1st BioSoft symposium Biophysical approaches to understand life at different scales Forschungszentrum Jülich – 6th November 2014

Within the scope of the graduate school program International Helmholtz Research School in Biophysics and Soft Matter (IHRS BioSoft), the fellows are pleased to organize the first networking event. We hope that our efforts help to foster a series of events in the near future.

This meeting brings together researchers using quantitative approaches towards the biophysical understanding of different physiological processes essential to life. The sheer complexity of living organisms poses a jumbled puzzle to solve when studying processes like the development of an early embryo or the maturation of pathogenic behaviors. Recent developments in biotechnology, molecular biology, biochemistry and biophysical modeling have fostered unprecedented approaches to unravel the multidimensional facets of such complex pathways.

Our workshop aims at providing an interdisciplinary platform to bring together the complementary skills of experimentalists and theoreticians (spanning as many involved fields as possible) in order to understand the role of forces, flow and fluctuations within biological systems. The 7 proposed talks specifically address 3 major themes:

1. Bioadhesion, cytoskeleton and cell motility 2. Tissue growth and morphogenesis 3. Collective behaviors in biological networks

The proposed themes focus on cellular behavior at different scales ranging from the single cell level to multicellular­aggregates like tissues, and even encompass complex biological networks.

A major goal is to provide a platform for students and young scientists to present and discuss their work with other students and expert researchers, in order to promote the mutual exchange of ideas and facilitate the development of novel research directions.

Looking forward to seeing in Juelich, Sabyasachi Dasgupta, Guglielmo Saggiorato, Gloria Fabris, and Melanie Balbach Schedule

9:00 ­ 9:10 Introduction to the symposium 9:10 ­ 10:05 Biomimetics (T1) A. Roux – Univ. of Geneva (Switzerland) Mechanics of protein coats in cell membrane traffic 10:05 ­ 11:00 Cytoskeleton (T2) J. Guck ­ TU Dresden (Germany) How cells feel ­ and why that's important 11:00 ­ 11:15 Coffee & tea break 11:15 ­ 12:15 Cell Adhesion (T3) E. Sackmann ­ TU München (Germany) Physics of Cell Adhesion 12:15­14:15 Poster Session @ Lunch 14:15 ­ 15:15 Collective Behavior (T4) P. Silberzan ­ Institut Curie, Paris (France) Imposing and releasing confinement to an epithelium 15:15 ­ 16:15 Bionetworks (T5) T. Mora ­ ENS, Paris (France) Inferring the statistical mechanics of collective phenomena 16:15 ­ 16:45 Coffee & tea break 16:45 ­ 17:45 Morphogenesis (T6) P. F. Lenne ­ IBDM, Marseille (France) Mechanics of cell contacts during tissue morphogenesis 17:45 ­ 18:45 Developmental Biology L. Hufnagel ­ EMBL Heidelberg (Germany) (T7) BioImaging across scales with light­sheet microscopy: from cells to embryos 18:45 ­ 19:00 Valedictory remarks Talk abstracts

T1. Aurelien Roux

Mechanics of protein coats in cell membrane traffic Proteins involved in membrane traffic transiently interact with lipid membranes in order to remodel them, i.e. to deform them, cut and fuse them. But lipid membrane are not passive in these processes, they are visco­elastic surfaces which require energy to be remodeled. In this talk I will review a few studies where we show that the elastic energy of the membrane impacts the function of protein assemblies in membrane traffic. In particular, we will show how membrane tension and rigidity competes with clathrin budding and dynamic fission reactions, and I will show how ESCRT proteins have evolved to deform membranes by buckling.

T2. Jochen Guck

How cells feel ­ and why that's important While most current biological research focuses on molecular, biochemical aspects of cell function, we are interested in the mechanical properties of cells and tissue and their importance for biological function. The mechanical strength of cells is largely determined by the cytoskeleton, an internal polymer hybrid network intricately regulated by many signaling pathways. This cytoskeleton evolves during physiological changes, such as differentiation, is involved in many cellular functions, such as migration, and is characteristically altered in pathologies, including cancer or inflammation. We can exploit the deformability of the cytoskeleton as a link between molecular structure and biological function to sensitively monitor these functional changes using an optical stretcher and a novel, high­throughput microfluidic technique. Our results indicate that the material properties of cells define their function, can be used as an inherent cell marker and could serve as target for novel therapies.

T3. Erich Sackmann

Physics of Cell Adhesion Cells migrate by ongoing formation of adhesion domains at the leading front and their dismantling at the trailing end. Protruding forces are generated by sequential generation of solitary actin gelation waves protruding form adhesion domains (AD). The AD are formed by interplay of generic and specific interfacial forces and act both as force transmitting feet and biochemical reaction centers controlling actin polymerization and actin­microtubule crosstalk. Actin polymerization serves the generation of protrusion forces while microtubules drive the motion of the cell body. The global polarization of migrating cells is mediated by actin microtubule crosstalk. The short range cell polarization is controlled by the competition of antagonistic GTPase controlled biochemical pathways. that promote actin gelation at the front of migrating cells and AD dismantling at the trailing ends , respectively. Insight into the actin microtubule crosstalk is gained by magnetic tweezer micro­rheometry. Microinterferometry (RICM) serves the observation of adhesion domains and the measurement of adhesion and transmission forces.

T4. Pascal Silberzan

Imposing and releasing confinement to an epithelium Epithelial tissues, for which cells maintain contacts with their neighbors, exhibit collective behaviors largely controlled by cell­cell interactions. In this context confinement and boundary conditions play an important role in the dynamics of these cell assemblies. Interestingly, many in vivo processes, including morphogenesis or tumor maturation, involve small populations of cells within a spatially restricted region. Cells confined on finite, population­sized domains exhibit both collective rotation with stochastic reversals and low­frequency radial displacement modes. When this boundary condition is removed, we observe the collective migration of these epithelia. In the first stages, the essential characteristics of these collective dynamics in these two situations are well described by the same model in which cells are described as persistent random walkers which adapt their motion to that of their neighbors. However, at late stages, cells in confined epithelia develop a tridimensional structure in the form of a peripheral cell cord at the domain edge. Epithelial confinement by itself is thus observed to induce morphogenetic­like processes including spontaneous collective pulsations and transition from 2D to 3D.

References: [1] Deforet, M., Hakim, V., Yevick, H. G., Duclos, G. & Silberzan, P. Emergence of collective modes and tridimensional structures from epithelial confinement. Nat. Commun. 5, 1–9 (2014). [2] Reffay, M. et al. Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells. Nat. Cell Biol. 16, 217 (2014). [3] Sepúlveda, N. et al. Collective cell motion in an epithelial sheet can be quantitatively described by a stochastic interacting particle model. PLoS Comput. Biol. 9, e1002944 (2013).

T5. Thierry Mora

Inferring the statistical mechanics of collective phenomena Collective phenomena are emergent events that cannot simply be explained as a sum of individual behaviors. They are relevant at many scales in biology, from the collective dynamics of neural networks to the concerted motion of bird flocks. Focusing on these two examples, I will show how the tools and concepts of statistical mechanics, when applied directly to experimental data, can be used to gain insight about the collective behavior of complex biological systems.

T6. Pierre ­ François Lenne

Mechanics of cell contacts during tissue morphogenesis Cell­generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell­cell and cell­extracellular matrix contacts to apply local forces on adhesive structures. Using quantitative imaging and force measurements in vivo, we study how cell­cell contacts are organized and how subcellular tensile forces are transmitted to drive tissue morphogenesis.

T7. Lars Hufnagel

BioImaging across scales with light­sheet microscopy: from cells to embryos Developmental processes are highly dynamic and span many temporal and spatial scales. A whole­embryo view of morphogenesis with subcellular resolution is essential to unravel the interconnected dynamics at the varying scales of development, from interactions within cells to those acting across the whole embryo. Bridging scales from the submicron to the millimeter range with a temporal resolution of several seconds (combined with a total imaging time of several hours) not only poses tremendous challenges for modern microscopy methods but also requires powerful computational approaches for data handling, processing and image analysis. I present a multiview selective­plane illumination microscope (MuVi­SPIM), comprising two detection and illumination objective lenses, that allows rapid /in toto/ fluorescence imaging of biological specimens with subcellular resolution. Campus Map Posters 01 Nanosecond protein dynamics studied by 02 Migration of red blood cells in microvessels, single­molecule FRET and a coarse­grained Katanov Dinar model, Gabba Matteo 03 Depletion interactions induced by fd virus: on 04 Numerical Study of (Dynamic) Light Scattering the limits of low density and Derjaguin by Red Blood Cells in Equilibrium and Flow, approximation, Johannes Mauer Desio Silvia 05 Added dimensionality: New approaches towards 06 Macromolecular crowding induces holo α ­ improved cell/chip coupling for multi­electrode lactalbumin aggregation by converting to its apo arrays, form, Ullmann Sabrina Mittal Shruti 07 Role of N­Acetylaspartate in Alzheimer disease, 08 PAH1 domain is responsible for the Warepam Marina thermodynamic and structural stabilization of hSin3B in low pH condition, Raj Tauheed Hasan 09 N­homocysteinylation Induces Different 10 Knowledge­based protein structure prediction, Structural and Functional Consequences on Hoffmann Falk Acidic and Basic Proteins, Gurumayum Suraj Sharma 11 Phase Differential Microscopy, a new method for 12 Conformational fluctuations of DNA hairpin­ measuring chemotactic fluxes, loops: dissecting the multiple impacts of Colin Remy macromolecular crowding, Stiehl Olivia 13 SPIM applications in organismal biology, 14 Mechanical cues during early embryogenesis of C. Struntz Philipp elegans, Fickentscher Rolf 15 Forces and restraints: a bottom­up approach to 16 Phase separation of self­propelled rods with studying cell shape, length bidispersity, Kurniawan Nicholas Abkenar Masoud 17 Potassium Binding to Excitatory Amino Acid 18 SMLM Imaging the Cytoskeleton, Transporters, Hendriks Johnny Kortzak Daniel 19 Local averaging of single particle cryo­electron 20 Structure and Dynamics of HIV­1 TAR RNA microscopy data, complex with a small molecule, Duraisamy Amudha Kumari Kolar Michal H. 21 Elements and relations of a Life Theory (LT), 22 Structural dynamics of a cyclic nucleotide­ Opielok Stephan binding domain during ligand binding, Mukherjee Shatanik 23 Super­resolution microscopy provides insights 24 Length matters: hydrophobic mismatch sorts into sea urchin sperm, SNARE proteins into distinct membrane Hamzeh Hussein domains, Milovanovic Dragomir 25 Forcing clathrin onto its knees, 26 Investigating the cross­talk between GBA1 and Platen Mitja GBA2 in Gaucher disease, Schonauer Sophie 27 Intracellular Calcium fluctuations modulate 28 Simulation of transport through biological directional migration of dendritic cells, networks, Guu Donald Denisov Dmitry 29 Dynamic properties of molecular motors moving 30 Simulation and Modeling for the Reconstruction along the cytoskeleton, of Nerve Fibers in the Brain by 3D Polarized Miedema Daniel Light Imaging, Menzel Miriam

31 Microrheology study of integrin dependent 32 Investigating dynamic processes in pathogenic mechanical properties of fibroblast cells, Acanthamoeba, Klein Jan Fenneke Selhuber­Unkel Christine

33 Neutron spectroscopic observation of fast 34 Artificial Tissue, ultra­soft elastomers for cell motions in ADH With and withoud NAD in mechanical investigation, aqueous solution, Heinrichs Viktor Michael Monkenbusch

35 Understanding and controlling cellular 36 Positive charged lipid bilayer formation on gold networks: Multicellular in vitro models for heart for neuronal cell culture, tissue, Choi Sung­Eun Benjamin Wolters 37 Monitoring dopaminergic signalling in the 38 A Novel Fusogenic Drug Delivery System, retina with genetically encoded sensor proteins, Braun Tobias Sieben Anna 39 The antimicrobial peptide [KIGAKI]3 perturbs 40 Solid State NMR Spectroscopy: Investigation of lipid membranes, the core arrangement in the prion domain of Schulte Marianne Sup35NM S17R mutant, Uluca Boran 41 Ab initio protein structure modeling with 42 PHAT: PHysarum Analysis Tool, density map, Dirnberger Michael Wang Zhe 43 Structure and dynamics of human Nedd4­1 44 Flow and Diffusion in Channel­Guided Cell WW3* domain, Migration, Panwalkar Vineet Marel Anna­Kristina 45 Supported lipid bilayer with incorporated fusion 46 Sorting cryo­EM images into classes of similar proteins: a platform for studying cell­cell molecular conformations, contact, Spiegel Michaela Afanasenkau Dzmitry 47 Mesoscale Modelling of Microparticle Flow in 48 Silicon Nanowire Structures For Biosensing, Deterministic Lateral Displacement Devices, Pud Sergii Henry Ewan 49 Simulation of single­molecule fluorescence data 50 Investigation of DPPC­Membranes in the gel with regard to Amyloid beta 42 detection, phase and its phase transition in Molecular Schneider Mario Dynamics Simulations Kowalik Bartosz 51 The acinar cage: molecule exchange and 52 Spline­like interpolation in particle tracking mechanical stability of breast glands are microrheology, determined by basement membranes, Tamás Haraszti Aljona Gaiko­Shcherbak, Gloria Fabris 53 Higher­order architecture of rhodopsin in 54 Effect of hydrophobic mismatch and rigidity of photoreceptors, proteins on the cluster formation of Anne V. Schulze transmembrane proteins in biomembrane, Hamidreza Jafarinia

Poster abstracts

P1. Matteo Gabba

Institute of Complex Systems 5 Forschungszentrum Jülich

Nanosecond protein dynamics studied by single­molecule FRET and a coarse­ grained model Well pronounced interdomain movements in 3­Phosphoglycerate kinase (PGK) are assumed to be crucial for the reversible phosphor transfer reaction catalyzed by this enzyme during glycolysis. Using a cysteine double mutant with fluorescent dyes attached at the distal ends of each domain of PGK from yeast [1], we performed single­molecule Förster Resonance Energy Transfer (smFRET) experiments[2]. The fast dynamics of the protein were simulated with an elastic network (EN) under a Multiparticle Collision Dynamics (MPC) approach, combined with an accessible volume (AV) description of the dye [3]. 2D­plots of the FRET­efficiency versus the donor lifetime [4] show that PGK is a highly flexible system with interdomain dynamics spanning from nanoseconds up to milliseconds. Here, slow interconversion between an extended state and a compact conformation of the domains take place. The internal dynamics of the compact state is faster than milliseconds. Starting from the compact state, hinge bending brownian fluctuations bring the ligand­free protein in the catalytically competent state. The character of this functional motion is encoded in the structural topology of PGK as shown by normal mode analysis (NMA). Upon addition of the substrates the expanded state depopulates, with a population shift mechanism selecting the compact conformation which better allows the functional relevant motions. The timescale of the interdomain motions is recovered by means of the mesoscale hydrodynamics simulation.

[1] T. Rosenkranz, R. Schlesinger, M. Gabba, J. Fitter, ChemPhysChem, 12, 704­710, 2011. [2] Matteo Gabba, Simon Poblete, Daryan Kempe, Antonie Schöne, Tina Züchner, Gerhard Gompper, Jörg Fitter, Biophysical Journal, Vol. 106, Issue 2, p253a [3] S. Sinbert et al., JACS, 133, 2463­2480, 2011. [4] E. Sismakis, A. Valeri, S. Kalinin, P.J. Rothwell, and C.A.M. Seidel, Methods in Enzymology, 475, 455­514, 2010. P2. Dinar Katanov

Institute of Complex Systems 2 Forschungszentrum Jülich

Migration of red blood cells in microvessels Blood flow resistance in microcirculation is affected by the distribution and migration of red blood cells (RBCs) in flow. RBCs in microvessels migrate toward the vessel center due to hydrodynamic interactions with the walls leading to a cell­free layer near the walls and to a decrease in blood flow resistance. However, the position of RBCs in flow is disturbed at vessel bifurcations and branches resulting in an increase of the flow resistance. Using mesoscopic hydrodynamics simulations of blood flow, we study the migration of RBCs toward the center of cylindrical vessels and monitor a change in the flow resistance. The RBC migration is investigated for different flow rates, tube diameters, hematocrit values, and RBC aggregation interactions. Our results show that the migration for different flow rates can be well described by a master curve using a proper time scale. RBC aggregation interactions lead to a significant decrease in flow resistance at low flow rates. Finally, the effect of flow disturbance at vessel branching sections on blood flow resistance is quantified. These results are also relevant for flow of a suspension with other deformable particles.

P3. Silvia Desio

Institute of Complex Systems 3 Forschungszentrum Jülich

Depletion interactions induced by fd virus: on the limits of low density and Derjaguin approximation Depletion interactions can determine the phase behavior of bi­colloidal or more complex suspensions, by which they play an important role in crowded biological systems as well as in colloidal formulations used in technical applications. The theory of Depletion interaction was first formulated in terms of pair­wise potentials by Asakura and Oosawa in the ’50 for the case of large colloidal spheres dispersed in a solution of non­interacting depletants, where the colloidal particles interact among each other and with the depletants solely by excluded volume, while the depletants are treated like an ideal gas (low density approximation) and they are much smaller than the spheres (Derjaguin approximation). The Asakura­Oosawa description has been proven right by many experiments and numerical calculations within its limits of low density and Derjaguin approximation. In this study we were exploring the limits of these approximations by willingly violating them for the case of Depletion potentials induced by rod­like depletants between a spherical probe and a planar wall. By means of total internal reflection microscopy (TIRM), we measured the Depletion potentials profiles between four differently sized probes in the micrometer range (1mm­4mm in diameter) and a planar wall which were induced by fd virus rods. The rods have a length L=880 nm, consequently we varied the size ratio L/R from 0.44 to 1.76. We applied suspensions with virus concentrations ranging from 0.06 mg/ml (just below the overlap value c*=0.07 mg/ml) up to 1 mg/ml (almost 15c*) to induce Depletion between the probes and the wall. We find that the low density approximation and the Derjaguin approximation hold surprisingly far beyond the expected limits. The low density approximation holds up to rod concentrations of approximately six to seven times larger than the overlap concentration, while Derjaguin approximation is violated only if the particle diameter is of the order of the rod length or smaller.

P4. Johannes Mauer

Institute of Complex Systems 2 Forschungszentrum Jülich

Numerical study of (dynamic) light scattering by red blood cells in equilibrium and flow We investigate light scattering properties of red blood cells in diffusion and flow conditions. The simulation results are expected to deepen the understanding of specific types of blood flow.

P5. Sabrina Ullmann

Institute of Complex Systems 6 Forschungszentrum Jülich

Added dimensionality: New approaches towards improved cell/chip coupling for multi­electrode arrays Multi­electrode arrays (MEAs) are gaining increasing importance for the investigation of signalling processes between electrogenic cells and the study of bionetworks. In contrast to patch clamping, the current gold standard for the investigation of cellular signals, MEAs are non­invasive and enable parallel, multi­site recording. Efficient cell­chip coupling for robust and long­term electrophysiological recording and stimulation, however, still remains a challenge. A possible approach for the improvement of the cell­electrode contact is the utilization of three­dimensional structures. Since the formation of a tight cell membrane/electrode contact is a prerequisite for a high sealing resistance and high signal amplitude, we aim to understand the geometrical conditions that facilitate a tight and stable interface. Here, we investigate various different designs such as mushroom­shaped 3D electrodes and nanocavities for their capabilities with respect to the recording of cellular signals.

P6. Shruti Mittal

Dr. B. R. Ambedkar Centre for Biomedical Research University of Delhi

Macromolecular crowding induces holo α ­lactalbumin aggregation by converting to its apo form Macromolecular crowding has been shown to have an exacerbating effect on the aggregation propensity of amyloidogenic proteins; while having an inhibitory effect on the non­ amyloidogenic proteins. However, the results concerning aggregation propensity of non­ amyloidogenic proteins have not been convincing due to the contrasting effect on holo α ­ lactalbumin (holo­LA), which despite being a non­amyloidogenic protein was observed to aggregate under crowded conditions. In the present study, we have extensively characterized the crowding­induced holo­LA aggregates and investigated the possible mechanism responsible for the crowding­induced aggregation process. We discovered that macromolecular crowding results in the loss/reduction in the calcium binding affinity of the holo­LA leading to aggregate formation. In addition, calcium is observed to act as a chaperone capable of inhibiting and dissociating crowding­induced holo­LA aggregates. The study has a direct implication to Alzheimer Disease as the results invoke a new mechanism to prevent Aβ fibrillation.

P7. Marina Warepam

Dr. B. R. Ambedkar Centre for Biomedical Research University of Delhi

Role of N­Acetylaspartate in Alzheimer disease Alzheimer’s disease (AD), one of the most common neurodegenerative disorder is well characterised by accumulation of a highly ordered protein aggregates in brain. Recently, with the advent of a technique (proton magnetic resonance spectroscopy) for monitoring changes in levels of brain metabolites during the disease progression, a decrease in N­Acetylaspartate (NAA) and an increase in Myoinositol (mI) levels have been most consistently detected in brains of AD patients. On such grounds, both of them were often regarded as a valuable marker for diagnosis of AD. A relationship between the formation of protein aggregates and changes in level of these two markers has not been studied. Therefore, it is important to investigate their roles on protein aggregation. In this study, we have investigated the effect of NAA and mI on aggregation profile of an aggregation model protein, carbonic anhydrase. Here, we found that while NAA with increasing concentration is able to inhibit aggregation of the protein, mI affect only the rate of aggregation. However, when mI is titrated with NAA, rate as well as magnitude of the protein aggregation is reduced. This suggests that importance of NAA level in brain cells AD patients to control the deposition of protein aggregations.

P8. Tauheed Hasan

Dr. B. R. Ambedkar Centre for Biomedical Research University of Delhi

PAH1 domain is responsible for the thermodynamic and structural stabilization of hSin3B in low pH condition Human Sin3B (hSin3B), is a scaffold protein that binds to different transcription factors and regulate transcription. It consists of six conserved domains that includes four Paired Amphipathic Helices (PAH 1­4), one histone deacetylase interaction domain (HID) and one highly conserved region (HCR). Sin3 has no DNA binding domain of its own; therefore it requires DNA binding transcription factors on PAH domains of Sin3 to regulate transcription of genes. Recent advances have proved that hSin3B is a stress protein and gets upregulated in various stress condition such as DNA damage, oncogenic stress, oxidative stress and low pH condition. As each of the PAH domains of hSin3B have different physiochemical properties such as pI (isoelectric point of protein) and hydropathy index. Therefore it might be possible that the PAH domains shows different thermodynamic and structure stability in stress condition. In the present communication we have investigated the effect of extreme pH on structure and thermodynamic stability of the different PAH domains of hSin3B. Our major finding is that PAH1 is structurally and thermodynamically more stable at pH 4 beyond which the protein gets unfolded whereas PAH2 and PAH3 domains get destabilized in low pH condition. This study indicates that PAH1 domain might responsible for stabilization of hSin3B in extreme of pH condition not the whole protein. P9. Suraj Sharma Gurumayum

Dr. B. R. Ambedkar Centre for Biomedical Research University of Delhi

N­homocysteinylation induces different structural and functional consequences on acidic and basic proteins One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε ­amino group of lysine residues of protein (called N­homocysteinylation). It has been believed that protein N­homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivities of HTL towards proteins with different physico­chemical properties and hence their structural and functional alterations were studied. We found that N­ homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N­homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration.

P10. Falk Hoffmann

Institute of Complex Systems 6 Forschungszentrum Jülich

Knowledge­based protein structure prediction We use the basin­hopping approach to global optimization for protein structure prediction. The basin­hopping approach is based on Monte Carlo with minimization and has already been employed to find the global minimum of peptides. However, for larger peptides and proteins the effective sampling of the high­dimensional conformational space has remained a challenge, thus necessitating the development of a guided basin­hopping approach. One such approach is to use NMR chemical shifts as structural restraints as they facilitate to determine near­native structures with very high accuracy. Furthermore, we implemented knowledge­based Monte Carlo moves which allow us to fold proteins from their primary to their tertiary structure without any restraints. Additionally, we also work on knowledge­based moves which are based on Ramachandran plots. We create a plot for every amino acid and change the dihedral angle at every move according to the Ramachandran plot of the corresponding amino acid.

P11. Remy Colin AG Sourjik Max Planck Institute for Terrestrial Microbiology

Phase Differential Microscopy, a new method for measuring chemotactic fluxes Recent developments in Fourier image analysis have significantly improved the statistics in measuring the dynamics of microorganisms. We designed a new image analysis method which allows the measurement of collective drifts, such as chemotactic drifts, with an unprecedented resolution (down to a few thousandths of the bacterial swimming speed), using video microscopy. The method is based on analyzing the Fourier components of the images of the film (more precisely their phase – hence the technique’s name) and does not require to actually track the cells, avoiding the errors specific to particle tracking. We applied this method to measuring the chemotactic velocity of populations of E. coli in response to linear gradients of attractants. The gradient is generated by connecting two large reservoirs with different attractant concentrations through a small channel. The motion of the bacteria is investigated in the center of this channel, where a linear gradient of the chemical forms. We were able to resolve both the linear and logarithmic gradient sensing regimes at respectively low and large concentrations of attractant, consistently with previous works. We derive a prediction for the chemotactic flux from the single­cell­level model for the chemotactic response, in excellent agreement with our measurements.

P12. Olivia Stiehl

Experimental physics IUniversity of Bayreuth

Conformational fluctuations of DNA hairpin­loops: dissecting the multiple impacts of macromolecular crowding Biochemical reactions in crowded fluids differ strongly from those in dilute solutions. Both, excluded volume interactions with surrounding macromolecules and an enhanced rebinding of the reaction partners due to a crowding­induced viscoelasticity and subdiffusion have been predicted to shift chemical equilibria towards the associate state. Using fluorescence correlation spectroscopy and UV absorption, we tested the stochastic opening and closing of single­stranded DNA hairpin­loops under crowded conditions. Our experiments reveal that crowding not only slows down the kinetics but also increases the steady­state fraction of closed hairpins significantly [1]. Exploiting varying degrees of diffusion anomalies with the same crowder and similiar occupied volume fractions, we could dissect the differential contributions of macromolecular crowding: Excluded volume already leads to an enhanced fraction of closed hairpins but this effect is strongly increased in crowded fluids that feature anomalous diffusion [2].

[1] O. Stiehl, K. Weidner­Hertrampf and M. Weiss: Kinetics of conformational fluctuations in DNA hairpin­loops in crowded fluids. New J. Phys. 15 (2013) 113010. [2] O. Stiehl, K. Weidner­Hertrampf and M. Weiss: Dissecting the multiple facets of macromolecular crowding on the diffusion and conformation of DNA hairpin­loops, submitted (2014).

P13. Philipp Struntz

Experimental physics I University of Bayreuth

SPIM applications in organismal biology Fluorescence imaging is a powerful tool for investigating the mechanisms of embryogenesis. However, common microscopy techniques like confocal imaging have the great disadvantage of high photobleaching which can interfere with the development of the embryo and hinder long­ term acquisitions of the sample due to signal loss and phototoxicity. To overcome these limitations we have designed and constructed a fully automated single­ plane illumination microscope (SPIM) for imaging embryos of the nematode Caenorhabditis elegans in the early stages of development [1]. The combination of rapid widefield detection with optical sectioning and reduced bleaching allows long­term, three­dimensional in vivo imaging with a high spatio­temporal resolution. Cell­movement and ­arrangment can be observed by imaging GFP­labeled nuclei of the embryo while SPIM­FCS measurements quantify the dynamics of labeled molecules in the three­dimensional environment of the living organism. SPIM therefore provides several applications within one setup and hence is a powerful tool for examining dynamics and pattern formation in organismal biology.

[1] Rolf Fickentscher, Philipp Struntz & Matthias Weiss: Mechanical cues in the early embryogenesis of Caenorhabditis elegans. Biophys. J, 105:1805 – 1811 (2013) P14. Rolf Fickentscher

Experimental physics I University of Bayreuth

Mechanical cues during early embryogenesis of C. elegans The impact of biochemical signaling on developmental processes has been studied intensively. To elucidate the role of mechanical cues during embryogenesis in the model organism Caenorhabditis elegans, we have used a custom­made light sheet microscope, which allowed for three­dimensional long­term imaging of living organisms with high spatiotemporal resolution [1]. We have imaged and analyzed C. elegans embryos in which nuclei or the plasma membrane were fluorescent. To obtain information on the migration of nuclei during early embryogenesis, we developed a specifically adapted tracking algorithm. In addition, we extracted information about cell­volumes, shapes and cell arrangements via a new segmentation algorithm based on seeded region growing. Following cell divisions and migration in three dimensions, we compared different individuals. Small deviations of cell trajectories indicated a robust cellular arrangement process. A simple mechanical model revealed that early cell organization is determined by the cells’ quest for a position with least repulsive interactions of their environment. The model also predicts key features of the developing tissue in agreement with experimental observations.

[1] R. Fickentscher, P. Struntz & M. Weiss: Mechanical cues in the early embryogenesis of Caenorhabditis elegans. Biophys. J, 105:1805 – 1811 (2013)

P15. Nicholas Kurniawan

Biological Soft Matter FOM Institute AMOLF

Forces and restraints: a bottom­up approach to studying cell shape Many important cellular functions, such as control of cell shape, mechanics, division, and migration, are governed by the dynamics of the cytoskeleton and its interactions with the cell membrane. To achieve this wide variety of functions, these interactions are highly regulated, both spatially and temporally, by a host of molecular players, making it difficult to disentangle the specific biochemical pathways. To address this problem, we have taken a bottom­up approach: an active, cross­linked cytoskeletal network in controlled confinements. We find that, in microchambers, myosin motors contract actin polymer networks to clusters with a scale­free size distribution. The switch between local and global contraction is governed by network percolation and restructuring by fascin cross­linkers. In cell­sized deformable liposomes, addition of fascin results in bundling of actin filaments, which can dramatically deform liposomes and form filopodia­like protrusions. Myosin motors further induce a variety of active structural reorganizations of actin bundles. Anchoring of actin to the membrane led to the formation of cortex­like structure on the inner surface of the membrane. These results provide valuable insights into the interplay between active intracellular forces and environmental confinement in determining cell shape.

P16. Masoud Abkenar

Institute of Complex Systems 2 Forschungszentrum Jülich

Phase separation of self­propelled rods with length bidispersity The collective behavior of microswimmers has gained considerable attention in the recent years. Examples of microswimmers span from biological cells to nanobots. Here, we propose a model for self­propelled rods in two dimensions that interact with a physical interaction. We model each rod by a number of beads to calculate the rod­rod interactions using a capped interaction potential [1,2].

Polydispersity is inevitable in most experimental studies of microswimmers. For the first time, we study the effect of geometric bidispersity in active rod suspensions, where the system is composed of short and long rods [3]. Depending on the density and the lengths of each rod species, we find a rich phenomenology for the bidisperse system: a disordered phase at low densities, a segregated phase with clusters of only the long rods, a phase where both long and short rods form giant clusters, and a "remixed" phase at very high densities. We also report on a phase where the presence of short rods imposes clustering of long rods in an otherwise homogeneous long­rod suspension.

[1] Yang et al., PRE 82, 031904 (2010). [2] Abkenar et al., PRE 88, 062314 (2013). [3] Abkenar et al., in preparation (2014). P17. Daniel Kortzak

Institute of Complex Systems 4 Forschungszentrum Jülich

Potassium binding to excitatory amino acid transporters Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission and prevent extracellular glutamate concentrations from reaching neurotoxic levels. EAATs couple the uptake of glutamate to the cotransport of three Na+ and one H+ and to the countertransport of one K+. They are not only secondary active transporters, but also anion­selective channels. The K+­dependent reactions are assumed to determine the rate of glutamate transport and represent promising pharmacological targets to modify EAAT function in disease conditions. Here we combine molecular dynamics simulations of a prokaryotic EAAT homologue and patch­clamp recordings to resolve how K+ binds to mammalian EAATs. Our simulations predict three distinct K+ binding sites, which we are currently validating experimentally. We propose that K+ and one Na+ ion share one overlapping binding site and that further, possibly transient, binding sites are responsible for K+­dependent glutamate transport.

P18. Johnny Hendriks

Institute of Complex Systems 4 Forschungszentrum Jülich

SMLM Imaging the cytoskeleton The cytoskeleton is built from structural elements (fibers, tubules) on the order of 10nm in width. As such it is an interesting subject of study for Single Molecule Localization Microscopy (SMLM).

P19. Amudha Kumari Duraisamy

Institute of Complex Systems 3 Forschungszentrum Jülich

Local averaging of single particle cryo­electron microscopy data Single particle cryo­EM is a powerful technique to study the structure of biomolecular assemblies that are often large, flexible and conformational heterogeneous. Most of the density maps obtained from cryo­EM experiments are limited in resolution by this conformational heterogeneity. Improving the resolution of the density maps, thus, requires to account for the structural heterogeneity. In principle, the resolution can be reached to the atomic level, if the images of the structures are aligned accurately [1]. In the biological macromolecules, the conformational motions leads to global structural changes, however there are often rather rigid domains. Those rigid domains could be used to align and average the density to reach higher resolution. This is analogous to NCS averaging used to improve the phase information in the field of X­Ray crystallography [2]. An algorithm is presented to average rigid domains and to improve the resolution of cryo­EM density maps.

[1] Henderson. R, Q. Rev. Biophys. 28 171­193 (1995). [2] Kleywegt. G and Read. R, Structure 5 1557­1569 (1997).

P20. Michal H. Kolar

Institute of Neuroscience and Medicine Forschungszentrum Jülich

Structure and dynamics of HIV­1 TAR RNA complex with a small molecule Trans­activating response element is a short non­coding viral RNA, which regulates transcription. Classical all­atom MD simulations of TAR­inhibitor complex are analyzed and compared with NMR data.

P21. Stephan Opielok

FU Berlin

Elements and relations of a Life Theory (LT) I propose a general theory about the evolving and nature of life. It seems to be based as a rule on interaction between symmetry and asymmetry (~ stability and flexibility) for equilibration. In that kind of multidisciplinary research is these study more generalize, fundamental, extended and interconnect to previous attempts to explain the question what is life.

Finally the data of this exploration show that nature of living systems is predictable by suitable methods for measurement like e.g. spectroscopy and probability. As a result, the way of life follow the principle of stationary friction by essential level of sufficiency energy use. Further informations under www.researchgate.com and google/author.

P22. Shatanik Mukherjee

Molecular Sensory Systems Forschungszentrum caesar Bonn

Structural dynamics of a cyclic nucleotide­binding domain during ligand binding Protein functions rely on protein motions. Protein structures, obtained with X­ray diffraction or NMR, reveal mostly static pictures and do not directly reveal structure­function relations. To connect structural changes of proteins with function, protein motions have to be analyzed in real time. I have analyzed an isolated bacterial cyclic nucleotide­binding domain (mlCNBD) to study the dynamics of receptor­ligand complex formation. cAMP binds to mlCNBD, resulting in a conformational change. Recent kinetic and NMR studies indicate that these structural transitions follow the “induced­fit” mechanism. However, the detailed mechanism of these structural rearrangements leading to channel activation remains elusive. Transient Electron Paramagnetic Resonance (tr­EPR) spectroscopy was used to resolve the dynamics of mlCNBD­cAMP complex formation. Cysteine residues were introduced at different sites in the protein to allow labeling with a paramagnetic reagent. Binding of cAMP to the labeled mutants is rapidly initiated either via photolysis of a caged­cAMP or through a micro­mixer. The tr­EPR data reveals that protein undergoes millisecond time scale motions. Collating data across the whole protein will enable us to reconstruct the steps from the apo to the holo state of the protein, thereby, answering whether “induced­fit” mechanism follows a concerted or sequential path from during conformational rearrangement. P23. Hussein Hamzeh

Minerva Research Group Forschungszentrum caesar Bonn

Super­resolution microscopy provides insights into sea urchin sperm Sea urchin sperm rely on chemotaxis to find their way to the egg cells of their own species within the vast ocean. Stochastic Optical Reconstruction Microscopy (STORM) and Stimulated Emission Depletion (STED) microscopy are employed to investigate the motility and distribution of proteins that are involved in the chemotactic signaling pathway. One of those proteins is the membrane­bound guanylyl cyclase, which plays a key role in the chemotaxis of the sea urchin Arbacia punctulata. Our analysis will provide invaluable insight into understanding chemotaxis at the molecular level.

P24. Dragomir Milovanovic Department of Neurobiology Max Planck Institute for Biophysical Chemistry

Length matters: hydrophobic mismatch sorts SNARE proteins into distinct membrane domains The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein­ protein interactions. Using plasma membrane­resident SNARE proteins as model, we now show that cholesterol­induced hydrophobic mismatch between the transmembrane domains and the membrane lipids not only suffices to induce clustering of proteins, but can also lead to the segregation of structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine­tuned by interactions with polyanionic phosphoinositides and proteins. Our findings demonstrate that the structural organization of membranes is governed by a hierarchy of interactions with hydrophobic mismatch emerging as one of the fundamental physical principles. P25. Mitja Platen

3rd Institute of Physics Georg August University Göttingen

Forcing clathrin onto its knees The assembly of triskelions (clathrin monomers) into a planar clathrin lattice is novel and assumingly of high value for nanotechnological approaches. Hence, a thoroughly characterization of its properties is crucial. In this study we are investigating the mechanics of and the triskelion orientation within these 2D lattices, using atomic force microscopy (AFM). Furthermore, we are identifying changes in the clathrin lattice caused by the absence of its natural occurring light chains. This, in addition, contributes to the discussion about the light chain’s overall function, which might be the stabilization of the triskelion’s knee­joints.

P26. Sophie Schonauer

Minerva Research Group Forschungszentrum caesar Bonn

Investigating the cross­talk between GBA1 and GBA2 in Gaucher disease The beta­glucosidases GBA1 and GBA2 both degrade glucosylceramide to glucose and ceramide. Mutations in the GBA1 gene cause Gaucher disease with different clinical subtypes in humans and mice. However, no genotype­phenotype correlation has been identified so far. Our results reveal a cross­talk between GBA1 and GBA2 in patients suffering from Gaucher disease, which might help in understanding the different phenotypes. A cross­talk between GBA1 and GBA2 was analyzed in dermal fibroblasts from Gaucher disease and control patients on an expression and activity level. Quantitative real­time PCR experiments and Western blot analyses indicated that GBA2 mRNA and protein levels were similar between control patients and patients carrying a mutation in GBA1. Our fluorescence­based beta­glucosidase activity assay revealed that not only GBA1, but also GBA2 activity was dramatically decreased in Gaucher disease patients. This effect could partially be rescued by overexpressing hGBA1. These results reveal a cross­talk between GBA1 and GBA2 on the activity level. Our findings add a new aspect to the complex nature of Gaucher disease and present an emerging target for its treatment. P27. Donald Guu

Molecular Sensory Systems Forschungszentrum caesar Bonn

Intracellular Calcium fluctuations modulate directional migration of dendritic cells Actin cytoskeletal dynamics and cell polarization are essential requirements for directional migration of dendritic cells (DCs) in the course of an efficient immune response. Pathogen encounter induces DCs to upregulate their expression of costimulatory molecules and their chemokine receptor CCR7 enabling them to migrate to draining lymphoid organs. Although Calcium­signalling processes have long been known to play important roles in regulating cell migration, in DCs the intracellular Calcium ­dynamics, their spatiotemporal coordination, and their interplay and integration with other migratory signals remain largely unknown. By the use of high­resolution video microscopy and a fluorogenic Calcium­sensitive dye we show that the chemokines CCL19 and CCL21 both induce a strong increase in intracellular Calcium levels of LPS­stimulated DCs in vitro. Interestingly, we found that DCs display intracellular Calcium­ oscillations which might be necessary to initiate directional migration and cell steering in a chemokine gradient. Furthermore we observed, that chemotaxis of DCs in three­dimensional collagen gels is strongly abrogated when intracellular Calcium levels were reduced by the use of BAPTA­AM chelating agent. Although the underlying mechanism remains unidentified, these data point to the fact that spatiotemporally coordinated calcium gradients are important in orchestrating directional migration of DCs.

P28. Dmitry Denisov

Soft Matter group University of Amsterdam

Simulation of transport through biological networks The transport of organelles and proteins is of vital importance for living cells. Besides passive transport by diffusion, active transport by molecular motors hopping over the cytoskeleton network is crucial for the survival of cells. We performed simulations using the Totally Assymetric Exlusion Process (TASEP), a paradigmatic model for nonequilibrium transport, to model the dynamics along the microtubule network. We found that the rules at the intersection of the network seem to be the key factor for the formation of traffic jams along the microtubule segments. The rate at which motors at crossing continue along the same microtubule or switch to the other microtubule appears to determine for the transport along the network. We found three different regimes of motor propagation through network depending on the average motor density. For example, for medium global densities the motor distribution through the network can be highly inhomogeneous and lead to a huge reduction of the transport current through the system, when larger part of the network will be in ‘virtual’ traffic jam.

P29. Daniel Miedema

Soft Matter group University of Amsterdam

Dynamic properties of molecular motors moving along the cytoskeleton We have developed an advanced analysis technique to extract quantitative motility parameters from the image sequences of (TIRF) microcoscopy of in vitro motility assays in an automated way.

P30. Miriam Menzel

Institute of Neuroscience and Medicine 1 Forschungszentrum Jülich

Simulation and modeling for the reconstruction of nerve fibers in the brain by 3d polarized light imaging 3D Polarized Light Imaging (3D­PLI) is a neuro­imaging technique that is able to reconstruct the three­dimensional pathways of nerve fibers in post­mortem brains at the micrometer scale. By transmitting polarized light through histological brain sections in a polarimeter, the birefringence of the nerve fibers is measured, thus revealing their spatial orientation. To learn more about the interaction of polarized light with brain tissue and to improve the fiber reconstruction, two complementary simulation approaches have been developed that simulate different fiber constellations and compare the measured fiber orientations with the underlying fiber model. The first simulation approach uses the Jones matrix calculus to simulate the intrinsic birefringence of the myelin sheaths which surround the nerve fibers. The fibers are generated in a 3D volume, discretized into small voxels, and transformed into a 3D vector field which indicates the orientation of the optic axes in the birefringent myelin sheath; the fibers are simulated with axial optic axes (macroscopic model) as well as radial optic axes (microscopic model). In order to compute a synthetic 3D­PLI image series, each myelin voxel is represented by the Jones matrix of a rotated wave retarder. The optical resolution of the polarimeter is simulated by applying blurring and rescaling to the resulting image series. The simulation of different fiber constellations has shown that the microscopic model transforms into the macroscopic model if the optical resolution of the polarimeter exceeds the diameter of single fibers. Thus, for lower optical resolutions, the fibers can be assumed to be uniaxially birefringent with the optic axes pointing in direction of the fibers. Another simulation approach uses a massively parallelized 3D Maxwell Solver to investigate other optical tissue properties. The fibers and the surrounding tissue are simulated as homogeneous isotropic materials with different refractive indices. By discretizing Maxwell's equations on a spatial grid, the electric field components of the transmitted light are calculated. The simulation of different fiber constellations has shown that the samples exhibit form birefringence and diattenuation, which reveals additional information about the spatial orientation of the fibers. Furthermore, it is possible to distinguish between different fibre constellations by the amount of scattered light. The presented simulation methods are valuable tools to better understand the interaction of polarized light with brain tissue and help to improve the reconstruction of the spatial fibre orientations by 3D­PLI.

P31. Fenneke Klein Jan

Institute of Experimental Physics Ulm University

Microrheology study of integrin dependent mechanical properties of fibroblast cells Physical forces are increasingly recognized as an important biological signal. The protein family of integrins are a key element in force sensing, functioning as a bidirectional force signalling protein. They link the cytoskeleton and the extracellular matrix, giving the cells the opportunity to respond to force by adapting the cytoskeletal filaments. However, how the different integrins cooperatively modulate the force response of the cytoskeleton is not understood.

To study the crosstalk between integrins avb3 and a5b1 we use mouse embryonic fibroblasts that express only the single integrin or a combination of both. We focused on the local mechanical properties of isolated cytoskeletal filaments using microrheology. Studying the influence of these integrins under static and shear stress conditions, and the effect of distinct substrate rigidity levels. Results show that the avb3 integrin is responsible for reinforcing the network under shear stress conditions. Without this integrin (a5b1 fibroblasts) the network is less elastic with a decreased elastic modulus under shear stress. While shear stress did not influence the elasticity of the other cell types. The substrate rigidity did not affect the elasticity of the cell types a5b1 and avb3a5b1.

P32. Christine Selhuber­Unkel

Biocompatible Nanomaterials University of Kiel

Investigating dynamic processes in pathogenic Acanthamoeba Acanthamoeba castellanii is a human pathogenic parasite that is wide­spread in our water reservoirs. It can, upon contact with the human eye, cause a severe keratitis. 90% of the patients with this painful disease are contact lens users, who got infected due to wrong contact lens care. Through small lesions of the outermost epithelial cell layer, the amoebae reach the cornea and start to destroy target cells by an extracellular killing mechanism. A crucial first step during this killing process is the adhesion between an Acanthamoeba and a target cell. This initial contact is supposed to be mediated by carbohydrates. Subsequently, intracellular granules move towards the contact site and release pore­forming molecules. These pore­forming molecules finally destroy the membrane of target cells. In our work we aim at understanding the biophysical processes involved in target­cell killing: we study the “killing kiss” between Acanthamoeba and target cells from their initial adhesion to intracellular transport processes and to target cell death. In particular, studying intracellular motion in Acanthamoeba is highly interesting, as Acanthamoebae are extremely motile, change their shape, and show fast intracellular motion. The final goal of our studies is to receive a comprehensive biophysical picture of Acanthamoeba pathogenicity.

P33. Michael Monkenbusch

Jülich Centre for Neutron Science 1 Forschungszentrum Jülich

Neutron spectroscopic observation of fast motions in ADH with and without NAD in aqueous solution P34. Victor Heinrichs

Institute of Complex Systems 1/7 Forschungszentrum Jülich

Artificial tissue, ultra­soft elastomers for cell mechanical investigation Cell morphology and protein expression are strongly influenced by the elasticity of cell environment. For investigation of cell mechanics, an elastic and biocompatible model substrate “artificial tissue” with precisely properties is required. Cross­linked polydimethylsiloxane is frequently used (nontoxic, easy to handle and commercially available). However, a Young’s modulus of 1 kPa (ultra­soft, necessary to model, e.g., brain or glial tissues) has not been achieved so far.

P35. Benjamin Wolters

Institute of Complex Systems 7 Forschungszentrum Jülich

Understanding and controlling cellular networks: Multicellular in vitro models for heart tissue We have developed a simple accurate method for ECM protein patterning on soft silicone substrates to generate muscle micro­tissues which can be analyzed regarding their physiological performance.

P36. Sung­Eun Choi

Institute of Complex Systems 7 Forschungszentrum Jülich

Positive charged lipid bilayer formation on gold for neuronal cell culture We show that positive charged lipid is essential for bilayer formation on gold surface. This charged surface is also useful for biomimetic neuronal cell culture environment. P37. Anna Sieben

Institute of Complex Systems 4 Forschungszentrum Jülich

Monitoring dopaminergic signaling in the retina with genetically encoded sensor proteins Second messengers play a major role in light adaptation processes. Two of these second messengers are calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) whose pathways are known to be closely interrelated. Dopamine (DA), which is released from dopaminergic amacrine cells upon light stimulation, was reported to influence the intracellular concentration of both messengers. The intracellular cAMP concentration ([cAMP]i) is affected in two opposed ways by DA: binding of DA to the D1 receptor family results in an increase in [cAMP]i, whereas the activation of the D2 receptor family causes a reduction in [cAMP]i. DA was also reported to cause a change in the intracellular concentration of Ca2+. Since DA is discussed as a main modulator in light adaptation processes, a better comprehension of the dopaminergic control of cAMP and Ca2+ homeostasis is mandatory.

P38. Tobias Braun

Institute of Complex Systems 7 Forschungszentrum Jülich

A novel fusogenic drug delivery system Liposomes based on neutral and cationic lipids combined with aromatic compounds are introduced as novel drug delivery systems. Such liposomes fuse highly efficiently with the cellular plasma membrane enabling the delivery of hydrophobic and amphipathic substances into the cellular plasma membrane.

P39. Marianne Schulte

Institute of Complex Systems 6 Forschungszentrum Jülich

The antimicrobial peptide [KIGAKI]3 perturbs lipid membranes The [KIGAKI]3 peptide is a designer antimicrobial peptide known to form amphiphilic β ­ strands on membrane surfaces. MD­simulations were carried out to investigate the interactions between the peptide and different lipid bilayers at atomistic detail. Three 200 ns simulations with different starting conformations of one peptide on the surface of POPC/POPG bilayer (4:1) as well as one 1 μs simulation of six [KIGAKI]3 peptides above the membrane surface were performed. The same simulations were repeated with a pure POPC membrane in order to compare the effects of charged lipids on the peptide­lipid interactions. We observed lysine residues as key interacting amino acids, because they form the majority of hydrogen­bonds with the membrane. An increase in the area per lipid and decrease in membrane thickness was observed. Experimental results suggest self­assembly of the peptides mainly on the bilayer surface, resulting in the formation of amyloid­like fibrils. Our assembly results indicate no aggregation in water during the 1 μs simulations, but most of the peptides were adsorbed on the lipid surface for both, the mixed POPC/POPG and the pure POPC systems. These results will contribute to the understanding of the mechanism of cell membrane disruption by antimicrobial peptides at molecular level.

P40. Boran Uluca

Institute of Complex Systems 6 Forschungszentrum Jülich

Solid State NMR Spectroscopy: Investigation of the core arrangement in the prion domain of Sup35NM S17R mutant The [PSI+] prion is a self­propagating amyloid form of Sup35 protein, which is a subunit of the translation termination factor [1]. Aggregation and the subsequently decreased level of soluble Sup35 lead to more frequent read­through of premature termination codons. Sup35 includes an N­ terminal domain (N), which is both necessary and sufficient for prion propagation, a charged domain (M) which increases the solubility of the protein and the C­terminal translation termination domain [2]. Previous solid state NMR structural studies on a construct containing the N and M domains, Sup35NM, have shown that it has a parallel in register β ­sheet core region whose length depends on the fibrillation temperature [3]. One single point mutation, the substitution of S17 by a positively charged R residue, is sufficient to prevent Sup35pS17R monomers from being co­fibrillated with Wild­type Sup35p [4]. However, the S17R monomers can form fibrils themselves at 4°C, as well as at 37°C.

We have used symmetry­based constant­time homonuclear dipolar recoupling [5] for distance measurements between backbone 13CO­13CO in amino­acid selectively 13CO labeled samples of WT and S17R Sup35pNM. We could confirm the in register paralel β ­sheet arrangement for WT fibrils. For the S17R mutant, our results indicate a different core region. P41. Michael Dirnberger

Algorithms and Compelxity Max Planck Institute for Informatics

PHAT: PHysarum Analysis Tool PHAT is a tool which takes a picture of a biological network as input and extracts the underlying structure as a weighted graph. This representation enables a detailed analysis based on Graph Theory.

P42. Zhe Wang

Institute of Complex Systems 6 Forschungszentrum Jülich

Ab initio protein structure modeling with density map Since the number of subnanometer resolution density maps obtained by cryo­EM is steadily increasing, methods for the interpretation of these data is highly demanded. To address this , we are trying to develop a method that can build up the protein backbone structure from a subnanometer resolution density map. The method is based on a combinatorial optimization algorithm, the Lin­Kernighan heuristic, which is used for solving the Euclidean traveling salesman problem. Meanwhile, the search of models with the Lin­Kernighan heuristic is biased with secondary structure and statistical potential restraints. With this we generate an ensemble of backbone traces, from which we determine the best trace with an appropriate structure averaging algorithm.

P43. Vineet Panwalkar

Institute of Complex Systems 2 Forschungszentrum Jülich

Structure and dynamics of human Nedd4­1 WW3* domain Nedd4‐ 1 binds and regulates the human epithelial sodium channel (ENaC) through interactions mediated with its WW domains. The third WW domain (WW3*) of Nedd4­1 binds the PY motifs of the α ENaC subunit with highest affinity. Impaired WW – PY motif interactions result in Liddle’s syndrome, an acute form of hypertension. Solution state NMR spectroscopy was employed to determine the solution structure and characterize dynamics of the WW3* domain. The solution structure shows that both the apo­ and α ENaC peptide bound WW3* domains have identical fold. Despite the identical fold, differences in side chain orientations were observed for residues in the vicinity of and at the peptide binding interface. 3J couplings recorded to measure the χ 1 torsion angle indicate presence of rotameric averaging in the apo­ WW3* domain. Model­free analysis, carried out on the 15N nuclear spin relaxation data, shows presence of similar ps­ns timescale dynamics in both apo­ and peptide­bound forms of WW3* domain. However, CPMG relaxation dispersion and model­free analysis reveal that the apo­ WW3* domain shows large motional contributions at µs­ms timescale and these motions are absent in the peptide­bound WW3* domain. Molecular dynamics simulations carried out on the apo‐ WW3* domain highlight the conformational fluctuations at the ns timescale. The results of this study indicate that conformational fluctuations along combined with the induced‐ fit mechanism are key factors that lead to high affinity binding of the WW3* domain to the cognate αENaC peptide. Thus, this study provides an understanding of how dynamics along with structural characteristics are key factors contributing to the high affinity binding of a protein‐ peptide complex.

P44. Anna­Kristina Marel

Biophysics Ludwig­Maximilians Universität München

Flow and diffusion in channel­guided cell migration Collective migration plays a notable role during wound healing, embryonic development and cancer progression. We studied the flow of epithelial cell sheets as they move through microchannels, using single cell tracking and PIV analysis. We found that a defined stationary cell current emerges when data is averaged over characteristic spatial and temporal scales. The averaged flow­field exhibits a velocity gradient in the direction of migration with a plug­flow­ like profile. The observed flow velocity can be decomposed into two contributions, a constant term stemming from directed cell migration and a diffusion­like contribution that scales with the density gradient. In order to connect diffusion mediated transport to underlying cellular motility, single cell trajectories and occurrence of vorticity were studied. We discovered that the directed large­scale cell flow alters fluctuations in cellular motion at short length scales: The formation of swirls is reduced compared to resting tissues. In addition, single­cell trajectories show persistent random­walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacement beyond neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short­range correlated motion that lead to a diffusion­like transport. P45. Dzmitry Afanasenkau

Institute of Complex Systems 3 Forschungszentrum Jülich

Supported lipid bilayer with incorporated fusion proteins: a platform for studying cell­cell contact In this work we study the possibility of incorporation of Fc­conjugated proteins in the lipid bilayer supported on a solid surface (in our case ­ glass) and using this system for culturing neuronal cells. Such system can, in some extend, mimic the properties and functions of the cell membrane and provide a model for studying cell surface receptor interactions with their ligands. We focused our studies on such membrane protein as Ephrin A5. Ephrin­EphR interactions result in diverse biological responses, such as neuronal cell adhesion, growth, migration, differentiation and axonal guidance. We used EphrinA5­Fc chimera incorporated in POPC bilayer and successfully cultured rat cortical neurons on it. The cells appeared to be functionally active and form mature synapses (which was checked with patch clamp recordings). The EphrinA5 was found to be accumulated under cell body and neurites. We believe that our approach can be extended to insert other membrane proteins to create a general culture platform for neurons and other cell types.

P46. Michaela Spiegel

Institute of Complex Systems 6 Forschungszentrum Jülich

Sorting cryo­EM images into classes of similar molecular conformations The resolutions of density maps which are reconstructed from single­particle cryo­electron microscopy (cryo­EM) images are often limited by the conformational heterogeneity of the biological macromolecules. To increase the resolution, the images have to be sorted into groups of similar conformations. The common approaches of sorting images typically compare densities (either in 2D or 3D). However, a large difference in conformation does not necessarily lead to a large difference in density.

We are developing a new sorting method that is based on comparing conformations. For this we are using a bootstrapping approach and refine pseudo­atomic models against an ensemble of bootstrapped density maps. These models capture the conformational variance and can be used for sorting the images. This procedure also reveals the conformational heterogeneity and is able to determine global conformational motions of large macromolecular structures and assemblies.

P47. Ewan Henry

Institute of Complex Systems 2 Forschungszentrum Jülich

Mesoscale modelling of microparticle flow in deterministic lateral displacement devices Deterministic lateral displacement (DLD) devices are a class of microluidic device tailored specifically for spatial sorting of microscale objects. A typical DLD device consists of a lattice of obstacles which form a bumper array with a characteristic critical radius. The bumper array separates particles by discriminating against incident objects with a larger than critical radius; causing lateral motion relative to smaller solutes which travel unhindered in the direction of flow. There are myriad possible configurations for the bumper array and accurate modelling provides an excellent tool for understanding the complex flow behaviour of solutes as well as realising optimal device configuration. We employ the Smoothed Dissipative Particle Dynamics method to model behaviour of polystyrene beads and red blood cells (RBCs) in a DLD device. RBCs are modelled as deformable cells represented by a viscoelastic spring­network encapsulating the appropriate mechanical and rheological properties. We compare the simulated behaviour of RBCs in complex flows through DLD devices with experimental observations and propose optimal DLD device configurations for efficient particle sorting.

P48. Sergii Pud

Institute of Complex Systems 8 Forschungszentrum Jülich

Silicon nanowire structures for biosensing We develop and fabricate silicon nanowire (NW) field effect transistors (FETs) for biosensing applications including biomolecule detection and extracellular monitoring of neuronal cell activity. P49. Mario Schneider

Institute for Physical Biology Heinrich­Heine University Düsseldorf

Simulation of single­molecule fluorescence data with regard to amyloid beta 42 In order to test different models and new data analysis approaches in single­molecule fluorescence spectroscopy, we developed a MATLAB based Brownian dynamics simulation of fluorescent particles. Typical photophysical phenomena like singlet and triplet state as well as non­photophysical phenomena like background are included. To test the simulations' reliability we utilize photon counting histogram (PCH), fluorescence cumulant analysis (FCA), fluorescence correlation spectroscopy (FCS), and number & brightness analysis (N&B). Furthermore we use Alexa Fluor 488 and Alexa Fluor 488­labeled Amyloid beta 42 (peptide in Alzheimers desease) as model systems and resimulate their experimental data, especially with regard to the detectability of different binary mixtures of amyloid beta 42. We show that simulated and experimental data are in good agreement.

P50. Batosz Kowalik

Institute of Theoretical Physics FU Berlin

Investigation of DPPC­Membranes in the gel phase and its phase transition in Molecular Dynamics Simulations Membranes in gel phases and melting transitions to the liquid phase have recently become a topic of increasing interest, as membranes in the outer part of our body are assumed to be in gel phase. Experiments and computer simulations have revealed sharp phase transitions in many physical quantities, which still are not completely understood. In our work, we investigate such a membrane consisting of dipalmitoylphosphatidylchloline (DPPC) that forms a lipid bilayer in an aqueous surrounding. We perform Molecular Dynamics Simulations, which allow us to look into atomistic detail of such systems. We examine in detail the thermodynamic phase transition. Also, we investigate the differences in structure, energy and order parameters. P.51 Aljona Gaiko­Shcherbak, Gloria Fabris

Institute of Complex Systems 7 Forschungszentrum Jülich

The acinar cage: molecule exchange and mechanical stability of breast glands are determined by basement membranes. The human mammary gland is a mechanically active tissue whose highly proliferative alveolar structures are surrounded by a continuous basement membrane (BM) scaffold. So far such BM has been poorly investigated, although, given its putative role as a barrier against metastatic invasion, it might prove of great importance to comprehend normal organ function and malignancy. Here we aimed at developing an optimized protocol for growing 3D in vitro MCF10A breast acinar structures and investigating the biophysical properties of their endogenously­secreted BM. By performing size dependent dextran tracer experiments, we observed that the permeation regulation depends not only on dextran size but also on the BM­ maturation grade. Collagen IV proved to be a trigger of this sieving effect. To try to assess the role played by the BM in ensuring the mechanical stability of the lobules, we performed AFM indentation experiments on native breast acini and on decellularized BM shells. Surprisingly, both structures could withstand loads of up to 30 nN without any loss of structural integrity, indicating the fundamental importance of the BM both for regulating acinar molecule influx and for providing mechanical stability.

P.52 Tamás Haraszti

MPI for Intelligent Systems Advanced Materials and Biosystems

Spline­like interpolation in particle tracking microrheology We present and prove method to convert the mean­squared displacement to the complex shear modulus using a segment­wise spline­like interpolation and conversion with a dynamical sampling to control the quality of the result. P.53 Anne V. Schulze

MNS Forschungszentrum CAESAR Bonn

Higher­order architecture of rhodopsin in photoreceptors

Photoreception begins with the absorption of photons and the transformation into an electrical response. The rod photoreceptor is outstanding in its sensitivity: it can respond to single photons. Key to this sensitivity is a specialised structure of the cell which harbours all necessary transduction elements: the outer segment. It houses a stack of several hundred disk membranes, in which rhodopsin molecules are highly concentrated. The oligomeric state and organisation of rhodopsin within these disk membranes has been discussed intensively. It is a matter of debate, whether rhodopsin diffuses freely within the disk membrane or is somehow ordered. A higher organisation of rhodopsin has been proposed to enable the ultimate sensitivity of the rod photoreceptor. We recently elucidated the supramolecular structure of rhodopsin in the dark­ adapted mouse retina using cryo­electron tomography (cryo­ET). This higher­order structure comprises of rows of rhodopsin dimers, which come in pairs and are aligned parallel to the disk incisure. Intriguingly, in light­adapted retinas, no organisation of rhodopsin could be identified. This raises the question, whether the supramolecular structure of rhodopsin falls apart upon light exposure. We, therefore, aim to analyse retinal tissue, in which various defined fractions of rhodopsin have been bleached. The bleaching of intact retinas can be analysed quantitatively via UV­VIS­spectroscopy. Cryo­ET will be used to analyse the organisation of rhodopsin in retinas with defined amounts of bleached rhodopsin prior to cryo­preservation. P.54 Hamidreza Jafarinia

Mechanical Engineering Sharif University of Technology

Effect of hydrophobic mismatch and rigidity of proteins on the cluster formation of transmembrane proteins in biomembranes

Membrane proteins aggregation is a very important biological phenomenon in a variety of cell functions. It has been suggested that aggregation behaviour of membrane proteins is influenced by the shape of the hydrophobic domain of the proteins, proteins hydrophobic mismatch and bilayer curvature. However, in this study by means of coarse grained membrane simulations it has been found that in thermal equilibrium, protein­protein interactions also depend on protein rigidity and structural strength. Based on simulation results, we have observed stable large clusters even in the absence of hydrophobic mismatch between lipids and proteins. Interestingly, our results also indicate that proteins with low structural strength aggregate to form two­dimensional large clusters. Proteins with high structural strength, by contrast, form one­dimensional structures. Considering different size of proteins with various length of hydrophobic part, our simulations show different kinds of aggregation behavior. But even in the presence of hydrophobic mismatch, flexibility of proteins plays a significant role on the shape and size of the clusters. We have also investigated the effect of protein rigidity on the diffusive mobility of the lipids. Travel Information

Forschungszentrum Jülich Wilhelm­Johnen­Strasse 52428 Jülich Germany

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The closest and most convenient airports to travel to Jülich are Cologne­Bonn (CGN) and Düsseldorf (DUS), from where you can reach Jülich by train in 1.5­2 hours. If arriving on an intercontinental flight, you might consider flying to Frankfurt/Main airport (FRA, two hours by train to Jülich if you use the high speed train to Cologne). Cologne main train station also has an IATA code (QKL). We do not recommend that you fly to Maastricht Aachen (MST), Dortmund (DTM) or Düsseldorf Weeze (NRN), because of the poor public transport connections to Jülich from these airports.

From Düsseldorf airport to Jülich: From the railway station “Düsseldorf Flughafen” at the airport, travel to Cologne main train station and then continue on to Düren. Some trains go directly to Düren whereas other connections involve a change at Cologne main train station. Continue from Düren as described in "By train".

From Cologne Bonn airport to Jülich: From the railway station “Köln/Bonn Flughafen” at the airport, take the S13 to Cologne main train station and then continue with a regional train (RE or RB) or S12 to Düren. Continue from Düren as described in "By train". By train:

To plan your train ride to Jülich, the website of DB Bahn will provide you with itineraries starting from many destinations within Europe. You can find the English version of the website here. Please enter “Jülich” (spelled with the German "umlaut": ü) when typing in your destination.

From Paris, Brussels, or Aachen:

From Aachen main train station, take a regional train (RE or RB) to Düren main station and change in Düren to the train to Jülich (Rurtalbahn (RTB) in the direction of Jülich or Linnich). For accommodation in Jülich town centre, leave the train at the main station in Jülich (Hauptbahnhof).

From or via Cologne:

Take the regional train (RE or RB) or S12 to the main station in Düren (Hauptbahnhof). Change in Düren for the train to Jülich (Rurtalbahn (RTB) in the direction of Jülich or Linnich). For accommodation in Jülich town centre, leave the train at the main station in Jülich.

External visitors (Important notes):

From Jülich to the Campus To arrive at the FZJ Research Campus you can either take the bus SB11 (direction FZJ) or the RurTalBahn (RTB) from Jülich to Jülich Forschungszentrum (direction Düren). For each RTB that arrives at the stop Juelich FZ, there’s a shuttle bus that brings you in the campus. If you are an external visitor, we suggest you to arrive at least 30 min before the beginning of the event. You must take off the bus before entering the campus and go to the visitor center to register (Volunteers from the organizing team shall be there). Our suggested connection is the following:

Bahnhof/ZOB (Bus), Th, dep 08:15 Bus Bus Direction: Jülich 06.11.14 SB 11 Forschungszentrum Seecasino, Jülich

Forschungszentrum Th, arr 08:28 Feuerwehr, Jülich 06.11.14 • Important : Please bring an National ID Card e.g. Passport will be required to enter the campus via the Main Gate.

From the Campus to Jüelich:

The symposium should finish around 19:00. If you have to come back to the town by public transport, the last connection leaves at 19.16. Earlier connections can be found at: bahn.com

The SB11 line brings you to the Campus' train station, and then you can take the small train to the central station. Beware, there are very few trains.

The bus stop is highlighted on the map. When you enter in the bus, please give your green Guest Visitor card to the driver. He will give it back at the entry point.

Forschungszentrum dep 19:16 Bus Direction: Seecasino, Jülich Bus SB11 Forschungszentrum Bahnhof Forschungszentrum arr 19:18 (Bus) Bahnhof (Bus), Jülich Jülich Forschungszentrum dep 19:37 RTB89837 (Train) Jülich arr 19:41

Participation certificate: If you need a participation certificate, please send an email to biosoft . symposium @ gmail .com with title “participation certificate” at least 2 days before the meeting.