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Visualizing the Competition between Gs and Gi Signaling at the Membrane
Saturday, February 11, 2017 47-Subg Structural Mechanisms of Mechanosensitivity in the TREK-2 K2P Potassium Channel Stephen J. Tucker. Department of Physics, University of Oxford, Oxford, United Kingdom. Mechanosensitive ion channels are gated open and shut by changes in membrane tension enabling the conversion of mechanical stimuli into changes in ionic composition and electrical signalling. However, the precise structural and biophysical mechanisms underlying these processes remain unclear. TREK-2 is a eukaryotic mechanosensitive ion channel that belongs to the Two-Pore Domain (K2P) family of Kþ channels and is widely expressed in both the central and peripheral nervous system. I will discuss our recent structural, functional and computational studies which examine how TREK-2 responds to stretch-induced changes in the large lateral forces that vary with depth across the bilayer, and in particular how the asymmetric structure of the channel contributes to this process. 48-Subg Membrane Mechanosensors Responsible for Touch and other Senses Miriam B. Goodman. Molecular & Cellular Physiology, Stanford University, Stanford, CA, USA. For decades, we have understood that ion channels are the first responders of touch sensation and other mechanical senses—converting the mechanical energy delivered in by sound, or by a touch, or by the bend of a limb into neural signals. Research in my group and others has identified at least three classes of proteins that form these so-called mechano-electrical transduction (MeT) channels in mammals and invertebrates: DEG/ ENaC sodium channels, TRP cation channels, and Piezo cation channels. We are working to increase knowledge of how MeT channels depend on the plasma membrane for function and the physics of force transfer. My talk will survey prior knowledge and discuss recent investigations combining the tools of genetic dissection with behavioral analysis and electrical recording from identified neurons to the biophysics of in vivo MeT channel activation using C. elegans nematodes as a tractable model. 49-Subg Visualizing the Competition between Gs and Gi Signaling at the Membrane Thomas Hughes. Montana Molecular, Bozeman, MT, USA. At the cell membrane, G-protein coupled receptors couple to a variety of signaling pathways. The Gs and Gi pathways compete to stimulate, or inhibit, adenyl cyclases at the membrane, regulating the levels of cAMP within the cell. Our goal was to create a genetically encoded, fluorescent biosensor to detect, in real time, this competition. We created a new generation of the cADDis cAMP biosensor which is remarkably bright and can be used to detect Gs signaling kinetics in fluorescence microscopes and fluorescence plate readers with a Z’ statistic of 0.9 or greater. The next step was to determine if cADDis could be used to detect Gi signaling. A variety of Gi-coupled receptors were expressed in HEK293 cells, but known agonists produced no detectable change in cADDis fluorescence. Reasoning that this was due to low basal adenylyl cyclase activity, we increased Gs signaling using the controlled coexpression of a constitutively active Gs mutant. In this context, the cADDis sensor showed large changes in fluorescence, reported EC50 values consistent with literature, and yielded high Z’ values in a standard automated fluorescence plate reader. To test whether cADDis can detect the competition between Gs and Gi, we first stimulated a Gs pathway and then followed with Gi-coupled receptor activation. The cADDis sensor reports the Gi mediated inhibition of the Gs response. The opposite experiment works as well. While the competition between Gs and Gi is easily measured, the competition between Forskolin and the Gi pathway produces inconsistent results with different kinetics. This was particularly true for the Adenosine 1 and D2-dopaminergic receptors, which produced robust Gi responses in HEK293 cells when cAMP was elevated with the Gs mutant or isoproterenol, but failed to produce a response in the presence of forskolin.
Membrane Structure and Assembly Subgroup 50-Subg Phases and Fluctuations in Biological Membranes Matthew B. Stone, Sarah A. Shelby, Marcos Nunez, Sarah Veatch. University of Michigan, Ann Arbor, MI, USA. Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma mem-
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brane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction. 51-Subg The Role of Cholesterol in Viral Spike Glycoprotein-Mediated Membrane Fusion Jinwoo Lee, Sung-tae Yang, Volker Kiessling, Lukas K. Tamm. Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA. Viral spike glycoproteins such as influenza HA, HIV gp120/gp41, and Ebola virus (EBOV) GP1/GP2 undergo major conformational transitions to facilitate cell entry of these viruses by membrane fusion. Moderately hydrophobic fusion peptides [or fusion loops (FL) in the case of EBOV] are protected by these proteins in their pre-fusion states, but become exposed and insert into cellular target membranes during fusion. We have solved the structures of the EBOV FL, membrane proximal external region (MPER) and transmembrane (TM) domains in membrane environments by NMR and studied their molecular interactions by NMR and fluorescence. A structural model of how these domains cooperate to open a fusion pore emerges from these studies. Viral membrane fusion is modulated by cholesterol in the viral and target cell membranes. Interestingly, in the case of HIV gp41-mediated fusion, membranes that consist of co-existing liquid-ordered (Lo) and liquid-disordered (Ld) bilayer domains are more fusogenic than either pure Ld or Lo lipid bilayers and lipid bilayer phase co-existence in both the viral and the target membrane synergistically helps fusion. Single particle experiments on supported bilayers and giant unilamellar vesicles show that the boundaries between the Lo and Ld regions are the sites of virosome and pseudovirus docking and fusion. It appears that line-tension that characterizes lipid phase boundaries is a major driving force of membrane fusion in cell entry of HIV and perhaps other enveloped viruses. Although these studies were performed using model systems with optically resolved lipid phase separations, it is plausible and likely that similar mechanisms operate in cell membranes that are also heterogeneous in lipid and protein composition and therefore also contain domain boundaries that are more frequent, finer grained, and more transient than in our model systems. 52-Subg Self-Organization and Dynamics of the Actin Cortex-Membrane Interface Gijsje Koenderink. FOM Institute AMOLF, Amsterdam, Netherlands. The main determinants of cell shape in mammalian cells are the plasma membrane and a thin polymer gel beneath it that is known as the actin cortex. The cortex controls the rigidity and mechanical stability of the cell surface and drives shape changes by means of myosin motors that contract the cortex. The actin cortex is linked to the cell membrane by noncovalent interactions, mediated by linker proteins that bind actin filaments to acidic phosphatidyl inositide (PIP) lipids. There is growing evidence that these interactions enable the membrane and the cortex to affect each other’s molecular organization. But given the molecular complexity of the cortexmembrane interface, it has been difficult to delineate the molecular mechanisms that mediate cortex-membrane crosstalk. To address this question, we build minimal cell models composed of planar lipid bilayers coupled to an active actin-myosin cortex. By integrating the bilayers with microfluidic flow devices we can sequentially flow in different cortical proteins and thus test their interdependence in forming a functional cortex. The 2D geometry of supported bilayers enables us to perform time-lapse imaging by total internal reflection microscopy and quantitative measurements of proteinmembrane binding by surface analytical techniques (QCM-d and ellipsometry). I will show that the composition of the membrane regulates the spatial organization and contractility of the cortex, while conversely, the cortex can influence membrane organization by sequestering PIP lipids and by restricting lipid diffusion.
Membrane Structure and Assembly Subgroup 50-Subg Phases and Fluctuations in Biological Membranes Matthew B. Stone, Sarah A. Shelby, Marcos Nunez, Sarah Veatch. University of Michigan, Ann Arbor, MI, USA. Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma mem-
9a
brane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction. 51-Subg The Role of Cholesterol in Viral Spike Glycoprotein-Mediated Membrane Fusion Jinwoo Lee, Sung-tae Yang, Volker Kiessling, Lukas K. Tamm. Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA. Viral spike glycoproteins such as influenza HA, HIV gp120/gp41, and Ebola virus (EBOV) GP1/GP2 undergo major conformational transitions to facilitate cell entry of these viruses by membrane fusion. Moderately hydrophobic fusion peptides [or fusion loops (FL) in the case of EBOV] are protected by these proteins in their pre-fusion states, but become exposed and insert into cellular target membranes during fusion. We have solved the structures of the EBOV FL, membrane proximal external region (MPER) and transmembrane (TM) domains in membrane environments by NMR and studied their molecular interactions by NMR and fluorescence. A structural model of how these domains cooperate to open a fusion pore emerges from these studies. Viral membrane fusion is modulated by cholesterol in the viral and target cell membranes. Interestingly, in the case of HIV gp41-mediated fusion, membranes that consist of co-existing liquid-ordered (Lo) and liquid-disordered (Ld) bilayer domains are more fusogenic than either pure Ld or Lo lipid bilayers and lipid bilayer phase co-existence in both the viral and the target membrane synergistically helps fusion. Single particle experiments on supported bilayers and giant unilamellar vesicles show that the boundaries between the Lo and Ld regions are the sites of virosome and pseudovirus docking and fusion. It appears that line-tension that characterizes lipid phase boundaries is a major driving force of membrane fusion in cell entry of HIV and perhaps other enveloped viruses. Although these studies were performed using model systems with optically resolved lipid phase separations, it is plausible and likely that similar mechanisms operate in cell membranes that are also heterogeneous in lipid and protein composition and therefore also contain domain boundaries that are more frequent, finer grained, and more transient than in our model systems. 52-Subg Self-Organization and Dynamics of the Actin Cortex-Membrane Interface Gijsje Koenderink. FOM Institute AMOLF, Amsterdam, Netherlands. The main determinants of cell shape in mammalian cells are the plasma membrane and a thin polymer gel beneath it that is known as the actin cortex. The cortex controls the rigidity and mechanical stability of the cell surface and drives shape changes by means of myosin motors that contract the cortex. The actin cortex is linked to the cell membrane by noncovalent interactions, mediated by linker proteins that bind actin filaments to acidic phosphatidyl inositide (PIP) lipids. There is growing evidence that these interactions enable the membrane and the cortex to affect each other’s molecular organization. But given the molecular complexity of the cortexmembrane interface, it has been difficult to delineate the molecular mechanisms that mediate cortex-membrane crosstalk. To address this question, we build minimal cell models composed of planar lipid bilayers coupled to an active actin-myosin cortex. By integrating the bilayers with microfluidic flow devices we can sequentially flow in different cortical proteins and thus test their interdependence in forming a functional cortex. The 2D geometry of supported bilayers enables us to perform time-lapse imaging by total internal reflection microscopy and quantitative measurements of proteinmembrane binding by surface analytical techniques (QCM-d and ellipsometry). I will show that the composition of the membrane regulates the spatial organization and contractility of the cortex, while conversely, the cortex can influence membrane organization by sequestering PIP lipids and by restricting lipid diffusion.