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T-Cells in Suspension Do Not Show Pre-Clustered LCK
570a
Wednesday, March 2, 2016
We find that acidic environment decreases the binding affinity of membranereconstituted CD47 to SIRPa. Finally, we discuss implications for in-vitro cancer models. Financial support from the Deutsche Forschungsgemeinschaft (DFG) via the IRTG 1524 is gratefully acknowledged. 2815-Pos Board B192 T-Cells in Suspension Do Not Show Pre-Clustered LCK Jorge Bernardino de la Serna1, Veronica T. Chang2, Dominic Waithe3, Ricardo A. Fernandes3, Marco Fritzsche3, Ana Mafalda Santos3, Dilip Shrestha3, James H. Felce3, Meike C. Assmann3, Simon J. Davis3, Christian Eggeling3. 1 Rutherford Appleton Laboratory, Science and Technology Facilities Council, Harwell-Oxford, United Kingdom, 2Wellcome Trust for Human Genetics, University of Oxford, Oxford, United Kingdom, 3MRC-Human Immunology Unit, University of Oxford, Oxford, United Kingdom. Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into ‘‘nano-domains’’ in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching nonactivating surfaces probably don’t resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels. 2816-Pos Board B193 Single-Molecule Fluorescence Imaging to Determine the Stoichiometry of the Twin-Arginine Translocase Hajra Basit1, Felicity Alcock2, Ben Berks2, Mark I. Wallace1. 1 Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom, 2Department of Biochemistry, University of Oxford, Oxford, United Kingdom. The twin-arginine translocation (Tat) machinery transports folded substrate proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. This mechanistically challenging machinery deploys three essential proteins, TatA, TatB, and TatC. TatA is known to form the protein translocating element of the Tat system. The size and the stoichiometry of the Tat translocation complex are thought to be dependent on the size of the substrate. Current models for Tat transport only predict the oligomeric state of TatA and if, and whether, this state changes during the transport cycle. We use single-molecule fluorescence techniques to probe into the stoichiometry of the translocase. 2817-Pos Board B194 DC-SIGN Mediated Dengue Virus Entry into Cells Kenneth Jacobson, Ping Liu, Marc Ridilla, Laurie Betts, Aravinda de Silva, Nancy L. Thompson. University of North Carolina, Chapel Hill, NC, USA. DC-SIGN (a single-pass, C-type lectin, transmembrane protein) is a receptor for dengue virus (DENV) and variety of other pathogens. It exists in small clusters on the plasma membranes of dendritic and other immune cells. Using NIH3T3 cells that express full length DC-SIGN or a mutant form lacking the cytoplasmic tail as well as primary human immature dendritic cells, we investigated DENV binding, surface transport, endocytosis and infection. We employed confocal microscopy, super-resolution imaging and
large-scale single particle tracking. After binding, the virus/receptor complexes co-migrate to clathrin coated pits where the complexes are endocytosed followed by release of the viral genome for replication. Suprisingly, dengue binding, even to a few DC-SIGN clusters, induces a global cellular response in which both loaded and unloaded DC-SIGN clusters exhibit dramatically increased lateral mobility, possibly to enhance encounters with coated pits. In addition, a small but significant fraction of DC-SIGN clusters in the plasma membrane undergo rapid, microtubule-based directed transport towards the cell center at velocities which can exceed 1mm/s. Since the measured velocities are increased after dengue binding, this activity may be required to bring captured pathogens from the leading margins back to perinuclear zone for processing by dendritic cells. A controversy exists as to whether DC-SIGN functions simply as an attachment factor for dengue viruses or whether it plays additional roles in viral entry. The absence of the DC-SIGN cytoplasmic region reduced both dengue binding and endocytosis. However, infection still occurred, albeit reduced, suggesting that DC-SIGN must be able to act in concert with a co-receptor containing cell entry motifs. Supported by NIH GM41402. 2818-Pos Board B195 Effect of Dengue Fusion Peptide in Langmuir Monolayers Thaı´s F. Schmidt1,2, Christian Salesse1, Karin A. Riske2. 1 Centre de recherche du CHU de Que´bec, De´partement d’ophtalmologie and Faculte´ de me´decine, Universite´ Laval, Que´bec, QC, Canada, 2Department of Biophysics, Universidade Federal de Sa˜o Paulo - UNIFESP, Sa˜o Paulo, Brazil. Membrane fusion peptides are part of Dengue virus system, serving as a gateway to the virus access into the cell. Studies on the mechanisms of action of these peptides to improve protocols for the treatment and achievement of new drugs to combat the dissemination of the virus are increasingly necessary. The Langmuir technique is very useful to study peptide-lipid interactions. Indeed, the utilization of lipid monolayers allows controlling several physical parameters such as lipid composition, surface pressure and many other parameters of interest. Here we study the interaction of the Dengue Flavivirus fusion peptide (FLAg) with Langmuir monolayers composed of different types of lipids, including POPG, (palmitoyl-oleoyl-phosphatydylglycerol). In order to assess peptide maximum insertion pressure (MIP) into the Langmuir model membrane, kinetic measurements were performed. The kinetics of FLAg binding onto the phospholipid monolayer was monitored until the equilibrium surface pressure (Pe) was reached. The MIP was determined by injecting FLAg at different initial surface pressures (Pi) of the lipid monolayers. To investigate the behavior of FLAg in Langmuir monolayers with different lipid composition, we also used in situ infrared spectroscopy (PM-IRRAS). Preliminary results showed an increase in surface pressure during adsorption kinetics experiments with POPG monolayers, revealing an electrostatic affinity that evidences the role of the membrane charge in the fusion process. Furthermore, surface activity of the peptide solution used as the subphase in the absence of lipid monolayers was also measured. PM-IRRAS measurements reveal how the peptide incorporates at the interface of POPG monolayers, showing changes in the spectra, besides indicating characteristic due to the presence of FLAg. Altogether, these data clearly demonstrate that useful information on the binding FLAg to membranes can be obtained by performing measurements of their monolayer binding parameters. 2819-Pos Board B196 Directing Membrane Pore and Stalk Formation in MD Simulations with Embedded Mechanical Devices Gregory Bubnis, Helmut Grubmuller. Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. Stalk and pore formation are two well-known examples of the membrane topological changes required in a host of cellular processes. Their intermediate states, however, involve rearrangements of only a few lipid and solvent molecules, making them hard to observe experimentally. Molecular simulations have suitable temporal/spatial resolution to observe these transitions and have suggested stalk(pore) formation paths where a hydrophobic(philic) bridge nucleates across a hydrophilic(phobic) slab. However, most simulations require biasing schemes that are susceptible to hysteresis near the topological transitions. Here we present a simulation method implementing a biasing scheme to reversibly direct pore formation and closure using a membrane embedded ‘mechanical device’. This ‘pore device’ biasing potential can be adjusted to open, close or restrain a pore, permitting exhaustive and rigorous sampling of intermediate states such as asymmetrical indentations and water wires
Wednesday, March 2, 2016
We find that acidic environment decreases the binding affinity of membranereconstituted CD47 to SIRPa. Finally, we discuss implications for in-vitro cancer models. Financial support from the Deutsche Forschungsgemeinschaft (DFG) via the IRTG 1524 is gratefully acknowledged. 2815-Pos Board B192 T-Cells in Suspension Do Not Show Pre-Clustered LCK Jorge Bernardino de la Serna1, Veronica T. Chang2, Dominic Waithe3, Ricardo A. Fernandes3, Marco Fritzsche3, Ana Mafalda Santos3, Dilip Shrestha3, James H. Felce3, Meike C. Assmann3, Simon J. Davis3, Christian Eggeling3. 1 Rutherford Appleton Laboratory, Science and Technology Facilities Council, Harwell-Oxford, United Kingdom, 2Wellcome Trust for Human Genetics, University of Oxford, Oxford, United Kingdom, 3MRC-Human Immunology Unit, University of Oxford, Oxford, United Kingdom. Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into ‘‘nano-domains’’ in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching nonactivating surfaces probably don’t resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels. 2816-Pos Board B193 Single-Molecule Fluorescence Imaging to Determine the Stoichiometry of the Twin-Arginine Translocase Hajra Basit1, Felicity Alcock2, Ben Berks2, Mark I. Wallace1. 1 Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom, 2Department of Biochemistry, University of Oxford, Oxford, United Kingdom. The twin-arginine translocation (Tat) machinery transports folded substrate proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. This mechanistically challenging machinery deploys three essential proteins, TatA, TatB, and TatC. TatA is known to form the protein translocating element of the Tat system. The size and the stoichiometry of the Tat translocation complex are thought to be dependent on the size of the substrate. Current models for Tat transport only predict the oligomeric state of TatA and if, and whether, this state changes during the transport cycle. We use single-molecule fluorescence techniques to probe into the stoichiometry of the translocase. 2817-Pos Board B194 DC-SIGN Mediated Dengue Virus Entry into Cells Kenneth Jacobson, Ping Liu, Marc Ridilla, Laurie Betts, Aravinda de Silva, Nancy L. Thompson. University of North Carolina, Chapel Hill, NC, USA. DC-SIGN (a single-pass, C-type lectin, transmembrane protein) is a receptor for dengue virus (DENV) and variety of other pathogens. It exists in small clusters on the plasma membranes of dendritic and other immune cells. Using NIH3T3 cells that express full length DC-SIGN or a mutant form lacking the cytoplasmic tail as well as primary human immature dendritic cells, we investigated DENV binding, surface transport, endocytosis and infection. We employed confocal microscopy, super-resolution imaging and
large-scale single particle tracking. After binding, the virus/receptor complexes co-migrate to clathrin coated pits where the complexes are endocytosed followed by release of the viral genome for replication. Suprisingly, dengue binding, even to a few DC-SIGN clusters, induces a global cellular response in which both loaded and unloaded DC-SIGN clusters exhibit dramatically increased lateral mobility, possibly to enhance encounters with coated pits. In addition, a small but significant fraction of DC-SIGN clusters in the plasma membrane undergo rapid, microtubule-based directed transport towards the cell center at velocities which can exceed 1mm/s. Since the measured velocities are increased after dengue binding, this activity may be required to bring captured pathogens from the leading margins back to perinuclear zone for processing by dendritic cells. A controversy exists as to whether DC-SIGN functions simply as an attachment factor for dengue viruses or whether it plays additional roles in viral entry. The absence of the DC-SIGN cytoplasmic region reduced both dengue binding and endocytosis. However, infection still occurred, albeit reduced, suggesting that DC-SIGN must be able to act in concert with a co-receptor containing cell entry motifs. Supported by NIH GM41402. 2818-Pos Board B195 Effect of Dengue Fusion Peptide in Langmuir Monolayers Thaı´s F. Schmidt1,2, Christian Salesse1, Karin A. Riske2. 1 Centre de recherche du CHU de Que´bec, De´partement d’ophtalmologie and Faculte´ de me´decine, Universite´ Laval, Que´bec, QC, Canada, 2Department of Biophysics, Universidade Federal de Sa˜o Paulo - UNIFESP, Sa˜o Paulo, Brazil. Membrane fusion peptides are part of Dengue virus system, serving as a gateway to the virus access into the cell. Studies on the mechanisms of action of these peptides to improve protocols for the treatment and achievement of new drugs to combat the dissemination of the virus are increasingly necessary. The Langmuir technique is very useful to study peptide-lipid interactions. Indeed, the utilization of lipid monolayers allows controlling several physical parameters such as lipid composition, surface pressure and many other parameters of interest. Here we study the interaction of the Dengue Flavivirus fusion peptide (FLAg) with Langmuir monolayers composed of different types of lipids, including POPG, (palmitoyl-oleoyl-phosphatydylglycerol). In order to assess peptide maximum insertion pressure (MIP) into the Langmuir model membrane, kinetic measurements were performed. The kinetics of FLAg binding onto the phospholipid monolayer was monitored until the equilibrium surface pressure (Pe) was reached. The MIP was determined by injecting FLAg at different initial surface pressures (Pi) of the lipid monolayers. To investigate the behavior of FLAg in Langmuir monolayers with different lipid composition, we also used in situ infrared spectroscopy (PM-IRRAS). Preliminary results showed an increase in surface pressure during adsorption kinetics experiments with POPG monolayers, revealing an electrostatic affinity that evidences the role of the membrane charge in the fusion process. Furthermore, surface activity of the peptide solution used as the subphase in the absence of lipid monolayers was also measured. PM-IRRAS measurements reveal how the peptide incorporates at the interface of POPG monolayers, showing changes in the spectra, besides indicating characteristic due to the presence of FLAg. Altogether, these data clearly demonstrate that useful information on the binding FLAg to membranes can be obtained by performing measurements of their monolayer binding parameters. 2819-Pos Board B196 Directing Membrane Pore and Stalk Formation in MD Simulations with Embedded Mechanical Devices Gregory Bubnis, Helmut Grubmuller. Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. Stalk and pore formation are two well-known examples of the membrane topological changes required in a host of cellular processes. Their intermediate states, however, involve rearrangements of only a few lipid and solvent molecules, making them hard to observe experimentally. Molecular simulations have suitable temporal/spatial resolution to observe these transitions and have suggested stalk(pore) formation paths where a hydrophobic(philic) bridge nucleates across a hydrophilic(phobic) slab. However, most simulations require biasing schemes that are susceptible to hysteresis near the topological transitions. Here we present a simulation method implementing a biasing scheme to reversibly direct pore formation and closure using a membrane embedded ‘mechanical device’. This ‘pore device’ biasing potential can be adjusted to open, close or restrain a pore, permitting exhaustive and rigorous sampling of intermediate states such as asymmetrical indentations and water wires