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Calculating Transmembrane Diffusivity
Wednesday, March 2, 2016 molecules could be trapped between nano-subdomains of densely packed saturated lipids, surrounded by high density of cholesterol. The nanoscopic membrane environment created by the nano-subdomains of rafts is thus unique, which illustrates the essentials of lipid rafts of Lo phase in cell membrane. 2810-Pos Board B187 Superdiffusive Motion of Membrane-Targeting Domains Diego Krapf1,2, Grace Campagnola3, Kanti Nepal2, Olve B. Peersen3. 1 Electrical and Computer Engineering, Colorado State University, Fort Collins, CO, USA, 2School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA, 3Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA. Membrane-targeting domains play crucial roles in the association of signaling molecules to the plasma membrane. For most peripheral proteins, the proteinto-membrane interaction is transient; but after proteins dissociate from the membrane they are observed to rebind following a brief excursion in the bulk solution. These membrane hops can have broad implications on protein dynamics. We study the diffusion of membrane-targeting domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behavior. However, the time-averaged MSD of individual trajectories is found to be linear with respect to lag time, as in Brownian diffusion. These observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution, rapidly increasing the area explored. Furthermore, this type of motion is characterized by a pronounced scattering in the time-averaged MSD of individual trajectories. 2811-Pos Board B188 Calculating Transmembrane Diffusivity Christopher N. Rowley, Ernest Awoonor-Williams, Kari Gaalswyk. Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL, Canada. The rate of unfacilitated diffusion of solutes through a lipid bilayer is an important factor in cell signaling, toxicology, and pharmacokinetics. The solubility-diffusion model allows the permeability coefficient of a solute to be calculated using molecular dynamics simulations [1]. This method requires only the potential of mean force and diffusion coefficient of the solute along the transmembrane axis. Accurate calculation of the transmembrane diffusion coefficient profile has proven difficult because the diffusivity depends strongly on the position of the solute in the membrane and converges slowly. Hummer proposed a simple method to calculate position-dependent diffusivity from the position-autocorrelation function of a restrained molecular dynamics simulations, but the values calculated in the lipid tail region can have large uncertainties and are sensitive to the method of integration, etc. We show that long-time correlations of the solute within the membrane are the cause of these issues. An alternative method based on the Laplace transform of the velocity autocorrelation function avoids this issue and provides a straightforward and reliable method of calculating transmembrane diffusion coefficient profiles. This method will facilitate calculation of the rates of unfacillitated membrane permeation. Our implementation of this method is available for free online [5]. [1] Marrink, Berendsen, J. Phys. Chem., 1994, 98, 4155, http://dx.doi.org/10. 1021/j100066a040. [2] Riahi, Rowley, J. Am. Chem. Soc., 2014, 136, 15111 http://dx.doi.org/10. 1021/ja508063s. [3] Hummer, New J. Phys. 2005, 7, 34, http://dx.doi.org/10.1088/1367-2630/7/ 1/034. [4] Woolf and Roux. J. Am. Chem. Soc. 1994, 116, 5916, http://dx.doi.org/10. 1021/ja00092a048. [5] GitHub: https://github.com/RowleyGroup/ACFCalculator. 2812-Pos Board B189 Probing Role of Cholesterol in Integrin Crosstalk and Complexation of Integrins with GPI-Anchored Urokinase Receptors and Gangliosides using Model Lipid Mixtures Yifan Ge1, Jiayun Gao1, Rainer Jordan2, Christoph Naumann1,3. 1 Chemistry and chemical biology, IUPUI, Indianapolis, IN, USA, 2 Makromolekulare Chemie, TU Dresden, Dresden, Germany, 3Integrated Nanosystems Development Institute, IUPUI, Indianapolis, IN, USA. It is now widely recognized that lipid composition may have a profound influence on membrane protein distribution and function. However, due to the complexity of plasma membranes, the underlying biophysical mechanisms of such a lipid-mediated regulation of membrane protein functionality
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remain elusive. Exemplary, there is still limited understanding about the role of cholesterol in the formation of functional complexes of membrane proteins with other membrane constituents, such as different membrane proteins and glycolipids. To address this potentially important topic, here we explore the influence of cholesterol content on the interaction behavior of different integrins (avb3 and a5b1), also known as integrin crosstalk, as well as the complexation of integrins with GPI-anchored urokinase plasminogen activator receptor (uPAR) and ganglioside GM3, respectively, using model lipid mixtures of well-defined compositions. Membrane protein complexation and distribution in the planar model membrane is analyzed using complementary confocal detection methods, including fluorescence correlation spectroscopy (FCS), confocal fluorescence intensity analysis, and photon counting histogram (PCH) method. Experimental results are presented, which not only confirm a5b1-avb3, avb3-uPAR, and a5b1-GM3 complexations in the fluid lipid bilayer, but also demonstrate the important role of cholesterol therein. They highlight the potential significance of cholesterol in the regulation of membrane protein complexes in biological membranes. 2813-Pos Board B190 Dynamics and Statics in Phase Separating, Adhering Lipid Membranes Orrin Shindell, Natalie Mica, Max Ritzer, Vernita D. Gordon. Department of Physics and Center for Nonlinear Dynamics, University of Texas Austin, Austin, TX, USA. When living cells adhere to one another or their environment they organize their cellular membrane components into physical structures responsible for biological functions. One striking example is the immunological synapse formed at the adhesion site of immune cells which exhibits dual heterogeneities—characterized by differences in proteins and lipids—that are at first dynamic for tens of seconds to minutes and then stable for tens of minutes to an hour (Grakoui et al., Science 285, 221-227 (1999)). We employ a minimalist biophysical approach to investigate the dynamic formation and static persistence of lipid and protein heterogeneities at the adhesion site of lipid membranes. In our experiments we adhere mixed-lipid model membrane vesicles to a supported bilayer substrate via biotin-avidin binding. In one set of experiments, our vesicle membranes are made of a lipid mixture near an Lo-Ld phase boundary but are kept above the miscibility transition temperature as measured in free floating vesicles (S. L. Veatch, S. L. Keller, Biophysical journal 85, 3074-3083 (2003)). When the membranes adhere, they form two coexisting heterogeneities at the adhesion site that are compositionally distinct from the non-adhered portion of the membrane: (1) a central region that excludes a membrane dye—which marks the Ld phase—and is devoid of binders and (2) a peripheral region that is enriched in the membrane dye and is dense with binders. In a second set of experiments, our vesicle membranes are made of binary lipid mixtures (DOPC and Cholesterol) that are reported to be miscible at all accessible temperatures. Remarkably, when these membranes adhere, they form domains that exclude the membrane dye at the adhesion site and the domains grow on seconds-to-minutes-long time scales. We suggest the domain growth in this system is a nonequilibrium phase transition. 2814-Pos Board B191 Specific Adhesion of Giant Plasma Membrane Vesicles to SurfaceImmobilized SIRPa by Membrane Reconstituted ‘‘Marker of Self’’ Signaling Protein CD47 Jan Steinku¨hler1, Cory Alvey2, Reinhard Lipowsky1, Rumiana Dimova1, Dennis Discher2. 1 Theory and Bio-Systems, Max Planck Institut of Colloids and Interfaces, Potsdam, Germany, 2Biophysical Engineering Laboratory, University of Pennsylvania, Philadelphia, PA, USA. Model membranes consisting of pure lipid components have clearly advanced the understanding of biological cell membranes. However, the reconstitution of membrane proteins into model lipid bilayers is not straightforward and requires specific strategies for each protein system. In contrast, membranes of living cells have a complex composition with a multitude of different lipid components, proteins and sugars. In this work, we show that membrane blebs derived from the plasma membrane of living cells, also called giant plasma membrane vesicles, can be used to study the interaction of the membrane proteins CD47 and SIRPa, which are involved in immune signaling. Membrane blebs are an interesting model because they exhibit the compositional heterogeneity of the plasma membrane but their morphological behavior can still be described by a few parameters such as bending rigidity, reduced volume and tension. Specifically, we study the interaction of CD47 reconstituted in blebs and surfaceimmobilized SIRPa. Using this model system we explore membrane-mediated cooperative binding effects under different biologically relevant conditions.
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remain elusive. Exemplary, there is still limited understanding about the role of cholesterol in the formation of functional complexes of membrane proteins with other membrane constituents, such as different membrane proteins and glycolipids. To address this potentially important topic, here we explore the influence of cholesterol content on the interaction behavior of different integrins (avb3 and a5b1), also known as integrin crosstalk, as well as the complexation of integrins with GPI-anchored urokinase plasminogen activator receptor (uPAR) and ganglioside GM3, respectively, using model lipid mixtures of well-defined compositions. Membrane protein complexation and distribution in the planar model membrane is analyzed using complementary confocal detection methods, including fluorescence correlation spectroscopy (FCS), confocal fluorescence intensity analysis, and photon counting histogram (PCH) method. Experimental results are presented, which not only confirm a5b1-avb3, avb3-uPAR, and a5b1-GM3 complexations in the fluid lipid bilayer, but also demonstrate the important role of cholesterol therein. They highlight the potential significance of cholesterol in the regulation of membrane protein complexes in biological membranes. 2813-Pos Board B190 Dynamics and Statics in Phase Separating, Adhering Lipid Membranes Orrin Shindell, Natalie Mica, Max Ritzer, Vernita D. Gordon. Department of Physics and Center for Nonlinear Dynamics, University of Texas Austin, Austin, TX, USA. When living cells adhere to one another or their environment they organize their cellular membrane components into physical structures responsible for biological functions. One striking example is the immunological synapse formed at the adhesion site of immune cells which exhibits dual heterogeneities—characterized by differences in proteins and lipids—that are at first dynamic for tens of seconds to minutes and then stable for tens of minutes to an hour (Grakoui et al., Science 285, 221-227 (1999)). We employ a minimalist biophysical approach to investigate the dynamic formation and static persistence of lipid and protein heterogeneities at the adhesion site of lipid membranes. In our experiments we adhere mixed-lipid model membrane vesicles to a supported bilayer substrate via biotin-avidin binding. In one set of experiments, our vesicle membranes are made of a lipid mixture near an Lo-Ld phase boundary but are kept above the miscibility transition temperature as measured in free floating vesicles (S. L. Veatch, S. L. Keller, Biophysical journal 85, 3074-3083 (2003)). When the membranes adhere, they form two coexisting heterogeneities at the adhesion site that are compositionally distinct from the non-adhered portion of the membrane: (1) a central region that excludes a membrane dye—which marks the Ld phase—and is devoid of binders and (2) a peripheral region that is enriched in the membrane dye and is dense with binders. In a second set of experiments, our vesicle membranes are made of binary lipid mixtures (DOPC and Cholesterol) that are reported to be miscible at all accessible temperatures. Remarkably, when these membranes adhere, they form domains that exclude the membrane dye at the adhesion site and the domains grow on seconds-to-minutes-long time scales. We suggest the domain growth in this system is a nonequilibrium phase transition. 2814-Pos Board B191 Specific Adhesion of Giant Plasma Membrane Vesicles to SurfaceImmobilized SIRPa by Membrane Reconstituted ‘‘Marker of Self’’ Signaling Protein CD47 Jan Steinku¨hler1, Cory Alvey2, Reinhard Lipowsky1, Rumiana Dimova1, Dennis Discher2. 1 Theory and Bio-Systems, Max Planck Institut of Colloids and Interfaces, Potsdam, Germany, 2Biophysical Engineering Laboratory, University of Pennsylvania, Philadelphia, PA, USA. Model membranes consisting of pure lipid components have clearly advanced the understanding of biological cell membranes. However, the reconstitution of membrane proteins into model lipid bilayers is not straightforward and requires specific strategies for each protein system. In contrast, membranes of living cells have a complex composition with a multitude of different lipid components, proteins and sugars. In this work, we show that membrane blebs derived from the plasma membrane of living cells, also called giant plasma membrane vesicles, can be used to study the interaction of the membrane proteins CD47 and SIRPa, which are involved in immune signaling. Membrane blebs are an interesting model because they exhibit the compositional heterogeneity of the plasma membrane but their morphological behavior can still be described by a few parameters such as bending rigidity, reduced volume and tension. Specifically, we study the interaction of CD47 reconstituted in blebs and surfaceimmobilized SIRPa. Using this model system we explore membrane-mediated cooperative binding effects under different biologically relevant conditions.