- Email: [email protected]
Mechanical Aspects of Mitochondrial Alterations in Apoptosis
Saturday, February 11, 2017
Bioengineering Subgroup 1-Subg Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting Isaac T.S. Li1, Yann R. Chemla2, Taekjip Ha3,4. 1 Chemistry, University of British Columbia, Kelowna, BC, Canada, 2 Department of Physics and Centre for Physics of Living Cells, University of Illinois Urbana-Champaign, Urbana, IL, USA, 3Department of Biophysics and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA, 4Howard Hughes Medical Institute, Baltimore, MD, USA. Rolling adhesion is the behaviour that leukocytes and circulating tumour cells exhibit as they passively roll along blood vessel walls under flow. It plays a critical role in capturing cells in the blood, guiding them toward inflammation sites, and activating cell signalling pathways to enable their subsequent transmigration. Rolling adhesion is mediated by catch-bond-like interactions between selectins expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 found at microvilli tips of leukocytes. Despite our understanding of individual components of this process, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behaviour remains poorly understood. Here, we developed 2 label-free methods that map the functional adhesion sites and their strength on a leukocyte surface. The first method relies on tracking the rotational angle of a single rolling cell, which confers advantages over standard methods that track the centre-of-mass alone. Constructing the adhesion map from the instantaneous angular velocity reveals that the adhesion profile along the rolling circumference is inhomogeneous. We corroborated these findings with a second method that allowed us to obtain a footprint of molecular adhesion events using DNA-based molecular force probes. Our results reveal that adhesion at the functional level is not uniformly distributed over the leukocyte surface as previously assumed, but is instead patchy. 2-Subg Interactions of Engineered Nanomaterials with Lipid Interfaces Amir Farnoud. Chemical and Biomolecular Engineering, Ohio University, Athens, OH, USA. The emergent applications of nanomaterials in food, cosmetics, bio-sensing, electronics, and medical products necessitates evaluation of their toxicity upon human exposure. Once inside the body, nanomaterials can interact with human cells. The cell membrane, a lipid bilayer that separates the cell from the outer environment, is the first cellular entity that ‘‘meets’’ the nanoparticles. Thus, understanding the interactions of nanoparticles with the cell membrane is expected to provide an understanding of the potential toxicity of such materials. However, despite a number of studies, a clear understanding of the mechanisms of nanoparticle-cell membrane interactions is still lacking and the role of nanoparticle physicochemical properties in such interactions remains ill-defined. In this talk, I will give an overview of my studies on the interactions of engineered nanoparticles with lipid interfaces, including lipid monolayers and bilayers representing simple models of biological membranes. Mechanisms of interactions between engineered polystyrene and silica nanoparticles and lipid interfaces will be discussed and an overview on how nanoparticle physicochemical properties might affect their interactions with lipid interfaces will be provided. 3-Subg Biomembrane Inspired Engineering Marjorie Longo. UC Davis, Davis, CA, USA. Since the invention of the optical microscope in the 17th century it has been observed that biological membranes compartmentalize the cellular machinery of life. Then, nearly 100 years ago, it was concluded, through surface science, that the cell membrane is a lipid bilayer. Since that time, many observations and theories have been put forward to try to explain how complex behavior emerges in living systems from the ubiquitous lipid bilayer structure and its integrated proteins and carbohydrates. We are now in a period of time when some of the most important paradigms posited in the last several decades for emergence of complex behavior in living cell membranes are being tested and questioned. These include the membrane (or lipid) raft hypothesis and mechanisms for generation of curvature and lipid asymmetry. An important outcome of this work may be the engineering of new micro- and nano-scale self-assembled systems and composites. This talk will focus upon our work in lipid phase behavior, crowding-induced mixing, biomembrane mechanical properties, nanoscale curvature generation, and bionanocomposites that may contribute toward the design of new biological membrane-inspired technology.
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4-Subg Physical Engineering of Behaviour and Function at the Cell and Tissue Levels Andrew Pelling. Department of Physics, University of Ottawa, Ottawa, ON, Canada. Living cells possess an exquisite ability to sense and respond to physical information in their microenvironment. Although this ability plays a key role in many fundamentally important physiological and pathological processes, it can also be exploited to control and manipulate biological behaviour and function. In recent years, the lab has become increasingly interested in created augmented biological systems by exploiting topographical, mechanical and physical cues to direct cellular organization, sorting and complex morphogenesis in three dimensions. This work has also yielded new insights into how cells respond to nano- and micro- scale physical information in highly artificial environments. I will review several projects in which cells are exposed artificial topographies to induce spontaneous cell-sorting in 3D, artificial mechanical stimuli that reveals unexpected physical properties of sub-cellular architecture, and plant-derived 3D scaffolds that can be used to create artificial hybrid mammalian tissues. These results provide insights in how key components of biological and physical feedback loops can be employed to control and govern the life of a cell. 5-Subg Optical Imaging of Protein Aggregation Reactions In Vitro and in Cells Clemens Kaminski. Dept. Chem Engineering and Biotech, Cambridge University, Cambridge, United Kingdom. The self-assembly of proteins into ordered macromolecular units is fundamental to a variety of diseases. For example, in Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), proteins that are usually harmless are found to adopt aberrant shapes; one says they ‘misfold’. In the misfolded state the proteins are prone to aggregate into highly ordered, toxic structures, called protein amyloids and these make up the insoluble deposits found in the brains of patients suffering from these devastating disorders. A key requirement to gain insights into molecular mechanisms of disease and to progress in the search for therapeutic intervention is a capability to image the protein assembly process in situ i.e. in cellular models of disease. In this talk I will give an overview of research to gain insight on the aggregation state neurotoxic proteins in vitro (1), in cells (2, 3) and in live model organisms (4). In particular, we wish to understand how these and similar proteins nucleate to form toxic structures and to correlate such information with phenotypes of disease (3). I will show how direct stochastic optical reconstruction microscopy, dSTORM, and multiparametric imaging techniques, such as spectral and lifetime imaging, are capable of tracking amyloidogenesis in vitro, and in vivo, and how we can correlate the appearance of certain aggregate species with toxic phenotypes of relevance to PD and AD (5-7). (1) Pinotsi et al, Nano Letters (2013) (2) Kaminski Schierle, et al, JACS (2011) (3) Esbjo¨rner, et al, ChemBiol (2014) (4) Kaminski Schierle, et al, ChemPhys Chem (2011) (5) Michel, et al, JBC (2014) (6) Pinotsi, et al, PNAS (2016) (7) Murakami, et al, Neuron (2015)
Mechanobiology Subgroup 6-Subg Mechanical Aspects of Mitochondrial Alterations in Apoptosis Ana J. Garcia-Saez. Membrane Biophysics, University of T€ubingen, T€ubingen, Germany. Bax permeabilization of the MOM during apoptosis proceeds via the opening of toroidal membrane pores that are large enough to allow the passage of proteins like cytochrome c. These pores are special in that lipids form part of the pore walls, where they bend to avoid exposure of the hydrophobic tails so that both monolayers form a continuous surface. As a result, toroidal pores are unstable structures whose lifetime depends on the balance between membrane tension, opening the pore, and line tension at the pore rim due to the very high lipid curvature, closing the pore. However, how Bax alters the mechanical properties of the membrane to induce the apoptotic pores remains unknown. We have shown that Bax forms large and stable pores, which are tunable in size. Moreover, it remodels membranes and stabilizes highly curved geometries. Atomic force microscopy data suggest that Bax does so by reorganizing the lipids and reducing the line tension at the pore edge. In addition, Bax accumulates at discrete foci, which are also enriched in Drp1, a dynamin-like protein responsible for mitochondrial fragmentation. Despite their relevant interplay during apoptosis, the mechanisms and mechanical consequences remain obscure. By combining FCS, superresolution microscopy and fluorescence exchange assays, we have discovered that Bax and Drp1 directly interact
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Saturday, February 11, 2017
at the MOM and that their association increases during apoptosis. We propose a new model how Bax and Drp1 mechanically cooperate to mediate mitochondrial alterations in apoptosis. 7-Subg Non-Equilibrium Phase Transitions in Actomyosin Cortices Nikta Fakhri. Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. Biological functions rely on ordered structures and intricately controlled collective dynamics. Such order in living systems is typically established and sustained by continuous dissipation of energy. The emergence of ordered patterns of motion is unique to non-equilibrium systems and is a manifestation of dynamic steady states. Many cellular processes require transitions between different steady states. Can general principles of statistical physics guide our understanding of such cellular self-organization? I will show that model actomyosin cortices, in the presence of rapid turnover, self-organize into three nonequilibrium steady states as a function of network connectivity. The different states arise from a subtle interaction between mechanical percolation of the actin network and myosin-generated stresses. All states show distinct dynamic order. Only the highest connectivity causes structural phase separation. We discover that the dominant mechanism defining the symmetries of the dynamic steady states is the emergence of ordered stress patterns. The marginally percolated state displays strong strain fluctuations, indicative of enhanced susceptibility. The striking dynamics in this model actomyosin cortex were revealed using fluorescent single-walled carbon nanotubes as novel probes. We propose self-organization of stress patterns as a new paradigm of biological function. 8-Subg Mechanical Signaling in Stem Cells: Self-Renewal and Ageing Kevin Chalut. University of Cambridge, Cambridge, United Kingdom. Stem cell culture has been characterised using soluble signals on tissue culture plastic, providing a biochemical foundation for self-renewal and differentiation. Nonetheless, most previous stem cell research has overlooked the role of the extracellular matrix (ECM) and mechanical signalling, despite increasing evidence that they both mediate self-renewal and differentiation. To investigate the role of ECM and mechanical signalling, we have developed a novel hydrogel protocol that can be mechanically tuned, ranging from embryo stiffness to skeletal stiffness, while maintaining control of ECM density. We can now present any combination of ECM molecules to cells with independent control over matrix density and stiffness. With our hydrogels, we have explored mechanical and ECM signaling in pluripotent stem cells and oligodendrocyte progenitor cells (OPCs). We have shown, in both mouse and human, that we can maintain optimal naı¨ve pluripotency using soft substrates with high ECM density, while stiff substrates with identical ECM density drives differences in the actuation of growth factor signaling pathways that drive heterogeneity and differentiation. We have also shown that we can reverse the loss of function associated with ageing and neurodegeneration in OPCs using soft substrates with high ECM density. We will present a number of functional studies and a quantitative analysis of RNA sequencing datasets to support the conclusion that mechanics is an essential regulator of stem cell identity. Ultimately, I will advance the hypothesis that mechanical sensing acts as a switch to modulate growth factor signaling to support either self-renewal or differentiation in stem cells. 9-Subg The Mechanical Control of Nervous System Development Kristian Franze. University of Cambridge, Cambridge, United Kingdom. During development and pathological processes, cells in the central nervous system (CNS) are highly motile. Despite the fact that forces are involved in any kind of cell motion, our current understanding of the mechanical interactions between CNS cells and their environment is very limited. We here investigated the mechanical control of neuronal growth in the developing brain. In vitro, growth and migration velocities, directionality, cellular forces as well as neuronal fasciculation and maturation all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, axon bundles turned towards soft substrates while glial cells migrated in the opposite direction. In vivo atomic force microscopy measurements revealed stiffness gradients in developing brain tissue, which axons followed as well towards soft. Interfering with brain stiffness and mechanosensitive ion channels in vivo both led to similar aberrant neuronal growth patterns with reduced fasciculation and pathfinding errors, strongly suggesting that neuronal growth is not only controlled by chemical signals - as it is currently assumed - but also by the tissue’s local mechanical properties.
10-Subg How do Single-Cell Properties Influence the Collective Mechanical Behavior of Confluent Tissues? Lisa Manning. Department of Physics, Syracuse University, Syracuse, NY, USA. Biological tissues involved in important processes such as embryonic development, lung function, wound healing, and cancer progression have recently been shown, via a systematic analysis of tissue mechanics and cell displacement statistics, to be close to a disordered liquid-to-solid or ‘‘jamming’’ transition. We expect these jamming transitions to be very important for biological function. For example, due to collective effects, cell migration is greatly reduced in solidlike tissues and enhanced in fluid-like tissues. Therefore, we would like to know how cells might regulate properties such as expression of cell-cell adhesion molecules and myosin activity in order to change the macroscopic properties of the tissue as a whole. I will discuss a new theoretical framework that predicts how the jamming transition is governed by single-cell mechanics and persistent cell motility. I will discuss how our a priori theoretical predictions with no fit parameters are precisely realized in cell cultures from human patients with asthma, and discuss how these ideas might also be applied in heterogeneous cell populations to understand processes in embryonic development and cancer progression. 11-Subg Mechanical Stretch Triggers Rapid Epithelial Cell Division Through the Stretch-Activated Channel Piezo1 Jody Rosenblatt. Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA. Despite acting as a barrier for the organs they encase, epithelial cells turnover at some of the fastest rates in the body. In order to maintain barrier function, cell division must be linked to cell death. How do the number of dying cells match those dividing to maintain constant numbers? We previously found that when epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. Conversely, what controls epithelial cell division to balance cell death at steady state? Here, we find that cell division occurs in regions of low cell density, where epithelial cells are stretched. Stretching epithelia either mechanically or by wounding causes cells to rapidly enter mitosis and this response also requires Piezo1. Stretchactivation of Piezo1 triggers a population of epithelial cells in early G2 that are poised for repair to produce cyclin B and enter mitosis following stretch. We find that Piezo1 localization varies depending upon epithelial cell density. In sparse regions, Piezo1 localizes to the plasma membrane, where it can mechanically sense stretch tensions to activate calcium currents. In cell dense regions, Piezo1 localizes to large cytoplasmic aggregates where it may better sense crowding to induce extrusion. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers by driving extrusion and apoptosis when cells are too crowded and cell division when they are too sparse.
Bioenergetics Subgroup 12-Subg Near-Neighbor Relationships of the Atypical Subunits that Form the Peripheral Stalk of the Mitochondrial ATP Synthase in Chlorophycean Algae Miriam Va´zquez-Acevedo1, Fe´lix Vega-DeLuna1, Lorenzo Sa´nchezVa´squez1, Lilia Colina-Tenorio1, Claire Remacle2, Pierre Cardol2, He´ctor Miranda-Astudillo2, Diego Gonzalez-Halphen1. 1 Institute of Cellular Physiology, National Autonomous University of Mexico, Mexico City, Mexico, 2Department of Life Science, University of Lie`ge, Lie`ge, Belgium. Mitochondrial F1Fo-ATP synthase (complex V) is an oligomeric complex that exhibits a molecular mass around 550 kDa. The F1Fo-ATP synthase of the green chlorophycean alga Chlamydomonas reinhardtii and of its close colorless relative Polytomella sp. is isolated as a highly-stable dimer of 1600 kDa after solubilization of algal mitochondria with lauryl-maltoside. Each monomer of the enzyme is constituted by 17 polypeptides, eight of which are the conserved subunits alpha, beta, gamma, delta, epsilon, a (Atp6), c (Atp9), and OSCP, plus nine atypical polypeptides, named Asa1 to Asa9, that seem to be present only in the chlorophycean lineage. The Asa subunits (named after ATP Synthase Associated subunits) form the very robust peripheral stalk that has been observed in several electron-microscope studies of the algal enzyme. We have addressed the close-neighbor relationships of the ASA subunits and their interactions with the orthodox, conserved subunits. For this purpose, we have generated sub-complexes after partial dissociation of the dimeric ATP synthase with high detergent concentrations or with amphiphilic polymers; detected several subunit-subunit interactions based on cross-linking experiments; reconstituted
Bioengineering Subgroup 1-Subg Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting Isaac T.S. Li1, Yann R. Chemla2, Taekjip Ha3,4. 1 Chemistry, University of British Columbia, Kelowna, BC, Canada, 2 Department of Physics and Centre for Physics of Living Cells, University of Illinois Urbana-Champaign, Urbana, IL, USA, 3Department of Biophysics and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA, 4Howard Hughes Medical Institute, Baltimore, MD, USA. Rolling adhesion is the behaviour that leukocytes and circulating tumour cells exhibit as they passively roll along blood vessel walls under flow. It plays a critical role in capturing cells in the blood, guiding them toward inflammation sites, and activating cell signalling pathways to enable their subsequent transmigration. Rolling adhesion is mediated by catch-bond-like interactions between selectins expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 found at microvilli tips of leukocytes. Despite our understanding of individual components of this process, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behaviour remains poorly understood. Here, we developed 2 label-free methods that map the functional adhesion sites and their strength on a leukocyte surface. The first method relies on tracking the rotational angle of a single rolling cell, which confers advantages over standard methods that track the centre-of-mass alone. Constructing the adhesion map from the instantaneous angular velocity reveals that the adhesion profile along the rolling circumference is inhomogeneous. We corroborated these findings with a second method that allowed us to obtain a footprint of molecular adhesion events using DNA-based molecular force probes. Our results reveal that adhesion at the functional level is not uniformly distributed over the leukocyte surface as previously assumed, but is instead patchy. 2-Subg Interactions of Engineered Nanomaterials with Lipid Interfaces Amir Farnoud. Chemical and Biomolecular Engineering, Ohio University, Athens, OH, USA. The emergent applications of nanomaterials in food, cosmetics, bio-sensing, electronics, and medical products necessitates evaluation of their toxicity upon human exposure. Once inside the body, nanomaterials can interact with human cells. The cell membrane, a lipid bilayer that separates the cell from the outer environment, is the first cellular entity that ‘‘meets’’ the nanoparticles. Thus, understanding the interactions of nanoparticles with the cell membrane is expected to provide an understanding of the potential toxicity of such materials. However, despite a number of studies, a clear understanding of the mechanisms of nanoparticle-cell membrane interactions is still lacking and the role of nanoparticle physicochemical properties in such interactions remains ill-defined. In this talk, I will give an overview of my studies on the interactions of engineered nanoparticles with lipid interfaces, including lipid monolayers and bilayers representing simple models of biological membranes. Mechanisms of interactions between engineered polystyrene and silica nanoparticles and lipid interfaces will be discussed and an overview on how nanoparticle physicochemical properties might affect their interactions with lipid interfaces will be provided. 3-Subg Biomembrane Inspired Engineering Marjorie Longo. UC Davis, Davis, CA, USA. Since the invention of the optical microscope in the 17th century it has been observed that biological membranes compartmentalize the cellular machinery of life. Then, nearly 100 years ago, it was concluded, through surface science, that the cell membrane is a lipid bilayer. Since that time, many observations and theories have been put forward to try to explain how complex behavior emerges in living systems from the ubiquitous lipid bilayer structure and its integrated proteins and carbohydrates. We are now in a period of time when some of the most important paradigms posited in the last several decades for emergence of complex behavior in living cell membranes are being tested and questioned. These include the membrane (or lipid) raft hypothesis and mechanisms for generation of curvature and lipid asymmetry. An important outcome of this work may be the engineering of new micro- and nano-scale self-assembled systems and composites. This talk will focus upon our work in lipid phase behavior, crowding-induced mixing, biomembrane mechanical properties, nanoscale curvature generation, and bionanocomposites that may contribute toward the design of new biological membrane-inspired technology.
1a
4-Subg Physical Engineering of Behaviour and Function at the Cell and Tissue Levels Andrew Pelling. Department of Physics, University of Ottawa, Ottawa, ON, Canada. Living cells possess an exquisite ability to sense and respond to physical information in their microenvironment. Although this ability plays a key role in many fundamentally important physiological and pathological processes, it can also be exploited to control and manipulate biological behaviour and function. In recent years, the lab has become increasingly interested in created augmented biological systems by exploiting topographical, mechanical and physical cues to direct cellular organization, sorting and complex morphogenesis in three dimensions. This work has also yielded new insights into how cells respond to nano- and micro- scale physical information in highly artificial environments. I will review several projects in which cells are exposed artificial topographies to induce spontaneous cell-sorting in 3D, artificial mechanical stimuli that reveals unexpected physical properties of sub-cellular architecture, and plant-derived 3D scaffolds that can be used to create artificial hybrid mammalian tissues. These results provide insights in how key components of biological and physical feedback loops can be employed to control and govern the life of a cell. 5-Subg Optical Imaging of Protein Aggregation Reactions In Vitro and in Cells Clemens Kaminski. Dept. Chem Engineering and Biotech, Cambridge University, Cambridge, United Kingdom. The self-assembly of proteins into ordered macromolecular units is fundamental to a variety of diseases. For example, in Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), proteins that are usually harmless are found to adopt aberrant shapes; one says they ‘misfold’. In the misfolded state the proteins are prone to aggregate into highly ordered, toxic structures, called protein amyloids and these make up the insoluble deposits found in the brains of patients suffering from these devastating disorders. A key requirement to gain insights into molecular mechanisms of disease and to progress in the search for therapeutic intervention is a capability to image the protein assembly process in situ i.e. in cellular models of disease. In this talk I will give an overview of research to gain insight on the aggregation state neurotoxic proteins in vitro (1), in cells (2, 3) and in live model organisms (4). In particular, we wish to understand how these and similar proteins nucleate to form toxic structures and to correlate such information with phenotypes of disease (3). I will show how direct stochastic optical reconstruction microscopy, dSTORM, and multiparametric imaging techniques, such as spectral and lifetime imaging, are capable of tracking amyloidogenesis in vitro, and in vivo, and how we can correlate the appearance of certain aggregate species with toxic phenotypes of relevance to PD and AD (5-7). (1) Pinotsi et al, Nano Letters (2013) (2) Kaminski Schierle, et al, JACS (2011) (3) Esbjo¨rner, et al, ChemBiol (2014) (4) Kaminski Schierle, et al, ChemPhys Chem (2011) (5) Michel, et al, JBC (2014) (6) Pinotsi, et al, PNAS (2016) (7) Murakami, et al, Neuron (2015)
Mechanobiology Subgroup 6-Subg Mechanical Aspects of Mitochondrial Alterations in Apoptosis Ana J. Garcia-Saez. Membrane Biophysics, University of T€ubingen, T€ubingen, Germany. Bax permeabilization of the MOM during apoptosis proceeds via the opening of toroidal membrane pores that are large enough to allow the passage of proteins like cytochrome c. These pores are special in that lipids form part of the pore walls, where they bend to avoid exposure of the hydrophobic tails so that both monolayers form a continuous surface. As a result, toroidal pores are unstable structures whose lifetime depends on the balance between membrane tension, opening the pore, and line tension at the pore rim due to the very high lipid curvature, closing the pore. However, how Bax alters the mechanical properties of the membrane to induce the apoptotic pores remains unknown. We have shown that Bax forms large and stable pores, which are tunable in size. Moreover, it remodels membranes and stabilizes highly curved geometries. Atomic force microscopy data suggest that Bax does so by reorganizing the lipids and reducing the line tension at the pore edge. In addition, Bax accumulates at discrete foci, which are also enriched in Drp1, a dynamin-like protein responsible for mitochondrial fragmentation. Despite their relevant interplay during apoptosis, the mechanisms and mechanical consequences remain obscure. By combining FCS, superresolution microscopy and fluorescence exchange assays, we have discovered that Bax and Drp1 directly interact
2a
Saturday, February 11, 2017
at the MOM and that their association increases during apoptosis. We propose a new model how Bax and Drp1 mechanically cooperate to mediate mitochondrial alterations in apoptosis. 7-Subg Non-Equilibrium Phase Transitions in Actomyosin Cortices Nikta Fakhri. Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. Biological functions rely on ordered structures and intricately controlled collective dynamics. Such order in living systems is typically established and sustained by continuous dissipation of energy. The emergence of ordered patterns of motion is unique to non-equilibrium systems and is a manifestation of dynamic steady states. Many cellular processes require transitions between different steady states. Can general principles of statistical physics guide our understanding of such cellular self-organization? I will show that model actomyosin cortices, in the presence of rapid turnover, self-organize into three nonequilibrium steady states as a function of network connectivity. The different states arise from a subtle interaction between mechanical percolation of the actin network and myosin-generated stresses. All states show distinct dynamic order. Only the highest connectivity causes structural phase separation. We discover that the dominant mechanism defining the symmetries of the dynamic steady states is the emergence of ordered stress patterns. The marginally percolated state displays strong strain fluctuations, indicative of enhanced susceptibility. The striking dynamics in this model actomyosin cortex were revealed using fluorescent single-walled carbon nanotubes as novel probes. We propose self-organization of stress patterns as a new paradigm of biological function. 8-Subg Mechanical Signaling in Stem Cells: Self-Renewal and Ageing Kevin Chalut. University of Cambridge, Cambridge, United Kingdom. Stem cell culture has been characterised using soluble signals on tissue culture plastic, providing a biochemical foundation for self-renewal and differentiation. Nonetheless, most previous stem cell research has overlooked the role of the extracellular matrix (ECM) and mechanical signalling, despite increasing evidence that they both mediate self-renewal and differentiation. To investigate the role of ECM and mechanical signalling, we have developed a novel hydrogel protocol that can be mechanically tuned, ranging from embryo stiffness to skeletal stiffness, while maintaining control of ECM density. We can now present any combination of ECM molecules to cells with independent control over matrix density and stiffness. With our hydrogels, we have explored mechanical and ECM signaling in pluripotent stem cells and oligodendrocyte progenitor cells (OPCs). We have shown, in both mouse and human, that we can maintain optimal naı¨ve pluripotency using soft substrates with high ECM density, while stiff substrates with identical ECM density drives differences in the actuation of growth factor signaling pathways that drive heterogeneity and differentiation. We have also shown that we can reverse the loss of function associated with ageing and neurodegeneration in OPCs using soft substrates with high ECM density. We will present a number of functional studies and a quantitative analysis of RNA sequencing datasets to support the conclusion that mechanics is an essential regulator of stem cell identity. Ultimately, I will advance the hypothesis that mechanical sensing acts as a switch to modulate growth factor signaling to support either self-renewal or differentiation in stem cells. 9-Subg The Mechanical Control of Nervous System Development Kristian Franze. University of Cambridge, Cambridge, United Kingdom. During development and pathological processes, cells in the central nervous system (CNS) are highly motile. Despite the fact that forces are involved in any kind of cell motion, our current understanding of the mechanical interactions between CNS cells and their environment is very limited. We here investigated the mechanical control of neuronal growth in the developing brain. In vitro, growth and migration velocities, directionality, cellular forces as well as neuronal fasciculation and maturation all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, axon bundles turned towards soft substrates while glial cells migrated in the opposite direction. In vivo atomic force microscopy measurements revealed stiffness gradients in developing brain tissue, which axons followed as well towards soft. Interfering with brain stiffness and mechanosensitive ion channels in vivo both led to similar aberrant neuronal growth patterns with reduced fasciculation and pathfinding errors, strongly suggesting that neuronal growth is not only controlled by chemical signals - as it is currently assumed - but also by the tissue’s local mechanical properties.
10-Subg How do Single-Cell Properties Influence the Collective Mechanical Behavior of Confluent Tissues? Lisa Manning. Department of Physics, Syracuse University, Syracuse, NY, USA. Biological tissues involved in important processes such as embryonic development, lung function, wound healing, and cancer progression have recently been shown, via a systematic analysis of tissue mechanics and cell displacement statistics, to be close to a disordered liquid-to-solid or ‘‘jamming’’ transition. We expect these jamming transitions to be very important for biological function. For example, due to collective effects, cell migration is greatly reduced in solidlike tissues and enhanced in fluid-like tissues. Therefore, we would like to know how cells might regulate properties such as expression of cell-cell adhesion molecules and myosin activity in order to change the macroscopic properties of the tissue as a whole. I will discuss a new theoretical framework that predicts how the jamming transition is governed by single-cell mechanics and persistent cell motility. I will discuss how our a priori theoretical predictions with no fit parameters are precisely realized in cell cultures from human patients with asthma, and discuss how these ideas might also be applied in heterogeneous cell populations to understand processes in embryonic development and cancer progression. 11-Subg Mechanical Stretch Triggers Rapid Epithelial Cell Division Through the Stretch-Activated Channel Piezo1 Jody Rosenblatt. Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA. Despite acting as a barrier for the organs they encase, epithelial cells turnover at some of the fastest rates in the body. In order to maintain barrier function, cell division must be linked to cell death. How do the number of dying cells match those dividing to maintain constant numbers? We previously found that when epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. Conversely, what controls epithelial cell division to balance cell death at steady state? Here, we find that cell division occurs in regions of low cell density, where epithelial cells are stretched. Stretching epithelia either mechanically or by wounding causes cells to rapidly enter mitosis and this response also requires Piezo1. Stretchactivation of Piezo1 triggers a population of epithelial cells in early G2 that are poised for repair to produce cyclin B and enter mitosis following stretch. We find that Piezo1 localization varies depending upon epithelial cell density. In sparse regions, Piezo1 localizes to the plasma membrane, where it can mechanically sense stretch tensions to activate calcium currents. In cell dense regions, Piezo1 localizes to large cytoplasmic aggregates where it may better sense crowding to induce extrusion. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers by driving extrusion and apoptosis when cells are too crowded and cell division when they are too sparse.
Bioenergetics Subgroup 12-Subg Near-Neighbor Relationships of the Atypical Subunits that Form the Peripheral Stalk of the Mitochondrial ATP Synthase in Chlorophycean Algae Miriam Va´zquez-Acevedo1, Fe´lix Vega-DeLuna1, Lorenzo Sa´nchezVa´squez1, Lilia Colina-Tenorio1, Claire Remacle2, Pierre Cardol2, He´ctor Miranda-Astudillo2, Diego Gonzalez-Halphen1. 1 Institute of Cellular Physiology, National Autonomous University of Mexico, Mexico City, Mexico, 2Department of Life Science, University of Lie`ge, Lie`ge, Belgium. Mitochondrial F1Fo-ATP synthase (complex V) is an oligomeric complex that exhibits a molecular mass around 550 kDa. The F1Fo-ATP synthase of the green chlorophycean alga Chlamydomonas reinhardtii and of its close colorless relative Polytomella sp. is isolated as a highly-stable dimer of 1600 kDa after solubilization of algal mitochondria with lauryl-maltoside. Each monomer of the enzyme is constituted by 17 polypeptides, eight of which are the conserved subunits alpha, beta, gamma, delta, epsilon, a (Atp6), c (Atp9), and OSCP, plus nine atypical polypeptides, named Asa1 to Asa9, that seem to be present only in the chlorophycean lineage. The Asa subunits (named after ATP Synthase Associated subunits) form the very robust peripheral stalk that has been observed in several electron-microscope studies of the algal enzyme. We have addressed the close-neighbor relationships of the ASA subunits and their interactions with the orthodox, conserved subunits. For this purpose, we have generated sub-complexes after partial dissociation of the dimeric ATP synthase with high detergent concentrations or with amphiphilic polymers; detected several subunit-subunit interactions based on cross-linking experiments; reconstituted