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The Equilibrium Spreading Tension of Pulmonary Surfactant
70a
Sunday, February 28, 2016
364-Pos Board B144 The Handedness of Nucleosomes is Governed by the Supercoiling of DNA Sung Hyun Kim1, Rifka Vlijm1, Paul de Zwart1, Jaco van der Torre1, Yamini Dalal2, Cees Dekker1. 1 BioNanoScience, Delft University of Technology, Delft, Netherlands, 2 National Cancer Institute, NIH, Bethesda, MD, USA. In eukaryotic cells, histone proteins constitute the basic building blocks for higher chromatin structures by forming a nucleosome where DNA wraps around a histone octamer. While it is widely accepted that the DNA is wound in left-handed manner around the histone core, recent experimental evidence supports the existence of right-handed nucleosomes in vivo and in vitro, especially for histone variant nucleosomes such as the centromere-specific histone variant CENP-A/CENH3. Furthermore, newer studies using high throughput sequencing demonstrated that large chromosomal domains exist with alternative topological states in vivo. Thus, how such states can support the lefthanded octamer remains unknown. Here, we use magnetic tweezers to examine chaperone-mediated nucleosome assembly, for canonical H3 and histone variant CENP-A nucleosomes, under various DNA supercoiling states. To our surprise, positive and negative DNA supercoiling applied to the DNA prior to nucleosome assembly, can be readily absorbed by H3 and CENP-A nucleosomes. Indeed, we observe that positive supercoiling of the DNA preceding assembly leads to the formation of right-handed H3 and CENP-A nucleosomes. Conversely, negative supercoiling of DNA results in left-handed H3 and CENP-A nucleosomes. These data support decades-old findings that nucleosomes with both types of chirality exist, and could potentially resolve controversy in recent literature surrounding the topological state of CENP-A nucleosomes in vivo. Finally, our observations have the important biological implication that individual nucleosomes can adapt their handedness as an adaptive feature in response to topological stress applied upon the DNA by biological forces such as transcription, replication, and mitosis. Our data support the possibility that excess supercoiling in large chromosomal domains may be relaxed through adaptive transformation of chirality embedded within the core of nucleosomes, and that this unusual facet of nucleosome behavior may underlie a general feature of the chromatin organization.
Membrane Physical Chemistry I 365-Pos Board B145 Migration of Vesicles and their Domains in a Thermal Gradient Emma Talbot, Lucia Parolini, Jurij Kotar, Lorenzo Di Michele, Pietro Cicuta. University of Cambridge, Cambridge, United Kingdom. Domains of coexisting liquid phases form on the surface of giant unilamellar vesicles of DiPhyPC/DPPC/cholestanol below the transition temperature. The domains are imaged by fluorescence microscopy and migrate towards higher temperatures when a thermal gradient is applied vertically across the vesicle. The migration is towards the hot side irrespective of whether the domains are formed from the Lo or Ld phase and regardless of the proximity to chamber boundaries. The arrangement of domains on each hemisphere is explored for both ‘‘normal’’ and ‘‘trapped’’ coarsening (for which long range repulsions keep domains apart). For ‘‘trapped’’ coarsening, the reduction in the coalescence rate enables clearer study of the spacing between domains. Domains crowd on the hotter side of the vesicle, depleting the cold side. Potential migration mechanisms are considered, including temperature preferences for each lipid, and favoured curvature for each phase. Observations are consistent with migration minimising line tension energy, due to the lower line interface energy closer to mixing. The motion of entire vesicles (here, sub-micrometer large unilamellar vesicles) composed of one lipid is also investigated in a temperature gradient. Thermophoresis accumulates vesicles towards the cold or hot, depending on the local mean temperature and the vesicle surface chemistry. A high degree of vesicle concentration is possible with a small temperature difference (chot/ccold~0.35 for DT=5 C). The magnitude and sign of the Soret coefficient (characterising the migration) depends on the chemical structure of the lipid headgroup. We use thermophoresis to separate vesicles by headgroup composition and explore the behaviour of ternary vesicles (formed from two lipids with different headgroups and cholestanol). The deviation of vesicles from a spherical shape below the transition temperature is observed to enhance the Soret coefficient.
366-Pos Board B146 Assessing Asymmetry in Detergent-Lipid Interactions with Isothermal Titration Calorimetry Helen Y. Fan1, Dew Das1, Heiko Heerklotz1,2. 1 University of Toronto, Toronto, ON, Canada, 2Albert-Ludwigs-Universita¨t Freiburg, Freiburg, Germany. Most of the extensively characterized detergents redistribute rapidly between the two membrane leaflets after partitioning into the membrane bilayer. Interactions between such detergents and the membrane are classically described by the three-stage model: with increasing detergent content, a saturation boundary marks the appearance of micelles and a solubilization boundary marks the absence of vesicles. These boundaries are commonly probed by methods such as turbidimetry, NMR, fluorescence spectroscopy, and isothermal titration calorimetry (ITC). However, many important detergents do not translocate easily in the bilayer and thus have slow flip-flop rates. Mechanisms such as ‘‘cracking in,’’ ‘‘micellar solubilization,’’ and ‘‘shedding’’ have been proposed to explain their solubilization of lipid membranes. The asymmetric distribution of detergents in the membrane has been quantified by ITC uptake and release experiments, NMR, and zeta potential measurements. Here we present ITC solubilization experiments as another approach to characterize detergents with such asymmetry. POPC and 12:0 lysophosphocholine are used as a model system where parameters such as the partition coefficient, enthalpy of detergent transfer from the aqueous to the bilayer phase, and the maximum fraction of detergent supported by the bilayer are determined. Understanding the interactions between these non-equilibrating detergents and the membrane is of fundamental importance as many of these detergents are involved in key biological processes and in membrane protein studies. 367-Pos Board B147 The Equilibrium Spreading Tension of Pulmonary Surfactant Maayan P. Dagan, Stephen B. Hall. Oregon Health & Science University, Portland, OR, USA. Monomolecular films at an air/water interface coexist at the equilibrium spreading tension with the bulk phase from which they form. For individual phospholipids, the spreading tension is single-valued, and separates conditions at which hydrated vesicles adsorb from tensions at which overcompressed monolayers collapse. With pulmonary surfactant, isotherms show that monolayers compressed on the surface of bubbles coexist with the threedimensional collapsed phase over a narrow range of surface tensions. Dispersed surfactant vesicles adsorb through this range to reach surface tensions at the lower end of the coexistence-region. Between the upper and lower ends of this range, rates of collapse for spread and adsorbed films decrease substantially. Changes during adsorption across this narrow region of coexistence between the two- and three-dimensional structures at least partially explain how alveolar films of pulmonary surfactant become resistant to collapse. 368-Pos Board B148 Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation Zachary I. Imam, Laura Kenyon, Jeanne Stachowiak. Biomedical Engineering, University of Texas at Austin, Austin, TX, USA. Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins thought to congregate in rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers may provide insights into the roles rafts play in cells. Multiple studies of PEG-conjugated lipids have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure. Furthermore, our recent work has shown that steric pressure on membrane surfaces can oppose the assembly of lipid domains. Based on these findings, this work investigates whether steric pressure among membrane-bound polymeric domains could oppose membrane phase separation. To address this question we created GUVs with a ternary composition (DPPC:DOPC: Chol), which separated into coexisting liquid ordered and disordered domains. DPPE-PEG lipids (1, 2, or 5 kDa PEG) were included at increasing molar concentrations. As the distance between PEG molecules on membrane surfaces decreased, the fraction of GUVs that phase separated decreased, suggesting that membrane-bound PEG chains opposed phase separation. Interestingly, for lipid mixtures with a Tm just above room temperature, domains were destabilized when the spacing among PEG chains was greater than their hydrodynamic diameter, suggesting
Sunday, February 28, 2016
364-Pos Board B144 The Handedness of Nucleosomes is Governed by the Supercoiling of DNA Sung Hyun Kim1, Rifka Vlijm1, Paul de Zwart1, Jaco van der Torre1, Yamini Dalal2, Cees Dekker1. 1 BioNanoScience, Delft University of Technology, Delft, Netherlands, 2 National Cancer Institute, NIH, Bethesda, MD, USA. In eukaryotic cells, histone proteins constitute the basic building blocks for higher chromatin structures by forming a nucleosome where DNA wraps around a histone octamer. While it is widely accepted that the DNA is wound in left-handed manner around the histone core, recent experimental evidence supports the existence of right-handed nucleosomes in vivo and in vitro, especially for histone variant nucleosomes such as the centromere-specific histone variant CENP-A/CENH3. Furthermore, newer studies using high throughput sequencing demonstrated that large chromosomal domains exist with alternative topological states in vivo. Thus, how such states can support the lefthanded octamer remains unknown. Here, we use magnetic tweezers to examine chaperone-mediated nucleosome assembly, for canonical H3 and histone variant CENP-A nucleosomes, under various DNA supercoiling states. To our surprise, positive and negative DNA supercoiling applied to the DNA prior to nucleosome assembly, can be readily absorbed by H3 and CENP-A nucleosomes. Indeed, we observe that positive supercoiling of the DNA preceding assembly leads to the formation of right-handed H3 and CENP-A nucleosomes. Conversely, negative supercoiling of DNA results in left-handed H3 and CENP-A nucleosomes. These data support decades-old findings that nucleosomes with both types of chirality exist, and could potentially resolve controversy in recent literature surrounding the topological state of CENP-A nucleosomes in vivo. Finally, our observations have the important biological implication that individual nucleosomes can adapt their handedness as an adaptive feature in response to topological stress applied upon the DNA by biological forces such as transcription, replication, and mitosis. Our data support the possibility that excess supercoiling in large chromosomal domains may be relaxed through adaptive transformation of chirality embedded within the core of nucleosomes, and that this unusual facet of nucleosome behavior may underlie a general feature of the chromatin organization.
Membrane Physical Chemistry I 365-Pos Board B145 Migration of Vesicles and their Domains in a Thermal Gradient Emma Talbot, Lucia Parolini, Jurij Kotar, Lorenzo Di Michele, Pietro Cicuta. University of Cambridge, Cambridge, United Kingdom. Domains of coexisting liquid phases form on the surface of giant unilamellar vesicles of DiPhyPC/DPPC/cholestanol below the transition temperature. The domains are imaged by fluorescence microscopy and migrate towards higher temperatures when a thermal gradient is applied vertically across the vesicle. The migration is towards the hot side irrespective of whether the domains are formed from the Lo or Ld phase and regardless of the proximity to chamber boundaries. The arrangement of domains on each hemisphere is explored for both ‘‘normal’’ and ‘‘trapped’’ coarsening (for which long range repulsions keep domains apart). For ‘‘trapped’’ coarsening, the reduction in the coalescence rate enables clearer study of the spacing between domains. Domains crowd on the hotter side of the vesicle, depleting the cold side. Potential migration mechanisms are considered, including temperature preferences for each lipid, and favoured curvature for each phase. Observations are consistent with migration minimising line tension energy, due to the lower line interface energy closer to mixing. The motion of entire vesicles (here, sub-micrometer large unilamellar vesicles) composed of one lipid is also investigated in a temperature gradient. Thermophoresis accumulates vesicles towards the cold or hot, depending on the local mean temperature and the vesicle surface chemistry. A high degree of vesicle concentration is possible with a small temperature difference (chot/ccold~0.35 for DT=5 C). The magnitude and sign of the Soret coefficient (characterising the migration) depends on the chemical structure of the lipid headgroup. We use thermophoresis to separate vesicles by headgroup composition and explore the behaviour of ternary vesicles (formed from two lipids with different headgroups and cholestanol). The deviation of vesicles from a spherical shape below the transition temperature is observed to enhance the Soret coefficient.
366-Pos Board B146 Assessing Asymmetry in Detergent-Lipid Interactions with Isothermal Titration Calorimetry Helen Y. Fan1, Dew Das1, Heiko Heerklotz1,2. 1 University of Toronto, Toronto, ON, Canada, 2Albert-Ludwigs-Universita¨t Freiburg, Freiburg, Germany. Most of the extensively characterized detergents redistribute rapidly between the two membrane leaflets after partitioning into the membrane bilayer. Interactions between such detergents and the membrane are classically described by the three-stage model: with increasing detergent content, a saturation boundary marks the appearance of micelles and a solubilization boundary marks the absence of vesicles. These boundaries are commonly probed by methods such as turbidimetry, NMR, fluorescence spectroscopy, and isothermal titration calorimetry (ITC). However, many important detergents do not translocate easily in the bilayer and thus have slow flip-flop rates. Mechanisms such as ‘‘cracking in,’’ ‘‘micellar solubilization,’’ and ‘‘shedding’’ have been proposed to explain their solubilization of lipid membranes. The asymmetric distribution of detergents in the membrane has been quantified by ITC uptake and release experiments, NMR, and zeta potential measurements. Here we present ITC solubilization experiments as another approach to characterize detergents with such asymmetry. POPC and 12:0 lysophosphocholine are used as a model system where parameters such as the partition coefficient, enthalpy of detergent transfer from the aqueous to the bilayer phase, and the maximum fraction of detergent supported by the bilayer are determined. Understanding the interactions between these non-equilibrating detergents and the membrane is of fundamental importance as many of these detergents are involved in key biological processes and in membrane protein studies. 367-Pos Board B147 The Equilibrium Spreading Tension of Pulmonary Surfactant Maayan P. Dagan, Stephen B. Hall. Oregon Health & Science University, Portland, OR, USA. Monomolecular films at an air/water interface coexist at the equilibrium spreading tension with the bulk phase from which they form. For individual phospholipids, the spreading tension is single-valued, and separates conditions at which hydrated vesicles adsorb from tensions at which overcompressed monolayers collapse. With pulmonary surfactant, isotherms show that monolayers compressed on the surface of bubbles coexist with the threedimensional collapsed phase over a narrow range of surface tensions. Dispersed surfactant vesicles adsorb through this range to reach surface tensions at the lower end of the coexistence-region. Between the upper and lower ends of this range, rates of collapse for spread and adsorbed films decrease substantially. Changes during adsorption across this narrow region of coexistence between the two- and three-dimensional structures at least partially explain how alveolar films of pulmonary surfactant become resistant to collapse. 368-Pos Board B148 Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation Zachary I. Imam, Laura Kenyon, Jeanne Stachowiak. Biomedical Engineering, University of Texas at Austin, Austin, TX, USA. Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins thought to congregate in rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers may provide insights into the roles rafts play in cells. Multiple studies of PEG-conjugated lipids have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure. Furthermore, our recent work has shown that steric pressure on membrane surfaces can oppose the assembly of lipid domains. Based on these findings, this work investigates whether steric pressure among membrane-bound polymeric domains could oppose membrane phase separation. To address this question we created GUVs with a ternary composition (DPPC:DOPC: Chol), which separated into coexisting liquid ordered and disordered domains. DPPE-PEG lipids (1, 2, or 5 kDa PEG) were included at increasing molar concentrations. As the distance between PEG molecules on membrane surfaces decreased, the fraction of GUVs that phase separated decreased, suggesting that membrane-bound PEG chains opposed phase separation. Interestingly, for lipid mixtures with a Tm just above room temperature, domains were destabilized when the spacing among PEG chains was greater than their hydrodynamic diameter, suggesting