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Imaging the Lipid Landscape within Focal Adhesions with Super-Resolution Microscopy
222a
Monday, February 13, 2017
Membrane Structure II 1096-Pos Board B164 Interaction of Daptomycin and Calcium with Lipid Bilayers Nicholas E. Charron1, Yuan-Pang Chang2,3, Ming-Tao Lee3,4, Huey W. Huang1. 1 Physics and Astronomy, Rice University, Houston, TX, USA, 2Department of Biophysics, National Central University, Jhongli, Taiwan, 3National Synchrotron Radiation Research Center, Hsinchu, Taiwan, 4Department of Physics, National Central University, Jhongli, Taiwan. Daptomycin is a cyclic lipopeptide antibiotic recently approved for clinical treatment of gram positive bacterial infections. It displays strong bactericidal behavior in the presence of excess calcium ions and in biomembranes containing negatively charged PG headgroups. Yet, the molecular mechanism of daptomycin, beyond the suggestion of ion channel formation and cellular depolarization, remains unknown. Recent data has shown results inconsistent with ion channel formation, prompting a more detailed investigation of Daptomycin and calcium interactions with lipid bilayers at higher structural resolutions. In order to fully characterize the mechanism of Daptomycin, it is necessary to understand the role of individual components that are integral to Daptomycin’s mode of action in the lipid bilayer. By using lamellar diffraction of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) multilayers, we examined the individual and combined effects of Daptomycin and calcium ions on bilayer structure. We found that calcium ions alone result in a slight thickening effect of the bilayer, partially supporting molecular simulations of calcium ions and PG bilayers that propose a calcium ordering effect at physiological calcium concentrations. Inclusion of daptomycin alone produced a bilayer thinning effect, suggesting that Daptomycin is capable of inserting into the headgroup region of the lipid bilayer during binding. Lastly, samples that contained both Daptomycin and calcium ions showed a bilayer thickening effect. These results suggest that Daptomycin binds to the membrane in the absence of calcium ions but induces a structural thickening of the bilayer in the presence of calcium ions. The apparent thickening effect of Daptomycin is unlike other membrane-active antibiotics, such as melittin or LL-37, which induce bilayer thinning. These studies may form the basis for more detailed investigations to characterize the molecular mechanisms of Daptomycin and other cyclic lipopeptides. 1097-Pos Board B165 Membrane Structural Analysis by Enhanced Raman Scattering Jason H. Hafner, James R. Matthews, Cyna R. Shirazinejad, Grace A. Isakson, Steven M.E. Demers. Physics & Astronomy, Rice University, Houston, TX, USA. Gold nanoparticles focus light to a molecular length scale, creating the possibility to visualize molecular structure. The high optical intensity at a nanostructure surface leads to surface enhanced Raman scattering (SERS) from molecules nearby. Due to the surface alignment and rapid decay of the electromagnetic near field, SERS spectra contain information on molecular position and orientation relative to the surface, but they are difficult to interpret quantitatively. Here we report a ratiometric analysis that combines SERS and unenhanced Raman spectra with polarizability tensors calculated by time dependent density functional theory (TDDFT), and electromagnetic (EM) near fields from numerical simulations. The analysis was applied to gold nanorods surrounded by fluid phase dioleoylphosphatidylcholine (DOPC) lipid bilayers that are only electrostatically bound to the nanorod surface. The DOPC bilayer structure was confirmed by analyzing the position and orientation of the carbon double bond stretch relative to the symmetric choline vibration in the headgroup. Tryptophan was introduced into the DOPC bilayer, and through analysis of three of its vibrational modes the tryptophan position and orientation were determined. The orientation was in good agreement with NMR and fluorescence measurements, while the position was found to be somewhat deeper into the acyl chain region of the lipid bilayer. This method offers a new approach to analyzing lipid membrane molecular structure under ambient conditions and without molecular labels. 1098-Pos Board B166 Mechanical Properties of Giant Plasma Membrane Vesicles Jan Steink€ uhler, Tripta Bhatia, Reinhard Lipowsky, Rumiana Dimova. Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Giant unilamellar vesicles are a popular membrane model because of their accessibility to many experimental methods. While the ability to precisely control the composition of such membranes is advantageous for basic physicochemical characterization of lipid membranes and their interactions, artificially formed GUVs suffer from some limitations. Only membranes of low compositional complexity, low protein densities and random protein orientation in the
bilayer can be obtained. In contrast, the plasma membrane of the living cell is highly complex in composition, crowded with proteins which have a defined orientation. As a missing link between these two extremes, chemically lysed plasma membrane vesicles have recently raised renewed interest. These vesicles, also called giant plasma membrane vesicles (GPMVs), have attracted attention because of their ability to exhibit optically resolvable liquid-liquid phase separation with micron-sized domains the formation of which is suppressed in the plasma membrane. In this work, we study the mechanical properties of GPMVs. We compare how various isolation conditions, such as chemical composition of solutions used to derive GPMVs, cell densities and cholesterol content, influence their bending rigidity. We also investigate how the local cellular environment modules the mechanical properties of extracted GPMVs. Furthermore, we measure the GPMV bending rigidity as a function of temperature and assess their phase state. Using micropipette aspiration we find a surprisingly large amount of hidden area which can be easily pulled out. We investigate the source and mechanism by which these area reservoirs appear. With these measurements, we hope to contribute to the understanding of the mechanical properties of the native plasma membrane. This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society. 1099-Pos Board B167 Imaging the Lipid Landscape within Focal Adhesions with SuperResolution Microscopy Julia T. Bourg, Sarah L. Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. Integrins are transmembrane adhesion receptors that partner with extracellular ligands and cytoskeletal proteins to form multi-protein adhesive complexes known as focal adhesions. These complexes span across large, micron-sized membrane domains and provide a link between the cell and its environment. Additionally, they serve as cell signaling hubs, regulating cell growth and motility by relaying mechanical and biochemical signals throughout the cell. Focal adhesion functionality is hypothesized to be sensitive to cell membrane composition, driven at least in part by the tendency for the plasma membrane to separate into ordered and disordered domains resembling liquid phases observed in model membranes. The impact of lipid heterogeneity on Integrin signaling is intrinsically difficult to study in cells because lipid domains are proposed to be small and dynamic. However, the dense clusters of Integrins found within focal adhesions may stabilize these domains, simplifying the detection of compositional heterogeneity in these sites. We use two-color super resolution microscopy to simultaneously image both Integrin receptors and membrane-anchored peptides in order to map the local lipid environment within focal adhesions. The peptides used in this study have previously demonstrated to preferentially sort into the liquid-ordered or liquid-disordered phases in model membranes and domains in intact cells, thereby serving as a marker of the physical state of the membrane. Imaging these peptides alongside Integrin receptors in mouse fibroblasts allows us to quantify the strength and lengthscale of their co-localization. Our long-term objective is to use this experimental framework to examine how chemical perturbations to the physical state of the membrane can impact Integrin activation and signaling responses. 1100-Pos Board B168 Investigation of Macro- and Nanoscopic Lipid Domains in Four Component Mixtures via SAXS and DSC Measurements Anna Weitzer1,2, Michal Belicka1,2, Drew Marquardt1,2, Peter Heftberger1,2, Georg Pabst1,2. 1 Institute of Molecular Biosciences (Biophysics Division), University of Graz, Graz, Austria, 2BioTechMed, Graz, Austria. We have studied lipid vesicles composed of DSPC/DOPC/POPC/cholesterol mixtures, which form, depending on the DOPC/POPC ratio, heterogeneities of varying size and complexity, such as coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains. The first aspect investigated was the dependence of the domain size on the DOPC/POPC ratio at a constant temperature, particularly the transition from macroscopic to nanoscopic domains with increasing POPC content. Small angle x-ray scattering (SAXS) data were collected at different synchrotron facilities and further analyzed with a global analysis program for multilamellar vesicles. This approach allows for parameters such as the bilayer thicknesses and the bending fluctuations to be determined. The results provided for macroscopic domains matched former investigations of similar systems, in the nanoscopic range new accessions were considered. Secondly, SAXS and differential scanning calorimetry (DSC) measurements monitored the temperature dependence of the transition between a domain structure (phase separation) and a homogeneous Ld phase. These data provided insight into the dissolving of Lo domains, showing a
Monday, February 13, 2017
Membrane Structure II 1096-Pos Board B164 Interaction of Daptomycin and Calcium with Lipid Bilayers Nicholas E. Charron1, Yuan-Pang Chang2,3, Ming-Tao Lee3,4, Huey W. Huang1. 1 Physics and Astronomy, Rice University, Houston, TX, USA, 2Department of Biophysics, National Central University, Jhongli, Taiwan, 3National Synchrotron Radiation Research Center, Hsinchu, Taiwan, 4Department of Physics, National Central University, Jhongli, Taiwan. Daptomycin is a cyclic lipopeptide antibiotic recently approved for clinical treatment of gram positive bacterial infections. It displays strong bactericidal behavior in the presence of excess calcium ions and in biomembranes containing negatively charged PG headgroups. Yet, the molecular mechanism of daptomycin, beyond the suggestion of ion channel formation and cellular depolarization, remains unknown. Recent data has shown results inconsistent with ion channel formation, prompting a more detailed investigation of Daptomycin and calcium interactions with lipid bilayers at higher structural resolutions. In order to fully characterize the mechanism of Daptomycin, it is necessary to understand the role of individual components that are integral to Daptomycin’s mode of action in the lipid bilayer. By using lamellar diffraction of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) multilayers, we examined the individual and combined effects of Daptomycin and calcium ions on bilayer structure. We found that calcium ions alone result in a slight thickening effect of the bilayer, partially supporting molecular simulations of calcium ions and PG bilayers that propose a calcium ordering effect at physiological calcium concentrations. Inclusion of daptomycin alone produced a bilayer thinning effect, suggesting that Daptomycin is capable of inserting into the headgroup region of the lipid bilayer during binding. Lastly, samples that contained both Daptomycin and calcium ions showed a bilayer thickening effect. These results suggest that Daptomycin binds to the membrane in the absence of calcium ions but induces a structural thickening of the bilayer in the presence of calcium ions. The apparent thickening effect of Daptomycin is unlike other membrane-active antibiotics, such as melittin or LL-37, which induce bilayer thinning. These studies may form the basis for more detailed investigations to characterize the molecular mechanisms of Daptomycin and other cyclic lipopeptides. 1097-Pos Board B165 Membrane Structural Analysis by Enhanced Raman Scattering Jason H. Hafner, James R. Matthews, Cyna R. Shirazinejad, Grace A. Isakson, Steven M.E. Demers. Physics & Astronomy, Rice University, Houston, TX, USA. Gold nanoparticles focus light to a molecular length scale, creating the possibility to visualize molecular structure. The high optical intensity at a nanostructure surface leads to surface enhanced Raman scattering (SERS) from molecules nearby. Due to the surface alignment and rapid decay of the electromagnetic near field, SERS spectra contain information on molecular position and orientation relative to the surface, but they are difficult to interpret quantitatively. Here we report a ratiometric analysis that combines SERS and unenhanced Raman spectra with polarizability tensors calculated by time dependent density functional theory (TDDFT), and electromagnetic (EM) near fields from numerical simulations. The analysis was applied to gold nanorods surrounded by fluid phase dioleoylphosphatidylcholine (DOPC) lipid bilayers that are only electrostatically bound to the nanorod surface. The DOPC bilayer structure was confirmed by analyzing the position and orientation of the carbon double bond stretch relative to the symmetric choline vibration in the headgroup. Tryptophan was introduced into the DOPC bilayer, and through analysis of three of its vibrational modes the tryptophan position and orientation were determined. The orientation was in good agreement with NMR and fluorescence measurements, while the position was found to be somewhat deeper into the acyl chain region of the lipid bilayer. This method offers a new approach to analyzing lipid membrane molecular structure under ambient conditions and without molecular labels. 1098-Pos Board B166 Mechanical Properties of Giant Plasma Membrane Vesicles Jan Steink€ uhler, Tripta Bhatia, Reinhard Lipowsky, Rumiana Dimova. Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Giant unilamellar vesicles are a popular membrane model because of their accessibility to many experimental methods. While the ability to precisely control the composition of such membranes is advantageous for basic physicochemical characterization of lipid membranes and their interactions, artificially formed GUVs suffer from some limitations. Only membranes of low compositional complexity, low protein densities and random protein orientation in the
bilayer can be obtained. In contrast, the plasma membrane of the living cell is highly complex in composition, crowded with proteins which have a defined orientation. As a missing link between these two extremes, chemically lysed plasma membrane vesicles have recently raised renewed interest. These vesicles, also called giant plasma membrane vesicles (GPMVs), have attracted attention because of their ability to exhibit optically resolvable liquid-liquid phase separation with micron-sized domains the formation of which is suppressed in the plasma membrane. In this work, we study the mechanical properties of GPMVs. We compare how various isolation conditions, such as chemical composition of solutions used to derive GPMVs, cell densities and cholesterol content, influence their bending rigidity. We also investigate how the local cellular environment modules the mechanical properties of extracted GPMVs. Furthermore, we measure the GPMV bending rigidity as a function of temperature and assess their phase state. Using micropipette aspiration we find a surprisingly large amount of hidden area which can be easily pulled out. We investigate the source and mechanism by which these area reservoirs appear. With these measurements, we hope to contribute to the understanding of the mechanical properties of the native plasma membrane. This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society. 1099-Pos Board B167 Imaging the Lipid Landscape within Focal Adhesions with SuperResolution Microscopy Julia T. Bourg, Sarah L. Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. Integrins are transmembrane adhesion receptors that partner with extracellular ligands and cytoskeletal proteins to form multi-protein adhesive complexes known as focal adhesions. These complexes span across large, micron-sized membrane domains and provide a link between the cell and its environment. Additionally, they serve as cell signaling hubs, regulating cell growth and motility by relaying mechanical and biochemical signals throughout the cell. Focal adhesion functionality is hypothesized to be sensitive to cell membrane composition, driven at least in part by the tendency for the plasma membrane to separate into ordered and disordered domains resembling liquid phases observed in model membranes. The impact of lipid heterogeneity on Integrin signaling is intrinsically difficult to study in cells because lipid domains are proposed to be small and dynamic. However, the dense clusters of Integrins found within focal adhesions may stabilize these domains, simplifying the detection of compositional heterogeneity in these sites. We use two-color super resolution microscopy to simultaneously image both Integrin receptors and membrane-anchored peptides in order to map the local lipid environment within focal adhesions. The peptides used in this study have previously demonstrated to preferentially sort into the liquid-ordered or liquid-disordered phases in model membranes and domains in intact cells, thereby serving as a marker of the physical state of the membrane. Imaging these peptides alongside Integrin receptors in mouse fibroblasts allows us to quantify the strength and lengthscale of their co-localization. Our long-term objective is to use this experimental framework to examine how chemical perturbations to the physical state of the membrane can impact Integrin activation and signaling responses. 1100-Pos Board B168 Investigation of Macro- and Nanoscopic Lipid Domains in Four Component Mixtures via SAXS and DSC Measurements Anna Weitzer1,2, Michal Belicka1,2, Drew Marquardt1,2, Peter Heftberger1,2, Georg Pabst1,2. 1 Institute of Molecular Biosciences (Biophysics Division), University of Graz, Graz, Austria, 2BioTechMed, Graz, Austria. We have studied lipid vesicles composed of DSPC/DOPC/POPC/cholesterol mixtures, which form, depending on the DOPC/POPC ratio, heterogeneities of varying size and complexity, such as coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains. The first aspect investigated was the dependence of the domain size on the DOPC/POPC ratio at a constant temperature, particularly the transition from macroscopic to nanoscopic domains with increasing POPC content. Small angle x-ray scattering (SAXS) data were collected at different synchrotron facilities and further analyzed with a global analysis program for multilamellar vesicles. This approach allows for parameters such as the bilayer thicknesses and the bending fluctuations to be determined. The results provided for macroscopic domains matched former investigations of similar systems, in the nanoscopic range new accessions were considered. Secondly, SAXS and differential scanning calorimetry (DSC) measurements monitored the temperature dependence of the transition between a domain structure (phase separation) and a homogeneous Ld phase. These data provided insight into the dissolving of Lo domains, showing a