny1, Hitoshi Morita1, Keisuke Sako1, Vanessa Barone1, Monika Ritsch-Marte2, Michael Sixt1, Raphael Voituriez4, Carl-Philipp Heisenberg1. 1 IST Austria, Vienna, Austria, 2Division of Biomedical Physics, Innsbruck Medical University, Innsbruck, Austria, 3Universite´ Paris-Diderot, Paris, France, 4Universite´ Pierre et Marie Curie, Paris, France. Cell migration is key for various biological processes and potentially leads to cancer dissemination if reactivated by tumour cells. Here we used zebrafish
Wednesday, March 2, 2016
3071-Pos Board B448 Mechanical Induction of the Tumorigenic b-Catenin Pathway by Tumour Growth Pressure in vivo Emmanuel Farge1,2. 1 Institut Curie, Paris, France, 2INSERM, Paris, France. The tumour microenvironment may contribute to tumorigenesis due to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the NotchþApcþ/1638N mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, associated with increased Ret and beta-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by acoustic strain imaging and shear wave elastography. Exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of beta-catenin on Tyr654, which impairs its interaction with the E-cadherin in adherens junctions, and which was followed by beta-catenin nuclear translocation after 15 days. As a consequence, elevated expression of beta-catenintarget genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic beta-catenin pathway, conserved from early embryos betacatenin dependent mechanical induction of patterning-genes expression, suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity. Fernandez-Sanchez, Barbier et al, Nature 2015, Jul 2;523(7558):92-5. http:// dx.doi.org/10.1038/nature14329. Epub 2015 May 11. 3072-Pos Board B449 Segregation of Mobile Nuclear Proteins Away from Chromatin when the Nucleus is Constricted Charlotte R. Pfeifer, Jerome Irianto, Dennis E. Discher. Molecular & Cell Biophysics Lab and Physics & Astronomy Grad Group, University of Pennsylvania, Philadelphia, PA, USA. As a cancer cell or adult stem cell squeezes through adjacent tissue or penetrates a basement membrane, its nucleus can exhibit very large deformations, but the effects of such constricted migration on chromatin organization and nuclear factors is poorly understood. Here, in micropipette aspiration and cell migration through rigid micropores, local densification and extension of chromatin is visualized together with a surprising squeeze-out of mobile factors. Hetero/euchromatin has been estimated to occupy f ~ 70 5 10% of the nucleus, but based on relative intensity of DNA and GFP-histones in a human osteosarcoma cell line drawn into a narrow constriction, f can easily increase locally to f* ~ 85%. A fluorescent locus within Chromosome-1 is also seen to re-orient into the constriction, consistent with chromosomal alignment and tighter packing upon lateral squeezing when compared to the usual fractal globule state of chromosomes. In contrast, any mobile protein in the nucleus, including a ~dozen proteins that function as either DNA repair proteins (e.g. BRCA1), transcription factors (e.g. RelA), or Cas9 nuclease, is seen to exhibit a reduced density within the constriction. A local change in free volume from (1 - f) ~ 30% to (1 - f*) ~ 15% within the constriction is consistent with a ~2-fold decrease in the relative intensity for all of these mobile protein within the constriction. Such a large decrease in mobile nuclear factors away from the constriction where DNA concentration is highest has important implications for regulating DNA repair, transcription, and nuclease-breakage, especially when combined with an increased tendency for rupture of the lamina and loss of the nuclear factors. 3073-Pos Board B450 A Chemo-Mechanical Model for Extracellular Matrix and Nuclear Rigidity Regulated Size of Focal Adhesion Plaques Xuan Cao1, Yuan Lin2, Tristian P. Driscoll3, Janusz Franco-Barraza4, Edna Cukierman5, Robert L. Mauck3,6, Vivek Shenoy1,6. 1 Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Mechanical Engineering, University of Hong Kong, Hong Kong, Hong Kong, 3Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA, 4 Cancer Biology Program, University of PennsylvaniaFox Chase Cancer Center, Philadelphia, PA, USA, 5Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA, 6Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
In this work, a chemo-mechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e. focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced back to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, the effective stiffness of the FA-ECM complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer ECM and/or nucleus, as well as a stronger chemo-mechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a 1D fiber does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model provides a framework in which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size. 3074-Pos Board B451 Spindle Micro-Fluctuations of Length Reveal its Dynamics Over Cell Division Benjamin Mercat1, Xavier Pinson1, Jonathan Fouchard2, Hadrien Mary2, Sylvain Pastezeur1, Zahraa Alayan1, Yannick Gachet2, Sylvie Tournier2, He´le`ne Bouvrais1, Jacques Pe´cre´aux1. 1 Team CeDRE, Institut de Ge´ne´tique et De´veloppement de Rennes, Rennes, France, 2Laboratoire de biologie cellulaire et mole´culaire du controˆle de la prolife´ration, Toulouse, France. The mitotic spindle ensures correct segregation of sister chromatids and correct partitioning in daughter cells. It comprises dynamical microtubules (alternating polymerizing and depolymerizing), a variety of molecular motors and their regulators. Although spindle structure is well known, the link to its functions remains elusive, calling for including the dynamics of its components and their interactions. This question was mostly investigated by in silico or in vitro approaches. But a detailed characterizing of spindle mechanics, in physiological conditions, is missing. We propose an image processing based, non invasive, method combined to an heuristic model to measure mechanical parameters along time. We tracked fluorescently labeled spindle pole at high temporal and spatial resolution and measured the micro-fluctuations of spindle length, in vivo. We computed its power density spectrum using short time Fourier transform (sliding window) — a blueprint of spindle mechanics. Such a spectrum is then fitted with a Kelvin Voigt model with inertia (a spring, a damper, an inertial element in parallel) convolved with the window. We validated this method by recovering the mechanical parameters over time from simulated data. Then we measured them, in vivo, in two model organisms, nematode C. elegans onecell embryo and fission yeast S. pombe. In control C. elegans embryos, metaphase appeared dominated by damping element, consistent with the slow spindle elongation observed. At anaphase onset, all three parameters collapsed, before increasing about 50s later to reach a regime where damping and inertia dominated, suggesting a dramatic rearrangement of the spindle. Using a gene candidate approach, we could relate these mechanical behaviors to known structures at work, overlapping and kinetochore microtubules during metaphase and centralspindle at anaphase. With such a tool, we will propose a model of the spindle accounting for the dynamics of its component. 3075-Pos Board B452 Actomyosin Network Contractility Triggers a Stochastic Transformation into Highly Motile Amoeboid Cells Verena Ruprecht1, Stefan Wieser2, Andrew Callan-Jones3, Michael Smutny1, Hitoshi Morita1, Keisuke Sako1, Vanessa Barone1, Monika Ritsch-Marte2, Michael Sixt1, Raphael Voituriez4, Carl-Philipp Heisenberg1. 1 IST Austria, Vienna, Austria, 2Division of Biomedical Physics, Innsbruck Medical University, Innsbruck, Austria, 3Universite´ Paris-Diderot, Paris, France, 4Universite´ Pierre et Marie Curie, Paris, France. Cell migration is key for various biological processes and potentially leads to cancer dissemination if reactivated by tumour cells. Here we used zebrafish