annual dutch meeting on molecular and cellular …...2009/09/09 · annual dutch meeting on...

Annual Dutch meeting on Molecular and Cellular Biophysics

List of contents

page I. Final programme

• Monday 28 September 2009 ------------------------------------------------------------ 2 • Tuesday 29 September 2009 ------------------------------------------------------------ 3

II. List of posters ------------------------------------------------------------------------------------- 5 III. Abstracts

• invited speakers ------------------------------------------------------------------------- 17 • oral presentations ----------------------------------------------------------------------- 25 • poster presentations -------------------------------------------------------------------- 37

IV. List of participants ---------------------------------------------------------------------------- 77

Practical organisation: Organisation committee: Ms. P.M.W. van Luling (FOM) Prof.dr. Nynke Dekker (TUD, chair)

Prof.dr. Herbert van Amerongen (WUR) Dr.ir. Sander Tans (AMOLF) Prof.dr. Antoinnette Killian (UU) Dr. Anna Akhmanova (EUR) Drs. Theo Saat (NWO-ALW) Dr. Rianne van Eerd (FOM)

e-mail: [email protected] phone: +31 (0)30 600 12 73 fax: +31 (0)30 601 44 06 Stichting FOM Postbus 3021 3502 GA UTRECHT

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Programme Monday 28 September 2009 09:30 - 10:30 Arrival / registration / coffee & tea Room: Kempenhal 10:35 - 10:45 Opening Room: Brabantzaal 10:45 - 11:15 I.1 T. Lee (Southampton, UK)

How lipids regulate membrane protein function Room: Brabantzaal

11:20 - 11:50 I.2 D. Rhodes (MRC, UK) Chromatin higher order structure and regulation of its compaction

Room: Brabantzaal

11:55 - 12:25 I.3 F. Taddei (INSERM, France) A systems approach to individual differences in longevity

Room: Brabantzaal

12:30 - 15:30 Lunch and Poster session 1 (odd numbers) Room: Kempenhal Room: session

Baroniezaal processes at the membrane-water interface

Brabantzaal mechanics of biomolecules

Diezehal microtubule-based transport

15:30 - 15:45 O.1 I. Kusters (RuG) Dual-color fluorescence-burst analysis (DCBFA): A new tool to study protein oligomerization at the membrane interface

O.4 A. Candelli (VU) Visualizing single-proteins on a single DNA molecule with Super-Resolution

O.7 J. Lindeboom (WUR) Arabidopsis cortical micro-tubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments

15:50 - 16:05 O.2 S. Bonnet (UU) Towards molecular transporters on lipid bilayers: Photocleavage of ruthenium complexes from model membranes, and thermal back-binding

O.5 F. Chien (LEI) Nucleosome stacking defines the structure of chromatin fibers

O.8 L. Kapitein (EMC) Sorting out neuronal polarity: mixed microtubu-les steer dynein-driven cargo transport into dendrites

16:10 - 16:25 O.3 R. Campen (AMOLF) Lipid hydration and its influence on membrane structure

O.6 J. Lipfert (TUD) The effect of torque on small-molecule binding to DNA

O.9 P. Shaklee (AMOLF) Collective dynamics of nonprocessive motors at the microtubule-membrane tube interface

16:30 - 17:00 Coffee and tea Room: Kempenhal Room: session

Baroniezaal polymerases in action

Brabantzaal cytoskeletal organisation

Diezehal membrane detection and signalling

17:00 - 17:15 O.10 M. Depken (VU) The nature of transcriptional pauses and their role in proofreading

O.13 C. Broederz (VU) Dynamics of transiently crosslinked cytoskeletal networks

O.16 S. de Keijzer (RU) Spatio-temporal analysis of membrane lipid remodeling during phagocytosis

17:20 - 17:35 O.11 T. Hoekstra (VU) Inducing T7 DNA polymer-ase to switch stochastically between exonuclease and polymerase activity

O.14 L. Laan (AMOLF) Reliable centering of dynamic microtubule asters in microfabricated chambers by pulling forces

O.17 T. van Zanten (IBEC) Direct nanoscale optical imaging of nucleation sites for cell adhesion

17:40 - 17:55 O.12 I. Vilfan (TUD) Real-time observation of transcription by RNA-dependent RNA polymerase transcription

O.15 M. Soares e Silva (AMOLF) Probing the structure and dynamics of actively contractile actin-myosin networks

O.18 F. van Hemert (LEI) Spatial restriction of GPCR-G protein mobility is essential for faithful chemotaxis

18:00 - 19:30 Dinner Room: Genderhal 19:30 - 20:00 Coffee Room: Kempenhal 20:00 - ... I.4 X. Zhuang (Harvard University, USA)

Nanoscopic imaging of biomolecules and cells Room: Brabantzaal


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Programme Tuesday 29 September 2009 Room: session

Baroniezaal protein-DNA interactions

Brabantzaal imaging cellular organisation

Diezehal protein folding and aggregation

09:00 - 09:15 O.19 M. Cristovao (EMC) Conformational changes in DNA and MutS during mismatch repair, analyzed by fluorescence spectroscopy

O.22 N. Dudkina (RuG) Dual-axis cryo-electron tomography: a powerful technique to explore intact mitochondria

O.25 K. Blank (RU) Triggering enzymatic activity with force

09:20 - 09:35 O.20 A. van der Heijden (LEI) Single basepair accuracy pre-diction of nucleosome posi-tioning in vitro and in vivo

O.23 M. Duits (UT) Spatiotemporal heterogeneous particle dynamics in living cells

O.26 D. Dekker (UT) Counterion induced higher order assemblies of alpha-synuclein fibrils

09:40 - 09:55 O.21 M. van Loenhout (TUD) Dynamics of RecA on single-stranded DNA

O.24 C. Tudor (UT) Fluorescence lifetime imaging of mechanical stress-activated signal transduction pathways in drosophila cells

O.27 X. Periole (RuG) Energetics insight into twisting a cross-b structure: implication for amyloid-fibrils polymorphism

10:00 - 10:30 Coffee and tea Room: Kempenhal Room: session

Baroniezaal nanoscale detection of biomolecules

Brabantzaal photosynthesis

Diezehal protein-protein interaction

10:30 - 10:45 O.28 X. Janssen (TU/e) The rotating magnetic parti-cles probe: A new technique to measure interactions between protein-coated particles and a surface

O.31 E. Romero (VU) Functional flexibility in the photosystem II reaction center: multiple charge separation pathways

O.34 R. Loris (VU Brussel) Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain

10:50 - 11:05 O.29 S. Kowalczyk (TUD) Detection of local protein structures along DNA using solid-state nanopores

O.32 T. Krüger (VU) Single-molecule spectroscopy reveals conformational switching in plant light-harvesting complexes

O.35 E. van Duijn (UU) Visualisation of protein complexes present during the chaperonin assisted folding cycle by ion-mobility mass spectrometry

11:10 - 11:25 O.30 M. Zevenbergen (TUD) Electrochemical detection of single molecules in nanofluidic devices

O.33 S. Ganapathy (LEI) Self-assembling natural and artificial light-harvesters

O.36 M. Merkx (TU/e) Protein conformational switches for intracellular imaging and diagnostics

11:30 - 14:00 Poster session 2 (even numbers) and lunch Room: Kempenhal 14:00 - 14:20 I.5 G. Wuite (VU)

Quantifying the physics inside the genome one molecule at a time Room: Brabantzaal

14:25 - 14:45 I.6 J. Herek (UT) Coherent control of natural and artificial photosynthetic light harvesting systems

Room: Brabantzaal

14:50 - 15:10 I.7 P. Bolhuis (UvA) Insights into protein conformational changes by multiscale modeling

Room: Brabantzaal

15:15 - 15:35 I.8 A. Akhmanova (EUR) 'Hitchhiking' to the microtubule plus end: identification of a molecular mechanism

Room: Brabantzaal

15:40 - 15:45 Closing remarks


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List of posters

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authors affiliation title

P.001 N. Laurens, C. Pernstich, R.W. Bellamy, L.E. Catto, Y.S. Kovacheva, S.E. Halford, G.J.L. Wuite

VU The impact of bending and twisting rigidity of DNA on protein induced looping dynamics

P.002 R. Buning, W.J.A. Koopmans, H. Neumann, J. Chin, T. Schmidt, J. van Noort

LEI Effect of histone acetylation on nucleosome dynamics revealed by spFRET microscopy

P.003 M. Tark, M. Bohn, D. Heermann, R. van Driel

UvA The third level of genome functioning: Chromatin folding

P.004 R. Vlijm, S. Hage, G.E. Chalkley, C.P. Verrijzer, C. Dekker

TUD Towards single-molecule experiments of chromatin-remodeling proteins

P.005 I. Vidic, C. Wyman, R. Kanaar, J. Lebbink

EUR Characterization of novel ssDNA binding proteins hSSB1 and hSSB2

P.006 J.F.A. van Vugt, M. de Jager, M. Murawska, A. Brehm, J. van Noort, C. Logie

RU ATP-dependent nucleosome repositioning is directed by the underlying DNA sequence

P.007 M.J. Wiggin, V. Tabard-Cossa, N.N. Jetha, C. Tropini, C. Feehan, A. Marziali

TUD Nanopore force spectroscopy

P.008 I.D.V. De Vlaminck, I.V. Vidic, J.L. Lebbink, M.T.J. van Loenhout, C.D. Dekker

TUD Torsional regulation of hRPA-induced dsDNA unwinding.

P.011 V. Raz, T.E. Abraham, S.M. van der Maarel, H. Tanke, R.W. Dirks

LUMC A multi-step aggregation of PABPN1

P.012 K. van Ommering, P.A. Somers, M. Koets, J.B.A. van Zon, J.J.H.B. Schleipen, L.J. van IJzendoorn, M.W.J. Prins

TU/e Tethered magnetic particle motion measured with evanescent wave microscopy

P.013 W.H. Roos, I. Gertsman, J. Johnson, G. Wuite

VU Breaking the chain mail: AFM study on the mechanical strength of a bacteriophage shell


List of posters

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P.014 A. Cambi, R. Diez-Ahedo, D. Normanno, C.G. Figdor, M.F. Garcia-Parajó

RU Biofunctional micropatterned surfaces to study the spatio-temporal organisation of integrins

P.015 C.A. Lopez, S.J. Marrink RuG MARTINI force field: extension to carbohydrates

P.016 T. Idema, C. Storm LEI A new perspective on the rich phase behavior of ternary lipid systems

P.017 H. van As, N. Homan, E. Gerkema, F.J. Vergeldt

WUR MRI of axial and radial water transport in plants

P.018 M.I. Snijder-van As, B. Rieger, V. Subramaniam, C.G. Figdor, J.S. Kanger

UT Membrane protein dynamics in single cell-cell contact

P.019 E.A. Golovina, H. van As, F.A. Hoekstra

WUR Membrane surface chemical stability correlates with desiccation tolerance and seed longevity

P.020 J. Zivkovic, L. Janssen, H.A. Heus, S. Speller

RU Atomic force spectroscopy on viral RNA - Protein system: RRE - Rev from HIV

P.021 B.F. van Oort, S. Witte, M.L. Groot, R. van Grondelle

VU Non-linear optical microscopy of leafs. First results and plans

P.022 K.K.M. Sweers, G.M.J. Segers-Nolten, K.O. van der Werf, M.L. Bennink, V. Subramaniam

UT Mechanical characterization of alpha-synuclein fibrils using AFM imaging and force spectroscopy

P.023 P. Gross, G.J.L. Wuite, E.J.G. Peterman VU Exploring the energy landscape of DNA melting during overstretching

P.024 C.H.J.M. Wintraecken WUR H-NS Polymerization in the absence of DNA


List of posters

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P.025 V.V. Pully, A.T.M. Lenferink, V. Subramaniam, A. van Blitterswijk, C. Otto

UT Dynamics of bone production by cells, as observed by Raman microscopy

P.026 J.M. Männik, P.J.G. Galajda, J.E.K. Keymer, C.D. Dekker

TUD The effect of physical confinement to the cellular structure in E.coli

P.027 G. Farge, N. Laurens, L. Dekker, M. Falkenberg, G.J.L. Wuite

VU Unravelling mitochondrial DNA organization

P.028 S. Shekhar, A. Klaver, C.G. Figdor, V. Subramaniam, J.S. Kanger

UT Studying the effect of manipulating phagosome routing on its maturation

P.029 C.C. Wu, H. Xu, C. Otto, D.N. Reinhoudt, R.G.H. Lammertink, J. Huskens, V. Subramaniam, A.H. Velders

UT Porous multilayer-coated AFM tips for dip-pen nanolithography of proteins

P.030 S. Brinkers TUD The dynamics of Tethered Particle Motion: How to interpret the mean squared displacement

P.031 L.H.F.M. Holtzer LEI Quantifying Dpp-endosome colocalization with Particle Image Cross Correlation Spectroscopy (PICCS)

P.032 A. Vila Verde, D.F. Frenkel AMOLF Formation of unusual micelles by bile salts

P.033 R.K.C. Campen, T.T.M. Ngo, M. Sovago, M. Bonn

AMOLF The chemical physics of gene transfection: Understanding the molecular mechanism of DNA/lipid interaction

P.034 M. van den Hout, G.M. Skinner, O.D. Broekmans, S. Hage, I.D. Vilfan, C. Dekker, N.H. Dekker

TUD Studying RNA using a combination of optical tweezers and solid-state nanopore

P.035 Y. Chung, I. Ruttekolk, P. Bovée-Geurts, M. Sinzinger, R. Brock

RU Quantitative analysis of molecular interactions in T cell receptor signaling by fluorescence correlation spectroscopy (FCS) and confocal microscopy


List of posters

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P.036 C. Tudor, A. Esseling-Ozdoba, C.G. Figdor, J. Kanger, V. Subramaniam

UT A fret-film study reveals the interaction between alcam and actin as a potential regulator of alcam binding activity

P.037 A. Pandit, P.K. Wawrzyniak, A.J. van Gammeren, F. Buda, S. Ganapathy, H.J.M. de Groot

LEI NMR secondary shifts of the light-harvesting 2 complex reveal functional motifs due to local frustration induced by its higher-order assembly

P.038 M.A. Schlager, L.C. Kapitein, I. Grigoriev, P.S. Wulf, N. Keijzer, A. Akhmanova, C.C. Hoogenraad

EMC Bicaudal-D like protein madmax-1 organizes dynein-dependent secretory transport and neural development

P.039 B.A. Berghuis, R.B.M. Koehorst, R.B. Spruijt, A. van Hoek, H. van Amerongen

WUR Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy

P.040 P.M. Dijkman, R.B.M. Koehorst, M. Mozzo, F. Passarini, R. Croce, H. van Amerongen

WUR Non-photochemical quenching in mutants of light-harvesting complex II

P.041 R.B.M. Koehorst, S. Laptenok, B. van Oort, A. van Hoek, R.B. Spruijt, I.H.M. van Stokkum, M.A. Hemminga, H. van Amerongen

WUR Profiling of polarity and dynamics in a protein-lipid system: A picosecond fluorescence study using streak camera detection

P.042 Z. Lansky, M. Janson WUR Focusing on ase1-mediated microtubule bundling

P.043 J.R. Alvarado, J. Nguyen, M. Soares e Silva, G. Koenderink

AMOLF Hard constraints on cellular organization

P.044 V. Breukels, G.W. Vuister RU Calcium binding of CBD2 is sensed structurally and dynamically throughout the molecule

P.045 F. Tostevin, P.R. ten Wolde AMOLF Mutual information between trajectories of biochemical networks

P.046 W.H. de Ronde, F. Tostevin, P.R. ten Wolde

AMOLF Information transmission for time-varying signals in biochemical networks


List of posters

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P.047 I.K. Piechocka AMOLF Structural hierarchy governs fibrin gel mechanics

P.048 J. Vreede, R.T. Dame UvA Conformations of the H-NS dimerization domain

P.049 M.H. Shabestari, I. Sepkhanova, M. Drescher, N.J. Meeuwenoord, R.W.A.L. Limpens, R.I. Koning, D.V. Filippov, M. Huber

LEI Monitoring Alzheimer amyloid peptide aggregation by EPR

P.050 J.M. Salverda, A.V. Patil, G. Mizzon, S. Kuznetsova, G. Zauner, N. Akkilic, J.J. Davis, G.W. Canters, H.A. Heering, T.J. Aartsma

LEI Fluorescent cyclic voltammetry of immobilized azurin: a direct observation of thermodynamic and kinetic heterogeneity

P.051 J. Teapal, E. Janson WUR Building microtubule networks in fission yeast

P.052 K. Wagner, G. Moolenaar, J. van Noort, N. Goosen

LEI Single-molecule analysis reveals two separate DNA binding domains in the Escherichia coli UvrA dimer

P.053 M.J. Jacobs, J. Zivkovic, M. Nassal, S. Speller

RU Different elastic properties of two types Hepatitis B virus capsids measured by AFM

P.054 M.H. Shabestari, F. Scarpelli, M. Drescher, H. Eerkens, T. Rutters-Meijneke, A. Holt, D.T.S. Rijkers, J.A. Killian, M. Huber

LEI Aggregation of transmembrane peptides studied by Spin-Label EPR

P.055 M. Escalante, L. Lenferink, Y. Zhao, N. Tas, J. Huskens, C.N. Hunter, V. Subramaniam, C. Otto

UT Energy transport in engineered nanometer arrays of light harvesting antenna complexes

P.056 J. Oomens, B. Redlich, J.D. Steill, N.C. Polfer, M.J. van Stipdonk

Rijnhuizen Peptide fragment structures in MS determined by IR spectroscopy

P.057 M. Schor, P.G. Bolhuis UvA Self-assembly of silk-based proteins


List of posters

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P.058 S.M.J. Kalisch, J. Husson, L. Laan, M. Dogterom

AMOLF Towards regulated microtubule dynamics under constant force

P.059 A. Andreoni, L.C. Tabares, A.W.J.W. Tepper, T.J. Aartsma, G.W. Canters

LEI Fluorescence correlation spectroscopy for the analysis of electron transfer in proteins

P.060 L.C. Tabares, M. Elmak, D. Kostrz, C. Dennison, T. Aartsma, G.W. Canters

LEI Picturing the activity of nitrite reductase at the single molecule level

P.061 A. Elmalk, L. Tabares, G. Canters, T. Aartsma

LEI Modification of single molecule fluorescence emission near gold nanoparticles

P.062 J.T. Holthausen, H. Sanchez, M. Modesti, T. Thorslund, S. West, C. Wyman, R. Kanaar

EMC Single-molecule analysis of RAD51 filament dissociation from double strand DNA

P.063 H.S. van der Honing, H. Kieft, N.C.A. de Ruijter, A.M.C. Emons, T. Ketelaar

WUR Does the actin filament bundling protein villin regulate cytoplasmic organization and stiffness in plant cells?

P.064 M. de Groot, B. Haslam, J.F. de Boer VU High NA optical coherence phase microscopy

P.065 A.P. Bechtluft, A. Kedrov, D.J. Slotboom, N. Nouwen, S. Tans, A.J.M. Driessen

AMOLF A mutant of the protein export-dedicated chaperone SecB defective in the holdase function

P.066 M. Skinner, M. van den Hout, O. Broekmans, C. Dekker, N.H. Dekker

TUD Distinguising double- and single-stranded nucleic acid molecules using solid-state nanopores

P.067 A. Pezzarossa, J. Piehler, T. Schmidt LEI Role of plasma membrane structuring on Interferon receptor assembly & signaling

P.068 D.H. de Jong, L.V. Schäfer, S.J. Marrink

RuG Coarse grained molecular dynamics simulations of transmembrane peptides in a model raft lipid membrane


List of posters

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P.069 T.L.J. Tian WUR Blue light induced NPQ in synechocystis PCC 6803

P.070 J. te Riet, A. Cambi, S. Speller, C.G. Figdor

RU AFM force spectroscopy to study the recognition of pathogens by immune cells

P.071 H. Sanchez, R. Kanaar, C. Wyman EUR Molecular recognition combining scanning force and fluorescence microscopy

P.072 B.S. Stuhrmann, F.H. Huber, C.V. Vogt, S.E. Ehrig, J.K. Käs

AMOLF Modeling and mimicking self-organized network growth in migrating cells

P.073 F. Passarini, E. Wientjes, R. Hienerwadel, R. Croce

RuG CP24, light harvesting and photoprotection

P.074 S.G.M. Calmat, H. van Herrikhuizen, C.F. Schmidt, E.J.G. Peterman, S.M. van der Vies

VU Chaperonin-assisted folding of the bacteriophage T4 major capsid protein

P.075 M. Baclayon, W. Roos, G. Shoemaker, C. Uetrecht, S. Crawford, M. Estes, V. Prasad, A. Heck, G. Wuite

VU Mechanical and structural properties of Norwalk virus capsid revealed by scanning force microscopy

P.076 I.E. Wientjes, H. van Amerongen, R. Croce

RuG Energy transfer in higher plant Photosystem I

P.077 I.I. Idriss, S.G.M. Calmat, E.J.G. Peterman, S.M. van der Vies

VUMC Visualization and characterization of dynamic events of the chaperonin folding machine by using Föster resonance energy transfer

P.078 A.M. de Jong, A. Jacob, A. El Fattahi, F. Lambert, X.J.A. Janssen, L.J. van IJzendoorn, M.W.J. Prins

TU/e Force induced dissociation of protein bonds

P.079 S.F. Fenz, R.M. Merkel, K.S. Sengupta LEI Biomembrane adhesion by mobile receptor-ligand pairs


List of posters

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P.080 M. Noutsou UMCU Critical domains in the tumor suppressor Axin1 are natively unfolded

P.081 F.P. Pedaci, S.K. Klijnhout, J.W.J.K. Kerssemakers, N.H.D. Dekker

TUD Optical torque wrench for single molecule studies

P.082 L. Hartsuiker, R.G. Rayavarapu, W. Petersen, S. Manohar, T.G. van Leeuwen, C. Otto

UT Raman imaging of single gold nanoparticles in live cancer cells

P.083 A. Janulevicius, M. van Loosdrecht, C. Picioreanu

TUD Effect of cell flexibility on alignment of model myxobacteria

P.084 V. Koning, N. Verhart, R. Purchase, R.J. Silbey, S. Völker

LEI Exciton dynamics and energy disorder in photosynthetic light-harvesting complexes

P.085 P.J. Laverman, J. Lipfert, I. Koster, M.A. Bjornsti, N.H. Dekker

TUD Single molecule magnetic tweezers study of topoisomerase IB activity in the presence of anti-cancer drugs

P.086 M.G.J. de Vos, F.J. Poelwijk, N. Tanyi, N. Battich, S.J. Tans

AMOLF The role of genotype by environment interactions in adaptive evolutionary trajectories

P.087 N. Hermans, C. Claire, R. Kanaar, J. Lebbink

EMC UvrD: Unwinding DNA in mismatch repair

P.088 S. Laptenok, S. Krumova, R. Koehorst, J.W. Borst, A. van Hoek, Z. Gombos, H. van Amerongen

VU Discriminating between chlorophyll and phycobilisome in FLIM images upon two-photon excitation.

P.089 M.A. Marosvolgyi, H.J. van Gorkom LEI Cost and colour of photosynthesis

P.090 S. Ramadurai, A. Holt, V. Krasnikov, J. Killian, B. Poolman

RuG Effect of hydrophobic mismatch and lipid composition on the mobility of bilayer spanning proteins and lipids


List of posters

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P.091 P.S. Singh, E.D. Goluch, M.A.G. Zevenbergen, B.L. Wolfrum, A.W.J. Tepper, H.A. Heering, G.W. Canters, S.G. Lemay

TUD Electrochemical sensing using nanofluidic devices

P.092 W.K. den Otter, M.R. Renes, W.J. Briels

UT Self-assembly of triskelions into closed polyhedral cages

P.093 N.J.M.S. Nusrat Jahan, A.M.E. Elmalk, H.H. Heering, G.W.C. Canters, T.J.A. Aartsma

LEI Probing the reactivity of nitrite reductase via dynamic protein-protein interaction

P.094 S.A. Muntean, A.V. Lyulin, M. Kemper, L.J. van IJzendoorn, M.W.J. Prins

TU/e Molecular dynamics simulations of polystyrene surfaces for biomedical applications

P.095 A.R. Hall, S. van Dorp, S.G. Lemay, C. Dekker

TUD Direct measurement of forces on protein-coated DNA molecules in solid-state nanopores

P.096 S. Lindhoud, A.H. Westphal, C.P.M. van Mierlo

WUR Multiple FRET studies reveal molecular features of the off-pathway folding intermediate of apoflavodoxin

P.097 S.W. Kowalczyk, L. Kapinos, R.Y.H. Lim, C. Dekker

TUD In vitro measurements of transport across a single biomimetic nuclear pore complex

P.098 T.R. Sokolowski, L.P. Bossen, T.W. Miedema, P.R. ten Wolde

AMOLF Using GFRD to study pattern formation due to the interplay of active and passive transport

P.099 N.B. Becker, R. Everaers AMOLF DNA nanomechanics: Analyzing DNA-protein tension

P.100 M.A.M. Franker, A. Candelli, M. Modesti, G. Wuite, E.J.G. Peterman

VU A novel assay to study Rad51 dynamics on ssDNA in real time

P.101 B.D. Drop, M. Birungi, E.J. Boekema, R. Croce

RuG Photosystem I of chlamydomonas reinhardtII


List of posters

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P.102 M. Kemper, L.J. van IJzendoorn, S.A. Muntean, A. Lyulin, M.W.J. Prins

TU/e Characterizing polystyrene for studying protein interactions

P.103 M.B.M. Meddens, A. Esseling-Ozdoba, J. Witsenburg, R. Brock, A. Cambi, C.G. Figdor

RU Microscopic evaluation of the role and dynamics of CD6 in T cell activation using microprinted surfaces

P.104 A. Mashaghi Tabari, P. Bechtluft, S.J. Tans

AMOLF Unraveling chaperonin activity of trigger factor at the single molecule level

P.105 M. Kogenaru, S. Tans AMOLF A synthetic regulatory cascade for quantitative studies

P.106 A.J.W. te Velthuis, S. Hage, S.H.W. van den Worm, E.J. Snijder, N.H. Dekker

LUMC Towards a reconstituted SARS-coronavirus replication complex at single molecule level

P.107 P. Galajda, R. Woolthuis, J. Mannik, F. Hol, C. Dekker, J. Keymer

TUD Cooperation and cheating in bacterial metapopulations

P.108 B. van der Vaart, A. Akhmanova, I. Grigoriev, S. Montenegro Gouveia, J. Demmers, N. Galjart

EMC SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that bind to EB1 and its homologues

P.109 Y. Nakasone, M. Avila-Pérez, J. Hendriks, K.J. Hellingwerf

UvA The biological relevance of photochemical parameters and microscopic rate constants of the light-sensor protein YtvA from Bacillus subtilis

P.110 S.M. Nabuurs, M. Boone, H. Venselaar, J.H. Robben, G. Vriend, G.W. Vuister, P.M.T. Deen

RU Aquaporin-2 binds LIP5: Insight into the structural interaction of a cargo protein to the ESCRT complex

P.111 Z. Huang, S.P. Donkers, N.H. Dekker TUD Zero-mode waveguides: A powerful tool for single-molecule optical studies

P.112 F. Hol, P.J. Galajda, J. Männik, C. Dekker, J.E. Keymer

TUD Evolution of antibiotic resistance in patchy ecosystems


List of posters

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P.113 J.H. Hendrix, Y. Engelborghs, Z. Debyser

KULeuven LEDGF/p75 switches from a dynamic to a tight chromatin interaction upon binding to HIV-1 integrase

P.114 A.W.J.W. Tepper LEI Electrical contacting of redox enzymes through DNA-directed self-assembly

P.115 P.J. Verschure, L.C.M. Anink, D.G.E. Piebes, M.C. Brink, A. Schwabe, F.J. Bruggeman

UvA Epigenetic gene regulation of the eukaryotic genome: A systems biology approach using synthetic cell systems

P.116 B. van den Broek, T.H. Oosterkamp, S.J.T. van Noort

LEI A multifocal 2-photon fluorescence setup for single-molecule tracking of chromatin dynamics in living cells

P.117 T.S. Shimizu, Y.T. Tu, H.C.B. Berg AMOLF Modular coupling in E.coli chemotaxis revealed by FRET with time-varying stimuli


List of posters

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Abstracts invited speakers

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I.1 How lipids regulate membrane protein function T. Lee Medicine, Health and Life Sciences, University of Southampton, SOUTHAMPTON, United Kingdom To what extent can our understanding of how water molecules interact with a water-soluble protein help us to understand how lipid molecules interact with a membrane protein? A first shell of water molecules is found covering the surface of a water-soluble protein, and water molecules are also found buried within the structure. Interactions of these water molecules with the protein help define its structure, and thus its function. Similarly, the surface of the transmembrane region of a membrane protein is covered with a first-shell of perturbed lipid molecules, referred to as the lipid annulus. Binding constants of lipids to these annular sites can be determined using a fluorescence quenching method, studying the quenching of the fluorescence of Trp residues in the protein by lipids with bromine-containing chains. Such studies show that the lipid annulus is heterogeneous - the mechanosensitive channel MscL, for example, contains a 'hot-spot' where anionic lipids bind with high affinity. Binding of anionic lipids to this hot-spot has a large effect on the flux through the MscL channel. Lipid molecules can also be found buried within the structure of a membrane protein, for example, at protein-protein interfaces in multimeric proteins. An example is provided by the homotetrameric potassium channel KcsA. The crystal structure of KcsA by MacKinnon shows an anionic lipid molecule bound at each monomer-monomer interface. Occupation of these sites by anionic lipid molecules is not required for tetramer formation, but is important for function. The open probability of the channel increases markedly with increasing anionic lipid content in the membrane, three of the four inter-subunit binding sites having to be occupied by anionic lipid for the channel to be open.



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I.2 Chromatin higher order structure and regulation of its compaction D. Rhodes MRC Laboratory of Molecular Biology, CAMBRIDGE, United Kingdom During the past decade it has become evident that histone and DNA modifications are key regulators of all nuclear processes whose substrate is DNA. Whilst the effects of, for instance, histone post-translational modification on transcription are well-documented, there is no mechanistic understanding of how such modification regulate chromatin condensation directly, or indirectly. Such an understanding is dependent on knowledge of the three-dimensional structure of chromatin. Although the structure of the first level of DNA folding, the nucleosome core, is known at atomic resolution, the structure of the second level of folding, whereby a string of nucleosomes folds into a fibre with an approximate diameter of 30 nm - the '30-nm' chromatin fibre, remains undetermined. I will describe our studies on the higher orders structure of chromatin with two primary aims: 1) Determination of the structure of the '30nm' chromatin fibre to provide an understanding of

fibre topology and nucleosome-nucleosome interactions. 2) Biophysical characterization of the effects of the linker histone and histone modifications on

the compaction and stability of chromatin higher order structure.



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I.3 A systems approach to individual differences in longevity F. Taddei Génétique Moléculaire Evolutive et Médicale, INSERM, PARIS, France The long-appreciated high variability of individual longevity is not trivially accounted for. While genetic and environmental differences play a major role, laboratory experiments demonstrated that even in absence of such differences, longevity distributions follow the same pattern as observed by demographers: an exponential increase in death probability with age that decays at old age. Our aim is to analyze and reconstruct key observations from human population in well-controlled quantitative large cohort experimental model systems with short generation time where each individual is followed throughout its lifespan. We therefore study in microbial systems and in nematodes the stochastic, molecular and dynamic phenomena underlying aging through iteration between observations, mathematical modelling and experiments to elucidate the mechanisms underlying aging kinetics and to develop new prevention or treatment strategies.



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I.4 Nanoscopic imaging of biomolecules and cells X. Zhuang Harvard University, Howard Hughes Medical Institute, CAMBRIDGE, United States of America When combined with a large repertoire of fluorescent probes, light microscopy allows the direct visualization of molecular processes in living organisms. The diffraction-limited resolution of fluorescence microscopy, however, leaves many biological structures too small to be observed in detail. We have recently developed a new imaging method, stochastic optical reconstruction microscopy (STORM), which breaks the diffraction limit and allows for super-resolution imaging. STORM uses single-molecule imaging and photo-switchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules, thereby breaking the diffraction limit. Using this approach, we have imaged cellular structures with a few tens of nanometer resolution in 3D. In this talk, I will discuss the general concept, recent technical advances, and biological applications of STORM. I will also describe our studies of protein-nucleic acid interactions using single-molecule fluorescence resonance energy transfer (FRET). Single-molecule FRET is a spectroscopy approach that allows us to probe nanoscopic dynamics within biomolecules or molecular complexes. We have used this approach to study various nucleic acid interacting enzymes. In this talk, I will describe our recent work on HIV reverse transcriptase, which catalyzes a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. Using a single-molecule FRET, we have observed highly dynamic interactions between RT and it nucleic acid substrates, which are not only critical for the function of the enzyme, but also suggestive of new mechanisms of anti-HIV drugs.



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I.5 Quantifying the physics inside the genome one molecule at a time G.J.L. Wuite VU University Amsterdam, Molecular Biophysics Group, AMSTERDAM, Netherlands The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in organizing, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA constantly rearranging it. In order to elucidate these kind of processes we perform single-molecule experiments on model systems such as restriction enzymes, DNA polymerases and repair proteins. The data we use to extract forces, energies and mechanochemistry driving these dynamic transactions. The results obtained from these model systems are then generalized and thought to be applicable to many DNA-protein interactions. In particular, I will report experiments that use a ccombination of fluorescence microscopy and optical tweezers to: (i) directly visualize the DNA overstretching transition and demonstrate that its origin is the cooperative melting of the two DNA strands. In the experiments we use intercalating dyes and fluorescently labeled single-stranded binding proteins to specifically visualize double- and single-stranded segments in DNA molecules undergoing the transition. Our data unambiguously show that the overstretching transition comprises a gradual conversion from double-stranded to single-stranded DNA, in agreement with the force-induced melting model. (ii) image single Rad51 monomers disassembling from nucleoprotein filaments formed on double-stranded DNA molecules. We quantified the fluorescence of disassembling nucleoprotein patches and found that disassembly occurs in bursts interspersed by long pauses. By applying external tension to these filaments, we find that disassembly slows down and can even be stalled. Upon relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results imply that tension-dependent Rad51 disassembly takes place only from filament ends, after tension-independent ATP hydrolysis.



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I.6 Coherent control of natural and artificial photosynthetic light harvesting systems J.L. Herek MESA+ Institute for Nanotechnology, Biomolecules and nanostructures, University of Twente, ENSCHEDE, Netherlands Coherent control aims to steer a quantum mechanical system toward a desired outcome by exploiting interference effects. In a closed-loop experiment combining adaptive femtosecond pulse shaping, molecular feedback, and a learning algorithm (see figure), even systems considered intractable on an ab initio quantum-mechanical level can be explored. The applicability of this approach to biomolecules has opened new access for spectroscopic studies that will provide insight into how nature designs systems for optimal performance. This talk will highlight studies of the spectroscopy, dynamics, and control of functional molecules for artificial photosynthesis, designed to mimic natural light harvesting antennas.



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I.7 Insights into protein conformational changes by multiscale modeling P. Bolhuis University of Amsterdam, Computational Physics and Chemistry, AMSTERDAM, Nederland Molecular dynamics simulation can in principle provide useful predictions of (sub)cellular processes on an atomistic level, that are complementary to experiment. However, in practice, molecular dynamics is far from fulfilling this promise due to the large size of a cellular system (1010 atoms) and the long times involved (at least on the order of seconds). The multiscale modeling framework aims to circumvent this problem by envisioning hierarchical levels of description, each appropriate for certain length and time scales of a biophysical system. The challenge for the computer simulator is to develop the right description for each length or time scales, to link different scales together and to develop efficient sampling algorithms. One approach is to start with the atomistic level molecular dynamics description and use this to define a coarser grained description. Another approach is to realize that many processes such as (un)folding and other conformational changes in proteins in fact are rare events caused by high free energy barriers between stable states. To overcome such barriers, many techniques have been developed, e.g. replica exchange, metadynamics, and transition path sampling. In this presentation I will introduce the multiscale modeling framework and several simulation methods. In addition, I will exemplify these methods on interesting applications such as conformational changes in (signalling) proteins, protein fiber self-assembly, and cooperativity in G-coupled receptors. In each case we can gain new insight from these simulations. In the first case, about the mechanism and reaction coordinate of protein conformational changes. In the second case, about possible fiber structures and assembly mechanisms. And in the last case, about a possibly novel physical origin for cooperativity in protein receptors.



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I.8 'Hitchhiking' to the microtubule plus end: identification of a molecular mechanism A. Akhmanova Department of Cell Biology, Erasmus Medical Center, ROTTERDAM, Netherlands Microtubules are dynamic polymers that control many aspects of cell polarization, migration and division. Growing microtubules accumulate at their ends a set of regulators collectively known as microtubule plus-end tracking proteins, or +TIPs, which include the tumour suppressor APC, microtubule-actin cross-linking factor MACF/ACF7 and microtubule stabilizers and destabilizers, such as CLASPs and MCAK. These proteins participate in the feedback between microtubules, cortical actin filaments and focal adhesions and are important for the regulation of microtubule dynamics and cell polarity. Recently, it became clear that many +TIPs recognize the ends of growing microtubules through End Binding (EB) proteins, such as EB1. Using biochemical and structural approaches as well as live cell imaging and in vitro reconstitution, we uncovered a common principle underlying the interaction between EB1 and its numerous binding partners. We identified a short sequence motif necessary and sufficient for tracking growing microtubule ends in an EB1-dependent manner both in vitro and in cells. This finding provides a rationale for introducing mutations to specifically disrupt microtubule tip localisation of different factors and thus determine the importance of microtubule tip association of these multifunctional proteins.


Abstracts oral presentations

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O.01 Dual-color fluorescence-burst analysis (DCBFA): A new tool to study protein oligomerization at the membrane interface I. Kusters1, G. van den Bogaart2, A. Kedrov1, V. Krasnikov3, F. Fulyani4, B. Poolman4, A. Driessen1

1Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands, 2Max Planck Institute for biophysical Chemistry, GÖTTINGEN, Germany, 3Department of Optical condensed matter physics, University of Groningen, Netherlands, 4Department of Biochemistry, University of Groningen, Netherlands DCBFA enables the measurement of protein binding to membrane receptors at low nanomolar concentration. Fluorescent bursts that reside from the fluorescently-labeled membrane proteins embedded in liposomes that diffuse through the focal volume of a confocal microscope will coincide with those from the bound proteins labeled with a second, spectrally well separated fluorophore. With DCFBA, single liposome resolution is obtained permitting investigations of protein oligomerization in membranes. We determined the oligomeric state of SecA bound to its membrane receptor SecYEG, the bacterial protein conducting channel. Different oligomeric states of SecYEG-bound SecA could be observed depending on the stage of protein translocation.

O.02 Towards molecular transporters on lipid bilayers: Photocleavage of ruthenium complexes from model membranes, and thermal back-binding S. Bonnet1, J.A. Killian2, R.J.M. Klein Gebbink1 1Debye Institute for Nanomaterials Science, UTRECHT, Netherlands 2Bijvoet Center For Biomolecular Research, UTRECHT, Netherlands Our goal is to use lipid bilayers as a support where individual molecules are transported unidirectionally. We have synthesized ruthenium complexes [Ru(tpy)(bpy)(L)]2+, where L is a ligand linked to cholesterol. Upon incorporation into LUVs, vesicles are obtained with the ruthenium cations at the lipid-water interface. Upon visible light irradiation, the Ru-L bond is cleaved, which releases ruthenium from the bilayer. The temperature and concentration dependence of the thermal back-binding of ruthenium to the membrane has also been studied. Overall, the (photo)coordination properties of ruthenium at the bilayer will be discussed, as well as the potential applications of such systems in the context of molecular motion.

O.03 Lipid hydration and its influence on membrane structure R.K.C. Campen, M. Sovago, A. Ghosh, M. Bonn FOM Institute for Atomic and Molecular Physics, AMSTERDAM, Netherlands Many of the properties of membranes depend on the structure and dynamics of water molecules at the membrane/water interface. We report a series of studies of the membrane/water interface with a non-invasive optical technique (vibrational sum frequency generation spectroscopy), that is sensitive only to a monolayer of water at the membrane/water interface. We demonstrate experimentally the complexity of the interaction between water and the lipid head groups and show this interaction depends intricately on lipid type; femtosecond dynamics studies reveal water heterogeneity not observed for other aqueous interfaces. These observations illustrate that, for fast structural dynamics, membrane-bound water is an inherent part of the membrane.



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O.04 Visualizing single-proteins on a single DNA molecule with super-resolution A. Candelli, P. Gross, G.J.L. Wuite, E.J.G. Peterman VU University Amsterdam, AMSTERDAM, Netherlands DNA-proteins interactions can be studied with great detail once precise and dynamic control over the mechanical state of a single DNA molecule can be achieved together with direct visualization of the biological process taking place. Our custom-built experimental setup combines optical trapping and wide-field epifluorescence microscopy and is capable of localizing DNA-bound proteins down to nanometer-accuracy while controlling the tension applied on the DNA at the PicoNewton level. We investigated the effect of the DNA dynamics on the maximum obtainable accuracy for the localization of single DNA-bound fluorescent proteins and we identified a force regime in which single-protein localization down to the nanometer-level is possible.

O.05 Nucleosome stacking defines the structure of chromatin fibers F. Chien1, M. Kruithof1, A. Routh2, D. Rhodes2, J. van Noort1 1LION, Leiden University, LEIDEN, Netherlands 2MRC Laboratory of Molecular Biology, CAMBRIDGE, United Kingdom Eukaryotic DNA and histones form dense 30 nm chromatin fibers. This compaction is driven by stacking of nucleosomes and has been implicated to regulate gene expression. We investigated the mechanical properties of reconstituted chromatin fibers with magnetic tweezers. The fibers can be characterized by three springs in series: a worm like chain (WLC) of the flanking DNA, a Hookean spring of the 30 nm fiber, and a WLC of the ruptured fiber. The data reveal internucleosomal interaction energy 17kT, which is salt dependent and correlated to the stiffness of the 30 nm fiber. The length fluctuations between 3 pN and 6 pN show multiple statistically independent unstacking events. The data are the first to reveal the folding of chromatin fibers.

O.06 The effect of torque on small-molecule binding to DNA J. Lipfert, S. Klijnhout, N.H. Dekker Kavli Institute of Nanoscience, DELFT, Netherlands DNA-binding small molecules are ubiquitous both in vivo and in vitro, e.g. as anti-microbial or chemotherapeutic drugs, biochemical probes, or imaging dyes. Here we use single molecule magnetic tweezers to discriminate between different types of small-molecule binding, exploiting their ability to control stretching force and torque while monitoring DNA length and twist. As a test case, we directly probe ethidium intercalation in real-time for different concentrations, and find that it unwinds DNA by 28 ± 3 degrees per ethidium molecule, is torque-dependent, and reduces the DNA torsional stiffness. In contrast, the minor groove binding anti-microbial drug netropsin overwinds the DNA, indicating that we can distinguish these mechanisms.



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O.07 Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments J.J. Lindeboom Wageningen University, WAGENINGEN, Netherlands Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. We provide evidence that cortical microtubules position the delivery of CESA complexes to the plasma membrane and interact with small CESA-containing compartments by a mechanism that permits motility driven by microtubule depolymerization. The association of CESA compartments with cortical microtubules was greatly enhanced during osmotic stress and other treatments that limit cellulose synthesis. On recovery from osmotic stress, delivery of CESA complexes to the plasma membrane was observed from microtubule-tethered compartments.

O.08 Sorting out neuronal polarity: mixed microtubules steer dynein-driven cargo transport into dendrites L.C. Kapitein1, M. Schlager1, M. Kuijpers1, P.S. Wulf1, M. van Spronsen1, F.C. MacKintosh2, C.C. Hoogenraad1 1Erasmus Medical Center, ROTTERDAM, Netherlands 2Vrije Universiteit, AMSTERDAM, Netherlands Neurons employ active transport driven by molecular motors to sort cargo between axons and dendrites. However, the basic traffic rules governing polarized transport are unclear. Here we show that the microtubule minus-end directed motor dynein is required for the polarized targeting of dendrite-specific AMPA receptors. To directly examine how dynein motors contribute to polarized dendritic cargo transport, we established a trafficking assay in hippocampal neurons to selectively probe specific motor protein activity. This revealed that, unlike kinesins, dynein motors drive cargo selectively into dendrites, governed by their mixed microtubule array. We propose that dynein establishes the initial sorting of dendritic cargo.

O.09 Collective dynamics of nonprocessive motors at the microtubule-membrane tube interface P.M. Shaklee1, L. Bourel-Bonet2, M. Dogterom3, T. Schmidt4 1AMOLF/ Leiden University, AMSTERDAM, Netherlands, 2Equipe de Biovectorologie, ILLKIRCH, France, 3AMOLF, AMSTERDAM, Netherlands, 4Leiden University, LEIDEN, Netherlands Effective intracellular transport relies on collective coordination of motors. Here, we directly examine collective motor dynamics of processive (P) and nonprocessive (NP) motors at the microtubule(MT)-membrane tube interface in vitro. FCS reveals directed movement as P motors walk along underlying MTs (<500nm/s) and accumulate at membrane tube tips. However, NP motors are purely diffusive, decorating the entire MT lattice with diffusion constants 100x smaller than that of freely diffusing lipid-motor complexes: 1µm2/s (from FRAP). Because NP motors dynamically bind and unbind they maintain an interaction and high density of motors on the MT, likely necessary to drive bidirectional tube dynamics reported previously (Shaklee et al 2009).



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O.10 The nature of transcriptional pauses and their role in proofreading M. Depken1, A. Galburt2, M.R. Parrondo3, W. Grill4 1VU University Amsterdam, AMSTERDAM, Netherlands, 2Washington University, ST. LOUIS, United States of America, 3Universidad Complutense, MADRID, Spain, 4MPI-PKS/CBG, DRESDEN, Germany The RNA polymerase is a protein molecular machine that transcribes genetic information from DNA into RNA with remarkably high fidelity. The growth of the RNA transcript is frequently interrupted by pauses, the detailed nature of which has remained controversial. Based on theoretical modeling and comparison to single molecule data, we argue that the majority of such pauses are due to a diffusive state where the polymerase slides backward along the transcript. We further show how these pause states can be used for proofreading the nascent RNA transcript, and discuss the inherent competition between velocity, efficiency, and fidelity during transcription.

O.11 Inducing T7 DNA polymerase to switch stochastically between exonuclease and polymerase activity T.P. Hoekstra, P. Gross, H. Geertsema, E.J.G. Peterman, G.J.L. Wuite Vrije Universiteit Amsterdam, AMSTERDAM, Netherlands T7 DNA polymerase (DNAp) can, besides catalyzing polymerization, proofread' and excise erroneously incorporated nucleotides which results in high fidelity replication. The incorporation and excision of nucleotides on a substrate DNA held in optical tweezers can be followed over time since the elastic properties of double- and single-stranded DNA differ drastically. At DNA tensions below 30 pN polymerase activity is observed while applying forces above 40 pN to the DNA results in DNAp performing exonucleolysis. Here we show that in between those forces, a region exists where DNAp switches back-and-forth between polymerization and exonucleolysis immediately revealing all the different states of the reaction pathway.

O.12 Real-time observation of transcription by RNA-dependent RNA polymerase transcription I.D. Vilfan1, S. Hage1, D.H. Bamford2, M.M. Poranen2, N.H. Dekker1 1Technical University of Delft, DELFT, Netherlands 2University of Helsinki, HELSINKI, Finland We examine real-time transcription kinetics of viral RNA-dependent RNA polymerases (RdRPs) using single-molecule techniques. During transcription, RdRP binds to the antisense strand of a dsRNA genome and synthesizes a new sense strand. As?RdRP progresses, it unwinds the dsRNA, in the process releasing the original sense strand in a single-stranded form. We have measured the rate of this release using magnetic tweezers, which represents the first application of this technique to RNA-dependent enzymatic processes. Surprisingly, in 10% of events a reversal of the transcription is detected. We present evidence linking this behavior to strand-switching by RdRP. Such strand-switching may provide clues to the mechanism of RNA recombination.



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O.13 Dynamics of transiently crosslinked cytoskeletal networks C.P. Broedersz, M.S. Depken, F.C. MacKintosh VU Amsterdam, AMSTERDAM, Netherlands Cells derive their mechanical properties largely from the cytoskeleton: a network of stiff biopolymers and associated proteins, capable of cross-linking and force generation. The short-timescale mechanical response of biopolymer gels has been studied extensively, while little is known about the biologically more relevant long-time behavior. It is on these longer timescales that the cytoskeleton remodels in response to internal and external cues. Here we present predictions for the viscoelasticity of semiflexible polymer networks cross-linked with physiological transient linkers. Our model allows us to elucidate the mechanisms by which the network can relax as a result of the constant breakage and formation of links in the network.

O.14 Reliable centering of dynamic microtubule asters in microfabricated chambers by pulling forces L. Laan1, N. Pavin2, G. Romet-Lemonne1, F. Julicher2, M. Dogterom1 1AMOLF, AMSTERDAM, Netherlands 2MPI-PKS, DRESDEN, Germany In cells, microtubules (MTs) interact with the cortex to generate pushing and/or pulling forces that position the organizing center correctly with respect to the cell's confining geometry. We investigate this positioning in in vitro experiments. MT asters are grown in squared microfabricated chambers. Pushing forces arise from MT polymerization and elastic restoring forces. Pulling forces are introduced by attaching motor proteins to the chamber walls. Surprisingly, we find that MT asters center more reliably by pulling and pushing forces together than by pushing forces alone. We developed a theoretical model in which pulling forces center an aster due to a MT-slipping induced anisotropic distribution of MTs in the microfabricated chamber.

O.15 Probing the structure and dynamics of actively contractile actin-myosin networks M. Soares e Silva1, M. Korsten1, G.H. Koenderink1, F.C. MacKintosh2 1FOM Institute AMOLF, AMSTERDAM, Netherlands 2Theoretical Physics, Vrije Universiteit, AMSTERDAM, Netherlands Living cells are a complex composite material subject to internal and external fluctuations.Myosin motors exert force on the actin cytoskeleton driving it out of equilibrium.Such tensions result in contractility that is essential for symmetry-breaking processes like cell division. We study how myosin activity induces patterning and contractility of the actin cortex.To this end we assembled a simplified in vitro model system of purified actin and myosin and varied myosin density and processivity.I will show that motor activity leads to large, nonthermal contractile fluctuations in the network with a coherence length that is sensitively tuned by the crosslink density.Moreover, myosin drives active assembly of ring-like actin structures.



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O.16 Spatio-temporal analysis of membrane lipid remodeling during phagocytosis S. de Keijzer1, D. Kilic1, C.G. Figdor1, S. Grinstein2, A. Cambi1 1Nijmegen Centre for Molecular Life Sciences-NCMLS, NIJMEGEN, Netherlands 2University of Toronto, TORONTO, Canada The signaling, targeting and trafficking during phagocytosis is dependent on lipid membrane remodeling. Lipids assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. Here we used fluorescently labeled biosensors based on K-ras and H-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during FcReceptor-mediated phagocytosis in live macrophages. The results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane.

O.17 Direct nanoscale optical imaging of nucleation sites for cell adhesion T.S. van Zanten1, A. Cambi2, M. Koopman3, B. Joosten2, C.G. Figdor2, M.F. Garcia-Parajo1 1IBEC-Institut de Bioenginyeria de Catalunya, BARCELONA, Spain 2Nijmegen Center for Molecular Life Sciences, NIJMEGEN, Netherlands 3University of Twente, ENSCHEDE, Netherlands Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their activity on a variety of cells. We have used single-molecule near-field optical microscopy (NSOM) to capture the spatiofunctional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Direct dual color nanoscale imaging revealed the existence of a GPI-AP subpopulation organized in nanodomains that further concentrated in regions smaller than 250nm, suggesting a hierarchical pre-arrangement of GPI-APs. In addition, integrin nanoclusters reside proximal to these GPI-AP nanodomains, forming hotspots on the cell surface. These hotspots seem to function as essential intermediates in nascent cell adhesion.

O.18 Spatial restriction of GPCR - G protein mobility is essential for faithful chemotaxis F. van Hemert1, M.D. Lazova1, B.E. Snaar-Jagalska2, T. Schmidt1 1LION, LEIDEN, Netherlands 2IBL Molecular Cell Biology, LEIDEN, Netherlands Gradient sensing is mediated by the GPCR cAR1 and its G protein (consisting of a Ga2 and a Gbg subunit). The development of a cell polarization requires amplification of the extracellular gradient signal leading to spatially restricted activation of intracellular signaling components. We investigated the mobility of cAR1, Ga2 and Gbg in unstimulated, global cAMP stimulated and chemotacting cells. We show that the precoupled GPCR - G protein complex dissociates upon activation and that Gbg immobilizes. In chemotacting cells, this F-actin and Ga2 dependent process is restricted to the leading edge. We hypothesize that F-actin functions as a scaffold for Gbg, locally enhancing signaling with downstream effectors.



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O.19 Conformational changes in DNA and MutS during mismatch repair, analyzed by fluorescence spectroscopy M. Cristovao Erasmus Medical Center, ROTTERDAM, Netherlands The mismatch repair (MMR) system is responsible for correcting replication errors that escape the proofreading function of DNA-polymerases. In E. coli, MutS, MutL and MutH are the key proteins of MMR. MutS recognizes the mismatch and recruits MutL in an ATP-dependent manner. In the process of mismatch recognition, MutS kinks mismatched DNA by 60 and this transition is an important trigger for the repair process. In our studies, both DNA and MutS were double labeled with donor and acceptor dyes. We used ensemble and single molecule fluorescence spectroscopy techniques to define dynamic conformational changes in DNA and MutS during binding, to reveal the mechanism of mismatch recognition controlled by nucleotide binding and hydrolysis.

O.20 Single basepair accuracy prediction of nucleosome positioning in vitro and in vivo A.H. van der Heijden1, J. van Vugt2, J. van Noort1 1Leiden University, LEIDEN, Netherlands 2Radboud University Nijmegen Medical Centre, NIJMEGEN, Netherlands Nucleosomes play an important role in the compaction and access of DNA in eukaryotic cells. Histone octamers favor binding to DNA sequences with specific periodically occurring dinucleotides. Specific pentameric sequences reduce nucleosome formation. Here we combined these two factors in a probability model to predict the binding positions of nucleosomes. The model has an unprecedented accuracy of a single bp. From these predictions we calculate the energy landscape for nucleosome positioning. The model not only accurately predicts the free energy of histone octamer binding, but also the genome wide localization of nucleosomes found in various organisms. Thus we revealed a simple DNA code that defines the organization of eukaryotic genomes.

O.21 Dynamics of RecA on single-stranded DNA M.T.J. van Loenhout1, T. van der Heijden1, C. Wyman2, R. Kanaar2, C. Dekker1 1Delft University of Technology, DELFT, Netherlands 2Erasmus Medical Center, ROTTERDAM, Netherlands We study the dynamics of binding and unbinding of RecA, the key protein in DNA double strand break repair, on its biologically relevant substrate, single-stranded DNA. Using magnetic tweezers to onitor the length of individual DNA molecules, we find that both ucleation and extension occur with a binding unit consisting of multiple RecA monomers. Further we show how the dynamics and mechanical properties of these filaments depend on nucleotide cofactor, ATPase activity, and force. These single-molecule measurements reveal that RecA filaments are dynamic and can contract and expand, providing an essential baseline for understanding the details of homologous recombination in vivo.



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O.22 Dual-axis cryo-electron tomography: a powerful technique to explore intact mitochondria N.V. Dudkina1, G.T. Oostergetel2, H.P. Braun3, E.J. Boekema2 1GBB, GRONINGEN, Netherlands 2University of Groningen, GRONINGEN, Netherlands 3University of Hannover, HANNOVER, Germany Dual-axis cryo-electron tomography (CET) is a novel technique for a 3D reconstructing of mitochondria under cryogenic 'life-like' conditions by tilting the specimen around a double-tilt axis. It helps to understand the overall organisation of mitochondrial proteins. Work on the green alga Polytomella, related to Chlamydomonas, showed superstructures of dimeric ATP synthases. The achieved resolution allowed to extract and average sub-volumes of dimeric ATP synthases, potential ribosomes and other complexes. Already after averaging of 84 sub-tomograms of dimeric ATP synthase the main features of this complex (headpieces, stalks, stators) were revealed. This is the first time that such resolution was achieved by CET on intact mitochondria.

O.23 Spatiotemporal heterogeneous particle dynamics in living cells M.H.G. Duits, Y. Li, S.A. Vanapalli, F. Mugele MESA+ Institute, ENSCHEDE, Netherlands Colloidal particles embedded in the cytoplasm of living cells often show remarkable heterogeneity in their amplitude of motion. However, consensus on the significance and origin of this phenomenon is still lacking. We did experiments on endogenous granules in Hmec-1 cells to reveal the particle dynamics as a function of both location and time. Mean squared displacements were found to vary strongly with location, but not according to a clear spatial pattern. Temporal variations in dynamics were much weaker. Our findings imply that 1) both individual particle dynamics and cell-averaged behavior can be studied, and 2) local dynamics can be revealed. For example, superdiffusive behaviors may be hidden under apparently diffusive global dynamics.

O.24 Fluorescence lifetime imaging of mechanical stress-activated signal transduction pathways in drosophila cells C. Tudor1, A. Pereira2, J. Kanger1, E. Martin-Blanco2, V. Subramaniam1 1University of Twente, ENSCHEDE, Netherlands 2Instituto de Biología Molecular de Barcelona , Parc Cientific de Barcelona, BARCELONA, Spain Cells use multi-protein kinase cascades to signal information from the cell membrane to the nucleus as a response to of external cues. One example of a signaling network is the Jun amino (N)-terminal kinase (JNK) cascade. This study aims to develop an unambiguous method to measure the activity of the JNK signaling pathway in Drosophila cells by evaluating the level of dJun phosphorylation using an intramolecular FRET sensor. The activity of the pathway was analyzed in different conditions by assessing conformational changes in the dJun-FRET module by using a combination of FRET and FLIM. We are able to follow the dynamics of the JNK signalling cascade in real-time in vivo, allowing the analysis of stress-mediated JNK-signaling responses.



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O.25 Triggering enzymatic activity with force K.G. Blank1, H. Gumpp2, E.M. Puchner2, J.L. Zimmermann2, U. Gerland3, H.E. Gaub2 1Institute for Molecules and Materials, NIJMEGEN, Netherlands 2LMU München, Chair for Applied Physics & Center for Nanoscience, MÜNCHEN, Germany 3LMU München, Arnold Sommerfeld Center for Theoretical Physics, MÜNCHEN, Germany Combining force spectroscopy with single molecule fluorescence allows for studying the influence of mechanical stain on enzymatic reactions. Using a TIRF-AFM set-up, we performed stretching and relaxation cycles on the enzyme CalB while recording the turnover of a fluorogenic substrate into a fluorescent product. These cycles were realized by using a covalent C-terminal and a reversible N-terminal attachment rupturing at a force of 60 pN. In this context, the rupture represents a trigger, which starts the relaxation process of the enzyme. After the trigger event a higher probability for enzymatic activity was observed at 1.7 seconds. This delay is explained with a newly developed theoretical model revealing a multistep relaxation process.

O.26 Counterion induced higher order assemblies of alpha-synuclein fibrils D.R. Dekker, M.M.A.E. Claessens, V. Subramaniam University of Twente, ENSCHEDE, Netherlands The neuronal protein alpha-synuclein (aS) assembles into amyloid fibrils. Aggregates of these fibrils are found in the Lewy bodies, a hallmark of Parkinson's disease. Here we report on the discovery of the in-vitro formation of higher order assemblies of aS fibrils. aS fibrils organize into well-ordered thin lamellar phases and form tubes with characteristic dimensions at physiological conditions. Alignment of aS fibrils into sheets is surprising given their large negative surface charge. Mono- or multi valent counterion association is necessary for the formation of suprafibrillar assemblies. The salt concentration required for sheet formation indicates that the details of the counterion interactions are important for the assembly process.

O.27 Energetics insight into twisting a cross-b structure: Implication for amyloid-fibrils polymorphism X.P. Periole University of Groningen, GRONINGEN, Netherlands It has been observed that amyloid-like fibrils present different morphologies and twists depending on their mode of preparation and even in a same sample. The results of a series of MD simulations of the cross-beta structure formed by the GNNQQNY peptide indicate that in the absence of crystal packing interactions there is no free energy barrier against twisting for the protofilament found planar in the crystal. We determined that there is only little free energy penalty (1 kT/peptide) in twisting the protofilament in the range of 0-12 degrees. The data also shows that the protofilament twist easily adapts to the different morphologies of a fibril and support an energetic basis for the different degrees of twist observed in amyloid fibrils.



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O.28 The rotating magnetic particles probe: A new technique to measure interactions between protein-coated particles and a surface X.J.A. Janssen1, A. van Reenen1, J.M. van Noorloos1, L.J. van IJzendoorn1, A.M. de Jong1, M.W.J. Prins2 1TU Eindhoven, EINDHOVEN, Netherlands, 2Philips Research, EINDHOVEN, Netherlands We demonstrate a new technique to measure interactions between protein-coated particles and a surface. The technique is based on the measurement of field-induced rotation of magnetic particles in contact with a surface. The parallel measurement of many particles with single-particle resolution gives reliable statistics in a short time. The interaction between streptavidin-coated particles and a glass substrate is measured for various buffer conditions. Binding increases for increasing ionic strength and decreasing pH. The results are found to be in agreement with calculations of the electrostatic interaction between a sphere and a wall. The rotational behavior is also measured for particles attached to a biologically functionalized surface.

O.29 Detection of local protein structures along DNA using solid-state nanopores S.W. Kowalczyk, A.R. Hall, C. Dekker Kavli Institute of NanoScience, DELFT, Netherlands Nanopores have been successfully employed as a new tool to rapidly detect single biopolymers, in particular DNA. Here, we examine the translocation of DNA with discrete patches of the DNA-repair protein RecA attached along its length. Using the fact that RecA-conjugated DNA and bare DNA yield very different current-blockade signatures, we demonstrate that it is possible to map the locations of the proteins along the length of a single molecule using a solid-state nanopore. This is achieved at high speed and without any staining. We currently obtain a spatial resolution of about 20 nm. This new capability of measuring local structures along DNA at high resolution will be of vital importance to next generation molecular diagnostics.

O.30 Electrochemical detection of single molecules in nanofluidic devices M.A.G. Zevenbergen, P.S. Singh, E.D. Goluch, B.L. Wolfrum, S.G. Lemay Kavli Institute of Nanoscience, DELFT, Netherlands We introduce a new single-molecule technique based on the electrical detection of electrochemically-active molecules in a nanofluidic device. The device consists of a thin, liquid-filled channel bounded by two electrodes. Molecules undergoing Brownian motion in the channel rapidly shuttle electrons between the electrodes, and can be detected as steps in the measured electrical current. We present the first experimental demonstration of this technique at the single-molecule level, and discuss ongoing application to the study of the enzyme tyrosinase.



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O.31 Functional flexibility in the photosystem II reaction center: Multiple charge separation pathways E. Romero1, V. Novoderezhkin2, I.H.M. van Stokkum1, J.P. Dekker1, R. van Grondelle1 1Vrije Universiteit Amsterdam, AMSTERDAM, Netherlands 2Moscow State University, MOSCOW, Russian Federation Energy and electron transfer reactions in Photosystem II reaction centers have been studied by femtosecond transient absorption-difference spectroscopy at 77 K in order to investigate the presence of different charge separation (CS) pathways. The Global analysis shows different timescales of Pheophytin anion formation (indication of CS): ultrafast, slow and very slow; in disagreement with a unique CS pathway. The Target analysis demonstrates that the spectral evolution can be described by the presence of two parallel CS pathways located in the active branch in charge separation, a slowly transferring peripheral Chlz cofactor, and two cofactors located in the inactive branch that transfer excitation energy to the active branch very slowly.

O.32 Single-molecule spectroscopy reveals conformational switching in plant light-harvesting complexes T.P.J. Krüger1, V.I. Novoderezhkin2, P. Horton3, E.I.E. Wientjes4, R. Croce4, R. van Grondelle1 1Vrije Universiteit Amsterdam, Netherlands, 2A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, MOSCOW, Russian Federation, 3University of Sheffield, SHEFFIELD, United Kingdom, 4Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Regulation of photosynthesis occurs by the rapid, reversible transformation of light-harvesting complexes from an efficient light-collecting state to a dissipative state, a process suggested to be induced by a distinct subtle protein conformational change. Using single-molecule spectroscopy we show for the first time how individual complexes have the inbuilt capacity to rapidly, reversibly and spontaneously switch into diverse dissipative states or various spectrally shifted emission states, with a rate and equilibrium dependent on the type of complex. The different functional states are structurally remarkably similar, indicating that subtle manipulation of the switching rates suffices to reproduce the various extents of quenching in vivo.

O.33 Self-assembling natural and artificial light-harvesters H.J.M. de Groot1, S. Ganapathy1, G. Oostergetel2, M. Reus3, A. Gomez Maqueo Chew4, F. Buda1, E.J. Boekema5, D.A. Bryant4, S. Sengupta6, P.K. Wawrzyniak1, V. Huber6 1Leiden Insitute of Chemistry, LEIDEN, Netherlands, 2RuG, GRONINGEN, Netherlands, 3MPI Bioanorganische Chemie, MUELHEIM A/D RUHR, Germany, 4The Pennsylvania State University, UNIVERSITY PARK, United States of America, 5University of Groningen, GRONINGEN, Netherlands, 6Universität Würzburg, WÜRZBURG, Germany, 7Martin-Luther-Universität, HALLE, Germany Solar power remains one of the most promising alternative clean energies. Solid state NMR spectroscopic techniques are combined with quantum mechanical calculations and cryo-EM imaging or X-ray diffraction to image the stacking and supramolecular organization of highly efficient chlorophyll antennae in chlorosomes from green bacteria and biomimetic systems. Chlorosomes are the largest and most efficient light-harvesting antennae found in nature, and they are constructed from hundreds of thousands of self-assembled bacteriochlorophyll (BChl) c, d or e pigments.



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O.34 Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain R. Loris, N. De Jonge Vrije Universiteit Brussel, BRUSSEL, Belgium Bacterial toxin-antitoxin modules ensure survival during episodes of nutritional stress. The ccd module codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA rejuvenates CcdB-gyrase complexes. The CcdA:CcdB ratio modulates repression of the operon. CcdA binds consecutively to two partially overlapping sites on CcdB. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. A second micromolar binding event regulates expression. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of CcdA.

O.35 Visualisation of protein complexes present during the chaperonin assisted folding cycle by ion-mobility mass spectrometry E. van Duijn, A. Barendregt, S.A. Synowsky, C. Versluis, A.J.R. Heck Utrecht University, UTRECHT, Netherlands Native mass spectrometry allows the structural investigation of intact protein complexes. Here we highlight the potential of a rather new approach within this field, ion mobility mass spectrometry (IMMS). IMMS allows the assessment of gas phase ion collision cross sections of protein complex ions, which can be related to overall shapes/volumes of protein assemblies. We studied several (intermediate) chaperonin complexes that can be present during substrate folding. Our results reveal that only genuine GroEL substrates are buried inside the chaperonins cavity. Additionally, we determined that the dimensions of the ternary chaperonin complex (GroEL- substrate-co-chaperonin), were similar to those of the empty GroEL-GroES complex.

O.36 Protein conformational switches for intracellular imaging and diagnostics M. Merkx Laboratory of Chemical Biology, EINDHOVEN, Netherlands Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of living cells. Inspired by the modular design of natural signaling proteins, we are pursuing a protein engineering approach in which functional protein domains are connected via flexible peptides. Controlling the conformational states in these chimeric proteins requires a quantitative description of domain-domain interactions and the conformational distribution of the linkers. We show that this approach allows rational design of FRET-based sensor proteins with a large change in emission ratio, including sensors that allow real time imaging of pM concentrations of zinc in single cells.


Abstracts posters

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P.001 The impact of bending and twisting rigidity of DNA on protein induced looping dynamics N. Laurens1, C. Pernstich2, R.W. Bellamy2, L.E. Catto2, Y.S. Kovacheva2, S.E. Halford2, G.J.L. Wuite1 1VU University, AMSTERDAM, Netherlands 2University of Bristol, BRISTOL, United Kingdom DNA-looping is a key regulatory mechanism involved in important processes in the cell. The relation between induced loop topology and DNA-protein dynamics is essential for understanding this process. In this investigation we use a tethered particle assay to determine the connection between the dynamics of DNA looping enzymes and the physical properties of DNA. Results are analyzed using a novel diffusive hidden Markov analysis. We show that both, the separation and orientation of the recognition sites have a profound influence on the formation and stability of these looped DNA-protein structures. The results are understood and modeled in terms of the helical pitch and the bending energy involved in protein induced loop formation.

P.002 Effect of histone acetylation on nucleosome dynamics revealed by spFRET microscopy R. Buning1, W.J.A. Koopmans1, H. Neumann2, J. Chin2, T. Schmidt1, J. van Noort1 1LION, LEIDEN, Netherlands 2Laboratory of Molecular Biology, Medical Research Council, CAMBRIDGE, United Kingdom Nucleosomes, besides condensing eukaryotic DNA, play a crucial role in gene regulation by modulating access to nucleosomal DNA for DNA-processing proteins. Accessibility is regulated by specific post-translational modifications to the histones. Histone acetylation at H3K56 for example has been shown to induce increased gene expression in vivo. We combined spFRET and Alternating Laser Excitation (ALEX) with gel electrophoresis and Fluorescence Correlation Spectroscopy (FCS) to diffusing nucleosomes and show that a single acetylation induces a more than 2-fold increase in DNA unwrapping of the first 20 basepairs. Using these techniques we aim to quantify and structurally interpret epigenetic changes in chromatin at the single molecule level.

P.003 The third level of genome functioning: Chromatin folding M. Tark1, M. Bohn2, D. Heermann2, R. van Driel1 1Swammerdam Institute of Life Science, AMSTERDAM, Netherlands 2Institute of Theoretical Physics, HEIDELBERG, Germany Recent high-throughput methods have provided an extensive amount of data which all suggest that chromatin folding is achieved by formation of chromatin-chromatin loops. The observed looping is highly dynamic and linked to cell differentiation. We propose that the chromatin folding constitutes a third level of genome functioning on top of the individual genes and their epigenetic control Our interest is to study the principles of chromatin folding in the cell nucleus and in particular how does chromatin folding relate to gene expression. In our approach we focus on the molecular mechanisms of loop formation, dynamics and ergodicity of looping as well as the relation of dynamic looping to genome function.


Abstracts posters

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P.004 Towards single-molecule experiments of chromatin-remodeling proteins R. Vlijm1, S. Hage1, G.E. Chalkley2, C.P. Verrijzer2, C. Dekker1 1Delft University of Technology, DELFT, Netherlands 2Erasmus University Medical Centre, ROTTERDAM, Netherlands For gene expressions the chromatin structure has to be unpacked, necessitating the remodeling of nucleosomes on DNA. One way to achieve accessible DNA is active relocation or removal of nucleosomes by ATP-dependent chromatin-remodeling proteins. We aim to study this remodeling with single-molecule techniques such as AFM and magnetic tweezers. The SWI/SNF family is an important and widely studied family of ATP-dependent chromatin remodelers. We are preparing to study the activity of Brahma (BRM), the motor protein of this protein family, on dsDNA with nucleosomes. In this poster, we will lay out the technical details of the experiment and show preliminary single-molecule results.

P.005 Characterization of novel ssDNA binding proteins hSSB1 and hSSB2 I. Vidic, C. Wyman, R. Kanaar, J. Lebbink Erasmus MC, ROTTERDAM, Netherlands Single-stranded DNA binding proteins (SSBs) are involved in many aspects of DNA metabolism, including DNA damage detection and repair. In addition to RPA, the human genome encodes two additional SSBs, hSSB1 and hSSB2. Cells depleted from hSSB1 or hSSB2 are hypersensitive to DNA-damage. hSSB1 and hSSB2 form separate heterotrimeric protein complexes (SOSS) with INTS3 and hSSBIP1. The existence of different ssDNA-binding complexes, RPA and SOSS, indicates a possible division of roles in DNA repair. We have characterized the binding of hSSB1 and hSSB2 to ssDNA. Binding kinetics of SSBs were analysed by EMSA and SPR measurements. In addition, the formation of DNA-protein complexes with hSSBs was visualized by scanning force microscopy.

P.006 ATP-dependent nucleosome repositioning is directed by the underlying DNA sequence J.F.A. van Vugt1, M. de Jager2, M. Murawska3, A. Brehm3, J. van Noort2, C. Logie1 1NCMLS, Radboud University, NIJMEGEN, Netherlands 2Leiden Institute of Physics, LEIDEN, Netherlands 3Institut für Molekularbiologie und Tumorforschung, MARBURG, Germany DNA accessibility and chromatin structure are influenced by the positions of nucleosomes along DNA. ATP-dependent chromatin remodelers are known to reposition nucleosomes. Recent studies reveal that 50-80% of in vivo nucleosome positions can be predicted by DNA sequence alone. This seemingly contrasts with remodeler induced nucleosome mobility. Our study on mononucleosomes repositioning on long DNA molecules by SNF2-type enzymes reveals that the DNA sequence can control the initial translocation direction, translocation distance and the newly adopted nucleosome positions upon enzymatic mobilization. We conclude that both remodeler intrinsic and DNA sequence specific properties interplay to define ATPase-catalyzed nucleosome repositioning.


Abstracts posters

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P.007 Nanopore force spectroscopy M.J. Wiggin1, V. Tabard-Cossa2, N.N. Jetha3, C. Tropini2, C. Feehan3, A. Marziali3 1TU Delft, DELFT, Netherlands 2Stanford University, STANFORD CA, United States of America 3University of British Columbia, VANCOUVER, BC, Canada Single molecule force spectroscopy (FS) provides a unique approach to probe energies of biomolecular structural transitions. Our group is developing force spectroscopy instruments from both organic and synthetic nanometer scale pores (nanopores). I will present the basic technique, and a number of projects we have been pursuing using nanopore force spectroscopy, including single molecule studies of prion protein structural transitions which have been implicated in diseases such as bovine spongiform encephalopathy; development of a rapid genotyping technique based on observations of DNA duplex dissociation kinetics, and detailed studies of DNA-nanopore interactions.

P.008 Torsional regulation of hRPA-induced dsDNA unwinding. I. De Vlaminck1, I. Vidic2, J. Lebbink2, M.T.J. van Loenhout1, C. Dekker1 1TU Delft, DELFT, Netherlands 2Molecular Radiation Biology group at the Erasmus Medical Center, ROTTERDAM, Netherlands Human Replication Protein A (hRPA) is the main human SSB and is essential for most aspects of cellular DNA metabolism. In vitro and at conditions of very low salt, RPA displays dsDNA binding and helix unwinding activity. Here we study the dynamics of this activity with a magnetic tweezers-based single molecule assay. We find that RPA-induced helix unwinding is strongly promoted by unwinding torque and suppressed by rewinding torque. Torque therefore provides a means for tight mechanical regulation. Torque generated through moderate stretching force (~0.5 pN) is sufficient to overcome the inhibitory effects of salt. A torque-based model explains the dynamics the unwinding activity and provides mechanistic insight.

P.011 A multi-step aggregation of PABPN1 V. Raz, T.E. Abraham, S.M. van der Maarel, H. Tanke, R.W. Dirks LUMC, LEIDEN, Netherlands Aggregation of protein is found in a wide spectrum of inherited degenerative diseases carrying an expansion mutation of Glu or Ala residues, and the mutant protein aggregates in inclusions. Patients with oculpoharyngeal muscular dystrophy carry an Ala expansion mutation in the poly(A) binding protein nuclear 1 (PABPN1). Here we studied PABPN1 aggregation in living cells using high-resolution microscopy of cells expressing either the WT or mutant alleles fused to YFP. Applying image quantification methods we show defined two sequential steps in PABPN1 aggregation, which differ in dynamics protein mobility and molecular organization. We show that aggregation of the mutant PABPN1 differs from the WT protein in the initial aggregation step.


Abstracts posters

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P.012 Tethered magnetic particle motion measured with evanescent wave microscopy K. van Ommering1, P.A. Somers2, M. Koets1, J.B.A. van Zon1, J.J.H.B. Schleipen1, L.J. van IJzendoorn2, M.W.J. Prins1 1Philips Research Eindhoven, EINDHOVEN, Netherlands 2Eindhoven University of Technology, EINDHOVEN, Netherlands We are investigating detection techniques for biosensing using superparamagnetic nanoparticles (diameter 500-1000 nm) and evanescent optical fields. The scattered light intensity of the particles strongly depends on the distance between particle and surface. We have built a set-up to record position and intensity information of biologically bound particles with exposure times down to 1 ms. As a model system we use dsDNA tethers with lengths up to 200 nm. We will show and discuss how the intensity and intensity fluctuations are influenced by magnetic particle type, magnetic field, and binding type.

P.013 Breaking the chain mail: AFM study on the mechanical strength of a bacteriophage shell W.H. Roos1, I. Gertsman2, J. Johnson2, G. Wuite1 1Vrije Universiteit, AMSTERDAM, Netherlands 2Scripps, LA JOLLA, CA, United States of America Bacteriophage HK97 consists of a nm-sized icosahedral protein shell which self-assembles as an empty particle. Subsequently DNA is packaged into this shell with the help of a molecular motor. Whereas the proteins in the empty shell need to be connected loosely to each other, in order to correct mistakes in self-assembly, the DNA-filled shell needs to be strong to withstand internal and external pressure. Shell strengthening is supposed to occur as a result of covalent cross-links that are formed during DNA packaging, giving the shell a chain mail (malinkolder) like appearance. By AFM-based nanoindentation experiments we elucidate the effect of this chain mail on the material properties of the bacteriophage shell.

P.014 Biofunctional micropatterned surfaces to study the spatio-temporal organisation of integrins A. Cambi1, R. Diez-Ahedo2, D. Normanno2, C.G. Figdor1, M.F. Garcia-Parajó2 1Nijmegen Centre for Molecular Life Sciences, NIJMEGEN, Netherlands 2Single Molecule Bionanophotonics group, Inst. for Bioengineering of Catalonia, BARCELONA, Spain Integrin adhesion depends on receptor occupancy and lateral organization on the cell membrane. To investigate the dynamic reorganization of integrins into high avidity clusters, surfaces of immobilized integrin ligands were fabricated by micro-contact printing. Monocytes were stretched over the patterns and the diffusion of the integrin LFA-1 was followed in time using single molecule TIRF microscopy. LFA-1 nanocluster trajectories showed an increased immobile fraction and a slower diffusion on the patterns compared to the ligand-free areas. Single-cluster intensity indicated a reorganization of LFA-1 nanoclusters in microclusters upon ligand binding, based on random ligand-encountering and binding rather than assisted diffusion.


Abstracts posters

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P.015 MARTINI force field: extension to carbohydrates C.A. Lopez, S.J. Marrink Groningen University, GRONINGEN, Netherlands We present an extension of the coarse grained (CG) MARTINI force field to carbohydrates. In line with the MARTINI force field development, the coarse grained model for carbohydrates has been systematically parameterized based on reproduction of experimental partitioning free energies in combination with mimicking the behaviour seen in atomistic simulations. Parameters were derived for all common mono- and disaccharides, considering the different ways of linking for monosaccharide units. The model has been tested on a number of small polysaccharides. For instance, the folding of a 26 (alpha 1-4) D-glucopyranose amylose chain was simulated both in a non-polar (nonane) and polar (water) environment.

P.016 A new perspective on the rich phase behavior of ternary lipid systems T. Idema1, C. Storm2 1Leiden Institute of Physics, LEIDEN, Netherlands 2Technische Universiteit Eindhoven, EINDHOVEN, Netherlands We study a ternary system consisting of cholesterol and 2 other lipids, mimicking the composition of the plasma membrane. The phase diagram of such a mixture exhibits a rich phase behavior with multiple phase coexistence regimes. Remarkably, phase separation even occurs when each of the three binary systems consisting of two of the three components is a uniform mixture. We have developed a model for the free energy of such mixtures, in which the phase separation of the ternary system is a consequence of an interaction between all three components. Using this model we can reproduce the phase diagrams and calculate the critical points, and also find the point of linear instability (the spinodal) and the line tension between coexisting phases.

P.017 MRI of axial and radial water transport in plants H. van As, N. Homan, E. Gerkema, F.J. Vergeldt Wageningen University, WAGENINGEN, Netherlands Hydraulic conductivity of axial and radial water transport provides key information to validate eco-biophysical plant models for evaporation. Flow in (woody) plants can be described by Darcy's law. However, there are many violations to Darcy's law when applied to living (woody) plants and key aspects of the relationships between pressure, flow/flow conducting area and water storage pools are not well understood. New intact plant MRI methods are now available to study water dynamics on different length and time scales, covering sub-cellular and long distance transport. Xylem dynamics and embolisms as a function of water potential and the interplay with storage pools during e.g. drought stress have been studied quantitatively.


Abstracts posters

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P.018 Membrane protein dynamics in single cell-cell contact M.I. Snijder-van As1, B. Rieger2, V. Subramaniam3, C.G. Figdor4, J.S. Kanger3 1MIRA, ENSCHEDE, Netherlands 2Delft University of Technology, DELFT, Netherlands 3MIRA, University of Twente, ENSCHEDE, Netherlands 4Radboud University Nijmegen Medical Centre, NIJMEGEN, Netherlands The dynamics of cell membrane proteins plays an important role in cell-cell interaction, for example cell to cell signaling. Previously, we reported that we developed a new method combining optical tweezers with total internal reflection fluorescence microscopy. Having validated the method and shown its versatility, we now have extended this method to monitor single cell to cell contact from t=0s onwards. In this way, we have studied the GFP-tagged membrane protein ALCAM in contact with its RFP-tagged ligand CD6. Here, we show the dynamics of these molecules and quantify the process of interaction.

P.019 Membrane surface chemical stability correlates with desiccation tolerance and seed longevity E.A. Golovina, H. van As, F.A. Hoekstra Wageningen University, WAGENINGEN, Netherlands Membranes are a primary target of desiccation injury and seed aging. Membrane surface motional /chemical stability was studied in dry POPC bilayer and in different dry organisms by FT-IR and spin probe ESR. POPC experiments showed the role of disaccharides (trehalose), monosaccharides (glucose) and extrinsic membrane proteins (poly-L-lysine) in membrane surface motional/chemical stabilization. In vivo experiments showed that membrane surface is more chemically stable environment and stronger immobilized in desiccation tolerant than in intolerant organisms, and in long-lived in comparison with short lived seeds. The data suggest a key role of membrane surface chemical stability in mechanisms of desiccation tolerance and seed longevity.

P.020 Atomic force spectroscopy on viral RNA - Protein system: RRE - Rev from HIV J. Zivkovic, L. Janssen, H.A. Heus, S. Speller Insitute for Molecules and Materials, NIJMEGEN, Netherlands Force spectroscopy has become a powerful tool for studying single molecule interactions:DNA-DNA, DNA-protein, ligand - receptor, etc.Nevertheless, RNA - protein interaction still remain unexplored in this field.RNA serves as a genome of many pathogenic viruses, like HIV, whose life cycle depends on few, very unique RNA - protein interactions: RRE- Rev and TAR-Tat.We focus on single molecule force spectroscopy of RRE fragment containing the high affinity binding site (stem loop IIB) and a Rev peptide that binds this stem loop specifically.Observed koff and potential width was found to be 3.5 s-1 and 3.43 respectively.Control measurements in the presence of neomycin yielded lower koff for this interaction.


Abstracts posters

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P.021 Non-linear optical microscopy of leafs. First results and plans B.F. van Oort, S. Witte, M.L. Groot, R. van Grondelle Vrije Universiteit, AMSTERDAM, Netherlands Understanding and modelling of photosynthesis is important for improving food and energy production. This requires describing the physics of the photosynthetic membrane at high resolution. Such a description is hampered by lack of data on energy migration, on functional heterogeneity, and on fluctuations due to disorder. Such data is not easily obtained by current experimental and theoretical methods. We present simultaneously recorded images of fluorescence and second- and third-harmonic generation in intact leafs, and plans for multi-pulse microscopy that may lead to a better understanding of photosynthesis on the membrane level.

P.022 Mechanical characterization of alpha-synuclein fibrils using AFM imaging and force spectroscopy K.K.M. Sweers, G.M.J. Segers-Nolten, K.O. van der Werf, M.L. Bennink, V. Subramaniam University of Twente, ENSCHEDE, Netherlands Alpha-synuclein (aS) monomers assemble into amyloid fibrils with diameters of around 8 nm. Here, atomic force microscopy (AFM) imaging and force spectroscopy are used to study the mechanical characteristics of these fibrils, which contain information on the underlying fibril formation mechanism. From AFM images we determined the bending rigidity of aS fibrils by analyzing the 2D fibril contours using statistical mechanical theory of semiflexible polymers. We also tested individual aS fibrils for bending using the AFM force-distance mode. From these, elastic properties such as Young's moduli are obtained and compared with the bending rigidities determined by AFM imaging. Typical bending rigidities found for these fibrils are 1.5e-24 Nm2.

P.023 Exploring the energy landscape of DNA melting during overstretching P. Gross, G.J.L. Wuite, E.J.G. Peterman Vrije Universiteit, AMSTERDAM, Netherlands DNA undergoes a phase transition at a stretching force of 65 pN, during which the polymer gains 70% of its contour length. The molecular basis of this overstretching transition has remained under debate. Here, we apply a combination of optical tweezers and fluorescence microscopy together with fluorescent markers for single-stranded DNA to visualize this structural transition. We find that DNA overstretching is a gradual conversion from double-stranded to single-stranded DNA. Furthermore, using equilibrium, high-resolution force-extension measurements, we show that force-induced melting occurs in a deterministic, burst-like manner, which is governed by local, sequence-specific DNA stability.


Abstracts posters

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P.024 H-NS Polymerization in the absence of DNA C.H.J.M. Wintraecken WUR, WAGENINGEN, Netherlands H-NS acts as a global thermo-sensitive repressor in bacteria by cooperative cross-linking of neighbouring DNA helices. Using Dynamic Light Scattering (DLS) we show that, at low temperatures and physiological ionic strength, H-NS polymerizes even without DNA, forming gel-like structures. At higher temperatures, the structures fall apart. The transition temperature of 32C is close to the temperature above which virulence genes are no longer repressed by H-NS. Mutations of two acidic residues in a highly conserved part of the H-NS linker lead to loss of temperature dependent H-NS self-association, highlighting the importance of H-NS protein-protein interaction for its interaction with DNA.

P.025 Dynamics of bone production by cells, as observed by Raman microscopy V.V. Pully, A.T.M. Lenferink, V. Subramaniam, A. van Blitterswijk, C. Otto University of Twente, ENSCHEDE, Netherlands Adult human bone marrow stromal cells are able to differentiate into the osteogenic lineage under the influence of osteo-inducing growth factors leading to in vitro mineralization, chemically similar to in vivo bone. The chemical transformations during cell differentiation and the ensuing preparation of extra-cellular matrix as well as the actual production of bone have been followed with chemically sensitive, high spatial resolution Raman microscopy over a 60 day period. The results show biomarkers that give a detailed picture of events of bone formation, maturation and carbonation, which have been obtained in conjunction with changes in the overall organic composition of the bone tissue with non-invasive and label-free optical analysis.

P.026 The effect of physical confinement to the cellular structure in E.coli J. Männik, P.J. Galajda, J.E. Keymer, C. Dekker Kavli Insitute of Nanoscience, DELFT, Netherlands Bacterial cells are usually considered mechanically rigid. Nevertheless, we have shown that E.coli bacteria can grow in nanofabricated constrictions where they are squeezed to about twice thinner than their typical diameters. Growth in such constrictions leads to flattened cells which laterally are considerably wider than regular E. coli. We will report on the cell growth, and spatial structure and dynamics of cytoskeletal proteins and nucleoid in these flat bacteria. Surprisingly, while the physical confinement has a profound effect on the cell shape, it has only a limited effect on the rate of cell division. Our study indicates that the cellular structure of bacteria has a high degree of plasticity in coping with physical confinement.


Abstracts posters

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P.027 Unravelling mitochondrial DNA organization G. Farge1, N. Laurens1, L. Dekker1, M. Falkenberg2, G.J.L. Wuite1 1Physics of Complex Systems, AMSTERDAM, Netherlands 2University of Gothemburg, GOTHEMBURG, Sweden Mitochondria contain a high copy, double-stranded circular DNA molecule which is organized in protein-DNA complexes termed 'nucleoids'. The organization in nucleoids is accomplished with the aid of DNA binding proteins, including the mitochondrial transcription factor A (TFAM). The dynamics of the TFAM-DNA interaction and the mechanism of DNA compaction by TFAM are still unclear. Here we use an array of single-molecule tools to address these questions. Using TPM, we observe clear compactive effect of TFAM on DNA. Moreover, the combined use of optical tweezers and fluorescence microscopy will provide further information on the dynamics of the protein induced DNA compaction, and will permit the direct visualization of the process.

P.028 Studying the effect of manipulating phagosome routing on its maturation S. Shekhar1, A. Klaver1, C.G. Figdor2, V. Subramaniam1, J.S. Kanger1 1University of Twente, ENSCHEDE, Netherlands 2Radboud University Nijmegen Medical Centre, NIJMEGEN, Netherlands We use magnetic tweezers to study the effect of manipulation of phagosomal routing on the maturation of phagosomes. Phagocytic cells are allowed to internalize 1-m pH sensing magnetic beads followed by application of an external magnetic force in order to influence the position of the bead-containing phagosomes. We have also developed a single pole magnetic tweezers capable of exerting forces of ~1nN on micron-sized magnetic particles. The effect of the manipulation on the phagosome maturation is characterized by measuring the pH in the phagosome by means of pH-sensing fluorescent dye coupled to the magnetic beads.

P.029 Porous multilayer-coated AFM tips for dip-pen nanolithography of proteins C.C. Wu, H. Xu, C. Otto, D.N. Reinhoudt, R.G.H. Lammertink, J. Huskens, V. Subramaniam, A.H. Velders MESA+, ENSCHEDE, Netherlands A simple method for fabricating nanoporous-structure-coated silicon nitride AFM tips for dippen nanolithography (DPN) by using the layer-by-layer (LbL) technique has been developed. The pore sizes can be adjusted by treating the LbL films coated onto the amino-terminated self-assembled monolayer (NH2-SAM)-functionalized tip surface with a base solution for different periods of time. This hydrophilic porous material can absorb biomolecules easily and also provides a larger-volume ink reservoir compared with a bare silicon nitride tip. Proof-of-concept of the porous AFM tip is demonstrated by using fluorescent proteins as ink molecules to fabricate protein patterns at the micrometer and submicrometer length scales.


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P.030 The dynamics of Tethered Particle Motion: How to interpret the mean squared displacement S. Brinkers Delft University of Technology, DELFT, Netherlands In TPM a particle is tethered to a substrate using a biopolymer. We image its motion here via darkfield microscopy onto a CCD camera. At short time scales (~ms) the particle exhibits free diffusion governed by diffusion constants of the particle and biopolymer. At longer time scales (~20 ms) the particle motion is restricted by the effective harmonic potential of the biopolymer. We characterize the motion by mean squared displacement plots. Experimentally we must exclude motion blur (short enough exposures) and capture the free diffusion time scale (high enough framerates). We quantify all these effects and show experimental results for gold nanoparticles (r=40nm) tethered by 900-2500bp dsDNA molecules that show good agreement with theory.

P.031 Quantifying Dpp-endosome colocalization with Particle Image Cross Correlation Spectroscopy (PICCS) L.H.F.M. Holtzer Leiden Institute of Physics, LEIDEN, Netherlands In living organisms individual molecules almost never operate on their own. We developed Particle Image Cross Correlation Spectroscopy (PICCS) to unambiguously measure molecule colocalization, even at large particle densities and down to a length scale of 10 nm. For demonstration of the method's power we studied the intracellular transport of the morphogen Dpp enclosed in endosomes. Dpp establishes a gradient in the wing imaginal disk of fruit fly larvae, providing positional information to cells. Using PICCS we found that 52% of apical Dpp is in early endosomes and that early endosomes contain 1.9 times as much Dpp as other endosomes. Our data suggests that Dpp resides shorter in early endosomes compared to late/recycling endosomes.

P.032 Formation of unusual micelles by bile salts A. Vila Verde, D. Frenkel AMOLF, AMSTERDAM, Netherlands Bile salts are biological surfactants, indispensable for the digestion of fats. They have a short and flexible tail and a rigid steroid ring system with distinct hydrophobic and hydrophilic faces. Their unusual geometry suggests that the structure of pure bile micelles may differ from that of micelles of standard surfactants. We investigate these issues using a minimal bile salt model and grand-canonical parallel tempering molecular dynamics simulations. We find that bile salts may pack in many different orientations in pure bile micelles, contrary to standard surfactants. This may explain why bile salts are better than other surfactants at forming small mixed micelles with the highly heterogeneous blends of fats typical of digestion.


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P.033 The chemical physics of gene transfection: Understanding the molecular mechanism of DNA/lipid interaction R.K. Campen, T.T.M. Ngo, M. Sovago, M. Bonn FOM Institute for Atomic and Molecular Physics, AMSTERDAM, Netherlands Gene therapy shows great promise but is technically challenging. Its successful implementation requires finding appropriate vectors (often lipids) for co-complexation with DNA such that the resulting structures can efficiently penetrate cell membranes, avoid intracellular degradation and are nontoxic. Understanding the molecular mechanism by which DNA interacts with vectors is critical to optimizing this process. Here we examine the interaction of DNA with two cationic and one zwitterionic lipid monlayer using interface specific sum frequency generation spectroscopy. We find, surprisingly, that the manner in which DNA interacts with each lipid is largely insensitive to molecular level details: it is governed by electrostatics.

P.034 Studying RNA using a combination of optical tweezers and solid-state nanopore M. van den Hout, G.M. Skinner, O.D. Broekmans, S. Hage, I.D. Vilfan, C. Dekker, N.H. Dekker TU Delft, DELFT, Netherlands In recent years, far-reaching discoveries about the functionality of RNA in biology have been made. With these developments has come an increasing need to know its structure. One appealing idea is the use of nanopores (nm-size holes in a SiN membrane) which could pass single-, but not double-stranded RNA. Here, we present experiments on single RNA molecules traversing nanopores, causing a temporary blockade of the ionic current through the pore. From the magnitude of this blockade, we show that we can distinguish different types of RNA molecules. We also present first steps towards using optical tweezers to arrest these, as well as more complex, molecules inside the nanopore, probing local features and forces.

P.035 Quantitative analysis of molecular interactions in T cell receptor signaling by fluorescence correlation spectroscopy (FCS) and confocal microscopy Y. Chung, I. Ruttekolk, P. Bovée-Geurts, M. Sinzinger, R. Brock Nijmegen Centre of Molecular Life Sciences, NIJMEGEN, Netherlands For a quantitative analysis of cellular signal transduction, the number of cellular signaling molecules, the fraction of molecules participating in signaling and the temporal and spatial organization of the signaling machinery have to be known. We quantitatively analysed the interaction of the linker for activation of T cells (LAT) and down-stream signaling proteins. First we developed a protocol combining FCS and Western Blots to determine endogenous signaling protein concentration. Then LAT-EGFP and mCherry-fusions of signaling proteins were expressed in cells, and stimulation-dependent interactions were quantitatively studied through a combination of confocal laser scanning microscopy, FCS and fluorescence cross-correlation spectroscopy.


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P.036 A fret-film study reveals the interaction between alcam and actin as a potential regulator of alcam binding activity C. Tudor1, A. Esseling-Ozdoba2, C.G. Figdor2, J. Kanger3, V. Subramaniam3 1University of Twente, ENSCHEDE, Netherlands 2Radboud University, NIJMEGEN, Netherlands 3Faculty of Science and Technology, University of Twente, ENSCHEDE, Netherlands Interactions between T cells and antigen-presenting cells represent the first step in the induction of an adaptive immune response.CD6 is a cell surface receptor expressed on mature T cells that specifically binds to ALCAM.CD6 and ALCAM are recruited to the antigen-induced DC-T cell contact zone and these interactions are required during the proliferative phase of the T cell response. The aim of the study is to reveal how the cytoskeleton dynamically regulates ALCAM binding activity at the cell-cell contact. This study demonstrates the interaction between ALCAM and Actin and opens the door for further investigation of the role of ALCAM-Actin interactions in the formation and stabilization of the immunological synapse.

P.037 NMR secondary shifts of the light-harvesting 2 complex reveal functional motifs due to local frustration induced by its higher-order assembly A. Pandit1, P.K. Wawrzyniak1, A.J. van Gammeren2, F. Buda1, S. Ganapathy1, H.J.M. de Groot1 1Leiden Institute of Chemistry, Leiden University, LEIDEN, Netherlands 2Klinisch Chemisch en Hematologisch Laboratorium, Amphia Ziekenhuis, BREDA, Netherlands Protein NMR secondary chemical shifts are widely used to predict the secondary structure and in solid-state NMR often the only unambiguous structural parameters available. However, the employed prediction methods are empirical and rely on the assumption that shifts are only affected by shielding effects of neighboring atoms. We analyzed the secondary shifts of a photosynthetic membrane protein with a very tight packing and found a correlation between secondary shift anomalies and pigment- or protein-protein contacts. We conclude that the higher-order interactions involve local backbone perturbations, revealing structural motifs for the role of the protein environment in tuning the pigment site energies and electronic transition states.

P.038 Bicaudal-D like protein madmax-1 organizes dynein-dependent secretory transport and neural development M.A. Schlager, L.C. Kapitein, I. Grigoriev, P.S. Wulf, N. Keijzer, A. Akhmanova, C.C. Hoogenraad Erasmus MC, ROTTERDAM, Netherlands Secretory trafficking is essential for proper cellular development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. We identified Madmax-1 as an Rab6 effector and as a regulator of dynein-dependent secretory trafficking. We show that Madmax-1 interacts with dynein motors, regulates Rab6 vesicle distribution, organizes secretory vesicle exocytosis and is required for neurodevelopment in zebrafish. Persistent expression of Madmax-1, as well as knockdown of both Rab6A and Rab6B suppresses neurite outgrowth in cultured neurons. Our results indicate an important role for Madmax-1 in neural development by serving as an organizer of dynein-dependent secretory trafficking.


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P.039 Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy B.A. Berghuis, R.B.M. Koehorst, R.B. Spruijt, A. van Hoek, H. van Amerongen Laboratory of Biophysics, WAGENINGEN, Netherlands A high-throughput FRET study was performed on the ~100 amino acids long N-terminal domain of the photosynthetic complex CP29. CP29 was singly mutated along its N-terminal domain, labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP29. Picosecond fluorescence experiments revealed fast energy transfer (~20 - 70 ps) from BODIPY to Chl a molecules in the hydrophobic part of the protein. From the transfer times distances were estimated between label and chlorophylls, using the Frster equation. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma, which was suggested before.

P.040 Non-photochemical quenching in mutants of light-harvesting complex II P.M. Dijkman1, R.B.M. Koehorst1, M. Mozzo2, F. Passarini2, R. Croce2, H. van Amerongen1 1Laboratory of Biophysics, WAGENINGEN, Netherlands 2Biophysical Chemistry, University of Groningen, GRONINGEN, Netherlands In highlight conditions, plants have to protect their photosynthetic machinery from photodamage that might arise through the formation of singlet oxygen. They possess a protective mechanism which dissipates excess excitation energy as heat: nonphotochemical quenching (NPQ). NPQ in light-harvesting complex II (LHCII) was investigated using steady state and time-resolved fluorescence measurements. Eight mutants of LHCII lacking a specific chlorophyll molecule were used to determine the role of these pigments in NPQ. The NPQ state was induced by aggregation in low detergent concentrations. Remarkably, all chlorophylls studied appear to be involved in the quenching process.

P.041 Profiling of polarity and dynamics in a protein-lipid system: A picosecond fluorescence study using streak camera detection R.B.M. Koehorst1, S. Laptenok1, B. van Oort1, A. van Hoek1, R.B. Spruijt1, I.H.M. van Stokkum2, M.A. Hemminga1, H. van Amerongen1 1Laboratory of Biophysics, WAGENINGEN, Netherlands 2Department of Physics and Astronomy, VU, AMSTERDAM, Netherlands Profiles of lipid-water bilayer dynamics were determined from ps time-resolved Streak Camera fluorescence images of membrane-embedded BADAN-labelled M13 coat protein. The protein was labelled at seven key positions, placing the label at well-defined locations from the water phase to the hydrophobic centre of the membrane. Analysis of the time-resolved fluorescence spectra provides the characteristic time constant for the twisting motion of BADAN, which is sensitive to the local flexibility of the protein-lipid environment. Also we obtain information about the mobility of water molecules at the membrane-water interface. The results provide an unprecedented nano-scale profiling of the dynamics and distribution of water in membrane systems.


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P.042 Focusing on ase1-mediated microtubule bundling Z. Lansky, M. Janson Wageningen University, WAGENINGEN, Netherlands Ase1p, the fission yeast homologue of eukaryotic PRC1, plays a key role in the construction of mitotic spindle by selectively crosslinking anti-parallel microtubules (MTs). Ase1p-crosslinking can slow down motor-dependent inter-MT sliding. Dimers of ase1p diffuse along the MT lattice and form non-diffusing multimers along MTs at sufficiently high concentrations. We investigate relative contributions of dimers and multimers to MT bundling and inter-MT-friction. We use an in-vitro assay based on single molecule TIRF microscopy to observe individual GFP-labeled ase1p dimers during a bundling event using both, stabilized and dynamic MTs. First experiments show that the formation of ase1-multimers is not required to initiate MT bundling.

P.043 Hard constraints on cellular organization J.R. Alvarado, J. Nguyen, M. Soares e Silva, G. Koenderink AMOLF, AMSTERDAM, Netherlands Cellular mechanics are primarily determined by the cytoskeleton, a rich network of polymers, cross-linking proteins, and molecular motors which exert forces through organized structures. Cytoskeletal polymers exhibit entropically driven organization when confined to cellular dimensions. We investigate this effect by confining cross-linked actin networks in biomimetic photolithographic microchambers. We control the size and shape of these chambers to mimic typical plant cell dimensions. We quantify the spatial distribution of actin filaments to investigate the interplay between entropically driven organization and the organization caused by different kinds of cross-linkers.

P.044 Calcium binding of CBD2 is sensed structurally and dynamically throughout the molecule V. Breukels, G.W. Vuister Institute for Molecules and Materials, NIJMEGEN, Netherlands We report the effects of calcium binding on the backbone relaxation rates and chemical shifts of the second calcium binding domain (CBD2) of the sodium-calcium exchanger (NCX) studied by NMR. Calcium not only induces structural and dynamical differences in the coordinating loops, but also at the opposite end of the molecule. Chemical shifts perturbations are found as far away as 20 angstrom from the binding sites and 15N relaxation studies show that the FG-loop located at the opposite site of the domain becomes more flexible. So the binding event is felt throughout the molecule and this suggests that the FG-loop is important in relaying the information of the binding event toward the membrane-spanning region of the NCX.


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P.045 Mutual information between trajectories of biochemical networks F. Tostevin, P.R. ten Wolde AMOLF, AMSTERDAM, Netherlands Cells continually respond to a wide range of time-varying signals. These signals are detected and processed by biochemical networks. Examples include circadian clocks and bacterial chemotaxis pathways. However, the reactions making up these networks are inevitably noisy. How reliably, then, can a signalling network transmit the information in these signals? We use information theory to quantify the signalling performance of networks for time-varying signals. We characterise signalling through some elementary reaction schemes. We also study how reliably the E. coli chemotaxis network can communicate the ligand concentration to the flagellar motor. Our results reveal a complex interplay between swimming behaviour and signalling performance.

P.046 Information transmission for time-varying signals in biochemical networks W.H. de Ronde, F. Tostevin, P.R. ten Wolde AMOLF, AMSTERDAM, Netherlands Numerous researchers have studied the relation between network topology and network characteristics, like the response time, the noise in the output or the gain of the network. These studies reveal interesting properties of different topologies, but lack a more general quantitative framework. We use the temporal mutual information, a measure for the temporal correlation between a signal and a response, as a general measure to quantify the behaviour of the network. The temporal mutual information can be seen as a generic signal-to-noise measure, and holds therefore nformation both on the gain and the noise strength in a network. We study the gain-to-noise ratio and characterize each motif for its ability for reliable signal transmission.

P.047 Structural hierarchy governs fibrin gel mechanics I.K. Piechocka AMOLF, AMSTERDAM, Netherlands Mechanical resistance to large shear forces is crucial for the biological function of blood clots in hemostasis and wound repair. Blood clot rigidity mainly depends on a crosslinked network of fibrin fibers, which is elastic and highly extensible. Here we investigate the physical origin of this remarkable mechanical behavior. We measure the rheological properties of reconstituted fibrin gels over a wide range of time scales and strain amplitudes. We find that increasing levels of shear strain lead to a succession of distinct elastic responses which reflect stretching events on different length scales. We present a theoretical model which explains these observations based on the unique hierarchical architecture of the fibers.


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P.048 Conformations of the H-NS dimerization domain J. Vreede1, R.T. Dame2 1Universiteit van Amsterdam, AMSTERDAM, Netherlands 2Universiteit Leiden, LEIDEN, Netherlands H-NS is a bacterial DNA organizing protein, comprising a DNAbinding domain and a dimerization domain. Dimerization occurs through the formation of a coiledcoil interaction motif, which is either parallel or anti-parallel, depending on the sequence length. These two conformations seem to have different functions in DNA organization, butlittle is known of the mechanisms underlying H-NSdimerization. Using molecular dynamics simulations we investigated the stability of the two structures, as well as the stability of the monomers in isolation.Our simulations enabled a thorough investigation of the dimerization domain and provide a startingpoint for advanced simulation methods.

P.049 Monitoring Alzheimer amyloid peptide aggregation by EPR M.H. Shabestari1, I. Sepkhanova1, M. Drescher1, N.J. Meeuwenoord2, R.W.A.L. Limpens3, R.I. Koning3, D.V. Filippov4, M. Huber1 1Molecular Nano Optics and Spins(MoNOS), LEIDEN, Netherlands 2Leiden Institute of Chemistry, LEIDEN, Netherlands 3Leiden University Medical Center, Department of Molecular Cell Biology, LEIDEN, Netherlands 4Leiden Institue of Chemistry, LEIDEN, Netherlands The aggregation of the -Amyloid (A) peptide into fibrils and plaques is the chief indicator of Alzheimer's disease. Specific interest in oligomers stems from the suggestion that small, oligomeric aggregates and protofibrils, rather than fully formed fibrils could be responsible for the toxicity of the A-peptide. We investigate the potential of EPR to detect early stages of the aggregation of the A-peptide. Continuous wave, 9 GHz EPR reveals three fractions of different spin-lable mobility. One attributed to monomeric A, one to a multimer (8 to 15 monomers), and the last one to larger aggregates or fibrils. The approach, allows detection of oligomers on the timescale of aggregation.

P.050 Fluorescent cyclic voltammetry of immobilized azurin: a direct observation of thermodynamic and kinetic heterogeneity J.M. Salverda1, A.V. Patil2, G. Mizzon2, S. Kuznetsova3, G. Zauner3, N. Akkilic1, J.J. Davis2, G.W. Canters3, H.A. Heering3, T.J. Aartsma1 1Leiden Institute of Physics LION, LEIDEN, Netherlands, 2Department of Chemistry, Oxford University, OXFORD, United Kingdom, 3Leiden Institute of Chemistry LIC, LEIDEN, Netherlands We report on fluorescent cyclic voltammetry (FCV) of an immobilized blue copper protein and its application to the direct resolution of dispersion in formal electrochemical potential and kinetics. Fluorescent cyclic voltammograms are shown to align with current based voltammograms, allowing for an equivalent analysis of electrochemical parameters. The spatial resolution of FCV is limited only by optical diffraction, which corresponds to quantities between 100 and 1000 labeled proteins for the measurements shown. On this scale the midpoint potential is found to sample a distribution of ~70 mV in width and the electron transfer rate varies by up to a factor of 100 over distances of less than 10 microns across the electrode surface.


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P.051 Building microtubule networks in fission yeast J. Teapal, E. Janson Wageningen University, WAGENINGEN, Netherlands The interphase bipolar array of MTs in fission yeast contains about 4 bundles of MTs in which MT minus ends overlap at the centre while plus ends point out towards the cell ends. It was shown previously that a two component system - the minus end directed motor klp2 and the static crosslinker ase1p- organizes these overlapping regions. We aim to modulate this two component system using genetics to investigate the molecular requirements for the design of microtubule networks. Here, we aim to express NCD, a minus-end directed motor out of Drosophila melanogaster, in a klp2D cell. In contrast to klp2p, which accumulates at plus ends, NCD is expected to bind and induce sliding forces along the full length of overlapping MTs.

P.052 Single-molecule analysis reveals two separate DNA binding domains in the Escherichia coli UvrA dimer K. Wagner, G. Moolenaar, J. van Noort, N. Goosen Universiteit Leiden, LEIDEN, Netherlands The UvrA dimer is the initial damage-recognizing protein in bacterial nucleotide excision repair. UvrA is an ATPase, but the function of ATP binding and hydrolysis is poorly understood. Here we show that the UvrA dimer is more stable with ATP than with ADP or ATP?S, suggesting that the active UvrA dimer contains ADP and ATP. UvrA prefers to bind DNA ends, independent on the cofactor present. A significant number of complexes were found where UvrA bridges two DNA ends implying the presence of two DNA binding domains. On DNA with a site-specific lesion damage-specific binding depends on the cofactor present; implying that damage recognition of UvrA depends on the ability of both monomers to interact with DNA flanking the lesion.

P.053 Different elastic properties of two types Hepatitis B virus capsids measured by AFM M.J. Jacobs1, J. Zivkovic1, M. Nassal2, S. Speller1 1SPM Group, Institute for Molecules and Materials, NIJMEGEN, Nederland 2Medicine II, Molecular Biology, University Hospital Freiburg, FREIBURG, Germany Viruses are pathogens that need host cells to reproduce. Apart from their disease bringing nature, their capsids are the smallest symmetric biological structures that appear naturally. They are designed to naturally enter cells and to survive large mechanical stress without damage. That makes them an interesting target for drug delivery in the body and the development of new resistant materials. We focus on the elastic behavior of Hepatitis B capsids. We study them with an AFM in force volume mode, what enables us to make elasticity maps. In this way we are able to distinguish two types of HBV capsids. There are also reasons to believe that the frequency of repeated indentations influences the measured elasticity of viral capsids.


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P.054 Aggregation of transmembrane peptides studied by Spin-Label EPR M.H. Shabestari1, F. Scarpelli1, M. Drescher1, H. Eerkens1, T. Rutters-Meijneke2, A. Holt2, D.T.S. Rijkers3, J.A. Killian2, M. Huber1 1Molecular Nano Optics and Spins(MoNOS), LEIDEN, Netherlands, 2Chemical Biology & Organic Chemistry, Bijvoet Center for Biomolecular Research, UTRECHT, Netherlands, 3Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical S, UTRECHT, Netherlands The structure and function of membrane proteins depends on the balance between protein-protein and protein-lipid interactions. Peptide-peptide aggregation is one of the possible consequences of unfavorable lipid-peptide interactions. We performed Electron Paramagnetic Resonance (EPR) on a spin-labeled model peptide (WALP) in the gel and the liquid-crystalline phases of saturated and unsaturated phospholipids. Results suggest that at 120 K WALP peptides form cluster aggregates in saturated phospholipids and line aggregates in unsaturated phospholipids. In the liquid-crystalline phase of both lipids, signatures of aggregation are absent, showing that in this phase the peptide can be accommodated by either lipid.

P.055 Energy transport in engineered nanometer arrays of light harvesting antenna complexes M. Escalante1, L. Lenferink1, Y. Zhao1, N. Tas1, J. Huskens1, C.N. Hunter2, V. Subramaniam1, C. Otto1 1MESA+ Institute for Nanotechnology, ENSCHEDE, Netherlands 2Molecular Biology and Biotechnology, SHEFFIELD, United Kingdom We report the first observation of long-range transport of excitation energy within a bio-mimetic molecular light-guide constructed from LH2 antenna complexes from Rhodobacter sphaeroides, organized vectorially into functional nanoarrays. Fluorescence microscopy of the emission of light after local excitation with a diffraction-limited light beam reveals long-range transport of excitation energy over micrometer distances, which is much larger than required in the parent bacterial system. The transport was established from the influence of active energy-guiding layers on the observed Point Spread Function (PSF) of the fluorescence emission.

P.056 Peptide fragment structures in MS determined by IR spectroscopy J. Oomens1, B. Redlich1, J.D. Steill1, N.C. Polfer2, M.J. van Stipdonk3 1FOM Rijnhuizen, NIEUWEGEIN, Netherlands 2University of Florida, GAINESVILLE, FL, United States of America 3Wichita State University, WICHITA, KS, United States of America Sequencing by collision induced dissociation (CID) of protonated peptides in a mass spectrometer is essential in the identification of proteins and peptides. However, the reaction chemistry underlying CID is still under much debate. The molecular structures of peptide fragments resulting from low-energy CID provide unique experimental information that is essential to corroborate theoretical models describing peptide fragmentation. The structure of peptide fragments in a mass spectrometer has been determined from IR spectra of mass isolated dissociation products, recorded using a free electron laser. We will present this novel method of ion spectroscopy and some of its applications in understanding the fundamentals of peptide sequencing.


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P.057 Self-assembly of silk-based proteins M. Schor, P.G. Bolhuis Universiteit van Amsterdam, AMSTERDAM, Netherlands Proteins can fold and assemble into long fibres that play a structural role. The natural silk protein has a repetitive amino-acid sequence and is known to form beta-sheet based fibers similar to amyloids. The formation of such fibers is poorly understood. We developed a coarse grained force field to study the fiber-forming behavior of an artificial silk protein (sequence (EGAGAGAG)x) with and without hydrophilic flanking regions, with MD simulations.

P.058 Towards regulated microtubule dynamics under constant force S.M.J. Kalisch1, J. Husson2, L. Laan1, M. Dogterom1 1AMOLF, AMSTERDAM, Netherlands 2Institute Curie, PARIS, France Microtubule (MT) dynamics is highly regulated by proteins, as well as by the forces generated when MTs grow against obstacles such as the cell cortex. However, the molecular mechanisms underlying this remain largely unknown. We therefore investigate the force-induced behavior of dynamic MTs in the presence of end-binding proteins by growing them against a rigid barrier in vitro. Our currently used optical tweezers technique allows us to achieve molecular resolution on the MT growth process, yet restricts us to increasing force conditions. To be able to exert constant forces we are currently integrating force-feedback into our setup and are testing it on our MT assay in the presence of the fission yeast proteins Mal3, Tea2, and Tip1.

P.059 Fluorescence correlation spectroscopy for the analysis of electron transfer in proteins A. Andreoni1, L.C. Tabares1, A.W.J.W. Tepper1, T.J. Aartsma2, G.W. Canters1 1Leiden Institute of Chemistry, LEIDEN, Netherlands 2Leiden Institute of Physics, LEIDEN, Netherlands The investigation of electron-transfer processes is very important to understand the mechanism of biochemical reactions. To investigate these processes we have developed a method that allows the detection of the redox state of a protein monitoring the fluorescence emitted by a label attached to it. This method is based on the Forster Resonance Energy Transfer (FRET) effect: the label is the fluorescence donor while an absorbing cofactor is the fluorescence acceptor. We brought the analysis of this processes to the single-molecule level using Fluorescence Correlation Spectroscopy (FCS). The model we are investigating is the well studied Cu(I)/Cu(II) Azurin dimer: the results shown are in good agreement with the data in the literature.


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P.060 Picturing the activity of nitrite reductase at the single molecule level L.C. Tabares1, M. Elmak2, D. Kostrz3, C. Dennison3, T. Aartsma2, G.W. Canters1 1Leiden Institute of Chemistry, LEIDEN, Netherlands 2Leiden Institue of Physics, LEIDEN, Netherlands 3Institute for Cell and Molecular Bioscience, NEWCASTLE UPON TYNE, United Kingdom By attaching a fluorescent dye to the Cu-containing protein it is possible to follow changes in the redox state of the following the changes in the intensity and fluorescence lifetime of the fluorophore. The method was applied to the study of copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) at the single molecule level by fluorescence lifetime imaging. Immobilized Atto-647N-AxNiR presented a lifetime of 3.7 ns in the reduced state and 1.1 ns in the oxidized state. Changing the concentration of nitrite it was possible to observe the enzyme turning-over in the picture. Unexpected, our results show that AxNiR can follow two distinct catalytic pathways that coexist in a population of proteins.

P.061 Modification of single molecule fluorescence emission near gold nanoparticles A. Elmalk1, L. Tabares2, G. Canters2, T. Aartsma1 1Leiden Institute of Physics, LEIDEN, Netherlands 2Leiden Institute of Chemistry, LEIDEN, Netherlands The hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described electron transfer protein, with gold nanoparticles has been investigated. Single-molecule measurements clearly show that labeled azurin molecules display increase in fluorescence intensity, with decrease in lifetime when they are assembled on gold nanoparticles, as compared to control glass slides. From intensity and lifetime histograms, it is clear that the intensities as well as lifetimes of individual azurin molecules on gold nanoparticles are more heterogeneously distributed than that on glass.

P.062 Single-molecule analysis of RAD51 filament dissociation from double strand DNA J.T. Holthausen1, H. Sanchez1, M. Modesti2, T. Thorslund3, S. West3, C. Wyman1, R. Kanaar1 1ErasmusMC, ROTTERDAM, Netherlands 2CNRS, MARSEILLE, France 3Cancer Research UK, LONDON, United Kingdom Homologous recombination is essential for preservation of genome stability, and thus preventing cancer. We focus on the core protein RAD51 driving DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators like BRCA2. We are determining the influence of recombination mediators on intrinsic aspects of RAD51-DNA interactions. For this purpose we visualize fluorescently labeled RAD51 interacting with dsDNA in a flow-cell, measuring loss of fluorescent signal over time. Here we study the effects of TR2, the C-terminal fragment of BRCA2, on the disassembly of RAD51 filaments in real-time.


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P.063 Does the actin filament bundling protein villin regulate cytoplasmic organization and stiffness in plant cells? H.S. van der Honing, H. Kieft, N.C.A. de Ruijter, A.M.C. Emons, T. Ketelaar Wageningen University, WAGENINGEN, Netherlands The organization of the cytoplasm is crucial for plant cell functioning, especially for plant cell growth. Actin filaments are involved in the generation of cytoplasmic organization: all cytoplasmic strands of interphase cells contain actin filaments, and when actin filaments are depolymerized, cytoplasmic strands disappear. Using optical tweezers, we show that stiffness is an actin-related property of plant cytoplasm. Since the actin filament bundling protein villin is known to play a role in cytoplasmic organization, we study the role of villin in the organization of the actin cytoskeleton and the cytoplasm in plant cells by localization studies, mutant analysis and tweezers studies.

P.064 High NA optical coherence phase microscopy M. de Groot1, B. Haslam2, J.F. de Boer1 1Vrije Universiteit Amsterdam, AMSTERDAM, Netherlands 2Massachusetts Institute of Technology, CAMBRIDGE MA, United States of America Spectral domain optical coherence phase microscopy is a low-coherence interferometric technique for quantitative phase imaging and detection of nanometer scale motion in living cells. It has been used to study the contraction of cardiomyocytes, cytoskeletal rheology of human breast cancer cells and to perform optical detection of action potentials in nerve bundles. The traditional implementation however, is not compatible with high NA optics and therefore lacks the high spatial resolution of other microscopy techniques. We describe a common path dual beam sample arm design that overcomes this problem. We demonstrate diffraction limited performance of a 1.2 NA objective and nanometer phase sensitivity.

P.065 A mutant of the protein export-dedicated chaperone SecB defective in the holdase function A.P. Bechtluft1, A. Kedrov2, D.J. Slotboom2, N. Nouwen2, S. Tans1, A.J.M. Driessen2 1AMOLF, AMSTERDAM, Netherlands 2RuG, GRONINGEN, Netherlands Preprotein secretion by the translocase of E. coli involves the chaperone SecB. By binding to hydrophobic sections of unfolded preproteins, SecB prevents their folding and maintains them in a translocation competent state. Here, we have investigated the effect of the single residue mutation leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB, on its chaperone function. The mutant retains its tetrameric state, binds to the motor domain of the translocase but is defective in its holdase function. Single molecule optical tweezers studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain.


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P.066 Distinguising double- and single-stranded nucleic acid molecules using solid-state nanopores M. Skinner, M. van den Hout, O. Broekmans, C. Dekker, N.H. Dekker TU Delft, DELFT, Netherlands Solid-state nanopores offer a promising method for rapidly probing the structural properties of biopolymers such as DNA and RNA. We have for the first time translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double-stranded RNA molecules and of single-stranded homopolymers poly(A), poly(U), poly(C). On the basis of their differential blockade currents, we can rapidly discriminate between both single- and double-stranded nucleic-acid molecules, as well as separate purine-based homopolymers from pyrimidine-based homopolymers.

P.067 Role of plasma membrane structuring on Interferon receptor assembly & signaling A. Pezzarossa1, J. Piehler2, T. Schmidt1 1Leiden University, LEIDEN, Netherlands 2Osnabruck University, OSNABRUCK, Germany We study type I interferon receptor which plays a key role in innate and adaptive immune responses. It comprises two proteins ifnar1 and ifnar2, present at low surface concentration. Upon ligand binding it forms a ternary complex and signaling pathways activate. Using single-molecule microscopy we follow individual receptor in the membrane of living cells. Through correlative analysis of Ifnar1 and Ifnar2 mobility we visualized association/dissociation events of the ternary complex, and obtained information on receptor's diffusion constants. We suggest a role for membrane nanostructure for differential signaling.

P.068 Coarse grained molecular dynamics simulations of transmembrane peptides in a model raft lipid membrane D.H. de Jong, L.V. Schäfer, S.J. Marrink GBB, GRONINGEN, Netherlands There is more and more experimental evidence for a large degree of lateral heterogeneity in biological membranes. Accordingly, small and highly dynamic membrane regions, known as rafts, are supposed to play important roles in a number of biological processes. We present the results from coarse grained MD simulations of WALP transmembrane peptides in a model raft that consists of a liquid-disordered (Ld) and a liquid-ordered (Lo) domain. All peptides partition to the Ld domain, irrespective of the hydrophobic mismatch. This agrees well with recent optical microscopy experiments on model vesicles. Our simulations yield detailed insights into the lateral organization of transmembrane peptides at near-atomic resolution.


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P.069 Blue light induced NPQ in synechocystis PCC 6803 T.L.J. Tian Wageningen University, WAGENINGEN, Netherlands To disclose the mechanism of blue-light induced non-photochemical quenching (NPQ) in cyanobacteria, picosecond fluorescence kinetics were measured in wild-type Synechocystis PCC 6803 and a mutant with over-expressed orange carotenoid protein (OCP) by using a streak-camera system. Time-resolved data obtained for both the unquenched and the quenched state were fitted by four decay components. The corresponding decay associated spectra (DAS) indicate that the mutant with extra OCP possesses the same quenching mechanism as wild-type Synechocystis. The quenching is mainly related to two components with ~280 ps and 1.5 ns lifetimes, which indicates that the NPQ mainly takes place in the allophycocyanin (APC) core of the phycobilisomes.

P.070 AFM force spectroscopy to study the recognition of pathogens by immune cells J. te Riet1, A. Cambi1, S. Speller2, C.G. Figdor1 1NCMLS, NIJMEGEN, Netherlands 2IMM, NIJMEGEN, Netherlands By measuring force-distance curves with AFM we can probe the interactions between a receptor on the cantilever and a ligand on a substrate. In particular we are studying Carbohydrate-binding proteins, which play an essential role in the recognition of specific glycosylation patterns of pathogens like viruses and fungi. However the exact mechanism, especially the specificity and strength of these interactions, is largely unknown at the single molecule level. By exploiting force spectroscopy of cell-cell interactions by AFM, we aim to probe the interaction forces between immune cells and pathogen cells and determine the affinity, avidity, and the stability of their interactions under dynamical conditions at the single-molecule level.

P.071 Molecular recognition combining scanning force and fluorescence microscopy H. Sanchez, R. Kanaar, C. Wyman Erasmus MC, ROTTERDAM, Netherlands Scanning force microscopy is a powerful technique for studying biomolecular structures with nanometer resolution. However, many proteins appear similar in shape an size in SFM images, limiting the information that can be obtained. Fluorescence techniques can successfully identify specifically labeled proteins in vivo and in vitro, also for dynamic studies. We show here the nanometer localization of single particles using a combined scanning force and fluorescence microscope. The set up is based on a conventional wide-field fluorescence microscope coupled to a commercially available scanner. Our experiments reveal the versatility of this method for identification and nanometer localization of different fluorescently labeled particles.


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P.072 Modeling and mimicking self-organized network growth in migrating cells B. Stuhrmann1, F. Huber2, C. Vogt2, S. Ehrig2, J. Käs2 1 AMOLF, AMSTERDAM, Netherlands 2University of Leipzig, LEIPZIG, Germany Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front. Aiming for an identification of physical organization principles, we reconstituted the active cell front in vitro, as a Monte Carlo simulation, and as a mathematical model. We demonstrate the system's ability to form two distinct networks, the lamellipodium and lamellum, by means of self-organization. We quantitatively investigate the influence of the actin modulators ADF/cofilin and tropomyosin, and of filament severing and annealing, on network properties. With our work we contribute to a fundamental understanding of how cells organize their molecular components to achieve movement.

P.073 CP24, light harvesting and photoprotection F. Passarini1, E. Wientjes1, R. Hienerwadel2, R. Croce1 1GBB, GRONINGEN, Netherlands 2Université d’Aix-Marseille II - LGBP, Dpt de Biologie, Fac de Sciences de Luminy, MARSEILLE, France CP24 is a chlorophyll-binding protein of Photosystem II of higher plants. It has been proposed to be involved in the process of excess energy dissipation that protects plants from photodamage in high-light condition. To search for the quenching site in CP24 and study the role of individual chromophores in light-harvesting and regulation of excitation energy distribution, we performed in vitro reconstitution of the protein combined with site-directed mutagenesis. The biochemical/spectroscopic analysis indicates that in CP24 the domain around a specific carotenoid binding site determines the stability of the protein and modulates its function allowing the conversion of the complex from a light-harvesting to a dissipative state

P.074 Chaperonin-assisted folding of the bacteriophage T4 major capsid protein S.G.M. Calmat1, H. van Herrikhuizen1, C.F. Schmidt2, E.J.G. Peterman1, S.M. van der Vies1 1VU University Amsterdam, AMSTERDAM, Netherlands 2University of Goettingen, GOETTINGEN, Germany Folding of bacteriophage T4 major capsid protein, gp23, is strictly dependent on E.coli chaperonin GroEL in conjunction with the T4-encoded co-chaperonin, gp31. Gp31 is structurally similar to the host homologue GroES, which cannot fold gp23 in conjunction with GroEL. GroEL-gp31 refolds gp23 in vitro via a cis-encapsulation mechanism. We investigate the kinetics of gp23 folding and show that it is complete after one minute. The folding rate of gp23 inside GroEL-gp31 is faster than that of other substrates of GroEL-GroES, and the folding of gp23 is very efficient. We conclude that the unique folding abilities of GroEL-gp31 in vivo are conferred by the substrate gp23 in addition to structural properties of the T4-encoded gp31.


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P.075 Mechanical and structural properties of Norwalk virus capsid revealed by scanning force microscopy M. Baclayon1, W. Roos1, G. Shoemaker2, C. Uetrecht2, S. Crawford3, M. Estes3, V. Prasad3, A. Heck2, G. Wuite1 1Vrije Universiteit Amsterdam, AMSTERDAM, Netherlands, 2Utrecht University, UTRECHT, Netherlands, 3Baylor College, HOUSTON, United States of America The Norwalk virus strain is responsible for human nonbacterial gastroenteritis. Its capsid is composed of 180 copies of a single protein, forming 38 nm diameter shells. The Norwalk capsids possess peculiar bridge-like structures on its surface and using Scanning Force Microscopy-based nanoindentation experiments we try to elucidate the mechanical impact of these structures on the overall capsid strength. For small indentations, the particle behaves elastically but at large indentations, the particle suffers irreversible damage. From the analysis of the force-indentation curves we determine the mechanical properties and structural strength of the capsid, described in terms of modulus of elasticity and breaking force.

P.076 Energy transfer in higher plant Photosystem I I.E. Wientjes1, H. van Amerongen2, R. Croce1 1Groningen University, GRONINGEN, Netherlands 2Wageningen University, WAGENINGEN, Netherlands Photosystem I (PSI) consists of a core moiety and an external light harvesting complex (LHCI), composed of four Lhca pigment-protein complexes, organized into two dimers Lhca1/4 and Lhca2/3. A remarkable spectroscopic characteristic of LHCI is the presence of chlorophylls which absorband fluoresce atlow energies (red-forms), mainly present in Lhca3 and Lhca4. Here we report a time-resolved-fluorescence study on PSI with a range of Lhca antenna size and composition. This allows us to investigate the energy transfer pathways in PSI-LHCI, which special attention for the red-forms. Our preliminary results indicate that the two dimers have similar transfer efficiencies and rates, and that the red-forms slow down the trapping rate.

P.077 Visualization and characterization of dynamic events of the chaperonin folding machine by using Föster resonance energy transfer I. Idriss, S.G.M. Calmat, E.J.G. Peterman, S.M. van der Vies VU University Medical Center, AMSTERDAM, Netherlands The E.coli, chaperonin machine (GroEL-GroES) is responsible for folding 15% of the newly-synthesized proteins i.e. those with complex 3D structures. During bacteriophage T4 infection, the E.coli chaperonin is altered in order to fold the T4 major capsid protein, by exchanging GroES for the phage-encoded gp31 protein. It is not fully understood why GroES is unable to assist the folding of gp23. Earlier work revealed some structural differences between the two chaperonin complexes. Here we report on the kinetics of eGFP folding with both chaperonin systems.


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P.078 Force induced dissociation of protein bonds A.M. de Jong, A. Jacob, A. El Fattahi, F. Lambert, X.J.A. Janssen, L.J. van IJzendoorn, M.W.J. Prins Eindhoven University of Technology, EINDHOVEN, Netherlands Proteins are important molecules for medical diagnostics and treatment. We investigate methods to rapidly quantify the binding strength of proteins. Fast quantification of protein bonds can be obtained by force induced dissociation. In this work we demonstrate and compare two methods: (i) using magnetic particles and magnetic fields, and (ii) using polystyrene particles and fluid flow. After incubation, a constant force is applied to the bound particles and their number on the surface is monitored in time. The biotin anti-biotin system and the IgG protein-G binding systems are studied. We propose a kinetic model to describe the dissociation of the biomolecular bonds.

P.079 Biomembrane adhesion by mobile receptor-ligand pairs S. Fenz1, R. Merkel2, K. Sengupta3 1University of Leiden, LEIDEN, Netherlands 2Research Centre Juelich, Institute of Bio- and Nanosystems, JUELICH, Germany 3University of Marseille, CINAM/CNRS, MARSEILLE, France We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific biotin-neutravidin interaction to a supported lipid bilayer (SLB). Based on a standard fluorescence microscope, a new set-up was developed that enables simultaneous imaging in RICM and fluorescence microscopy. GUVs adhering to SLBs were characterized with respect to their inter-membrane distance and fluctuation amplitude. Fluorescent imaging of receptors yielded their distribution and concentration. We present both static and dynamic analysis of the inter-membrane distance and bond ordering for the limiting cases of dense and dilute bonds.

P.080 Critical domains in the tumor suppressor Axin1 are natively unfolded M. Noutsou UMC Utrecht, UTRECHT, Netherlands The Wnt pathway mediates crucial cell growth and differentiation responses during development and is frequently mutated in cancer. The tumour suppressor Axin performs a central role in the Wnt pathway by scaffolding a complex of APC, GSK3 and CK1 to direct degradation of -catenin. Structural information on critical parts of Axin is lacking. Using biophysical approaches (fluorescence spectroscopy, NMR and CD) we demonstrate that the central domain of Axin, which binds GSK3 and -catenin, is natively unfolded yet carries residual helical structure. With our ongoing work we aim to understand how the intrinsically disordered region of Axin contributes to its tumour suppressor activity in the Wnt pathway.


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P.081 Optical torque wrench for single molecule studies F. Pedaci, S. Klijnhout, J.W.J. Kerssemakers, N.H. Dekker TUDelft, DELFT, Netherlands At the molecular level, the torque applied to biopolymers plays a central role in many processes involving conformational changes and interactions with proteins. We will study the torque-sensitivity of important biological systems using a novel optical tweezers configuration termed the optical torque wrench[1]. This technique will allow us to control in real-time both force and torque in single biopolymers, with high temporal and spatial resolution. Here we present a first characterization of our instrument. This will allow us to acquire fundamental insight into the torque-sensitivity and dynamics of nucleic acids, DNA packaging, polymerase activity, and related biological processes. [1] LaPorta and Wang, PRL 92, 190801, 2004.

P.082 Raman imaging of single gold nanoparticles in live cancer cells L. Hartsuiker, R.G. Rayavarapu, W. Petersen, S. Manohar, T.G. van Leeuwen, C. Otto University of Twente, ENSCHEDE, Netherlands Gold nanorods (GNR) are rapidly emerging for use in biomedical applications due to their biocompatibility and their favourable optical properties, e.g. enhanced absorption cross-section. GNR surface plasmon resonance bands can be tuned to the near-infrared region (NIR) by changing the rods aspect ratio, enabling increased penetration for NIR optical imaging. Raman images are made to monitor the interaction of different GNR with SKBR3 breast carcinoma cells. Raman spectra show both broad-band and narrow-band features related to the gold nanorods inside the cells. We are therefore able to non-invasively detect gold nanorods with different coatings in live cancer cells.

P.083 Effect of cell flexibility on alignment of model myxobacteria A. Janulevicius, M. van Loosdrecht, C. Picioreanu Delft University of Technology, DELFT, Nederland Myxobacteria are social bacteria that exhibit a complex life cycle which culminates in the formation of multicellular fruiting bodies. Myxobacteria cells are elongated flexible rods that move on a substratum by gliding. During the development, myxobacteria often form multicellular structures from aligned cells. It is commonly suggested that cell flexibility helps mechanically interacting myxobacteria cells to align. However, we are not aware of any experimental or theoretical evidence to support this idea. We created a computational mass-spring model of a flexible rod-shaped cell that glide on a substratum and investigated how cell flexibility affects the alignment of a population of several hundreds mechanically interacting cells.


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P.084 Exciton dynamics and energy disorder in photosynthetic light-harvesting complexes V. Koning, N. Verhart, R. Purchase, R.J. Silbey, S. Völker Leiden University, Leiden, Nederland Light-harvesting (LH) complexes of purple bacteria have strongly interacting pigments which cause delocalization of the electronic excitation. The degree of this delocalization remains a subject of debate. To clear up the contradictions in the literature and to get a better understanding of the interplay between coherence and energy disorder, we performed hole burning experiments on a number of purple bacteria. From the depth of the holes measured as a function of wavelength, we determined the lowest k = 0 exciton states distribution. By comparing the experimental results with simulations, we could estimate the amount of energy disorder and electronic coupling in the various bacteria. Problems with the theoretical model will be addressed.

P.085 Single molecule magnetic tweezers study of topoisomerase IB activity in the presence of anti-cancer drugs P.J. Laverman1, J. Lipfert1, I. Koster1, M.A. Bjornsti2, N.H. Dekker1 1TU Delft, DELFT, Netherlands 2Department of Pharmacology and Toxicology School of Medicine University of Alaba, BIRMINGHAM, United States of America Magnetic tweezers (MT) are a powerful tool to study how single topoisomerase IB (topIB) molecules uncoil supercoiled DNA. Recent MT studies have revealed how wt topIB activity is dramatically affected by the presence of camptothecin-class (CPT) topIB inhibitors, used clinically as anti-cancer drugs. Here, we use MT to study how CPT influences the activity of the mutant G365C which has been shown to be drug-resistant in vivo. In the presence of CPT, the wt exhibits a marked difference in the speed of supercoil (sc) removal for positive and negative coils. In contrast, sc removal by the G365C is slow and shows similar velocities for coils of either sign. These results will help to distinguish several models of CPT induced cytotoxicity.

P.086 The role of genotype by environment interactions in adaptive evolutionary trajectories M.G.J. de Vos, F.J. Poelwijk, N. Tanyi, N. Battich, S.J. Tans AMOLF, AMSTERDAM, Nederland Organisms live in variable environments. In constant environments the role of genotype x genotype interactions in adaptive trajectories has been investigated. The role of genotype x environment interactions in evolution remains unclear. To investigate adaptation in periodical fluctuating environments, we used a synthetic model system of a selective operon under the control of the lac repressor in E. coli. Novel (inverse) regulatory variants of the lac repressor had been identified. We reconstructed all mutational intermediates and determined their phenotype and fitness. We show that not all trajectories towards optimal fitness were equally favourable in each environment. We can explain these effects at the phenotypic level: each mutation optimised the regulation in one environment, whereas the expression in the other environment was largely unaffected.


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P.087 UvrD: Unwinding DNA in mismatch repair N. Hermans, C. Claire, R. Kanaar, J. Lebbink Erasmus MC, ROTTERDAM, Netherlands DNA mismatch repair is responsible for the correction of DNA replication errors, and crucial in maintaining genomic stability. In Escherichia coli, the protein MutS mediates recognition a mismatch, and recruits MutL. MutL activates MutH, which nicks the newly synthesized DNA strand at a GATC motif. After nicking, DNA helicase II (UvrD) starts unwinding the double helix from 3' to 5', from the nick in the direction of the mismatch. What determines the direction of excision in MMR? This question is assessed by partly reconstituting the MMR system in vitro and visualizing the subsequent steps by using AFM, and by studying the interaction between the MMR proteins and DNA by SPR and EMSA.

P.088 Discriminating between chlorophyll and phycobilisome in FLIM images upon two-photon excitation. S. Laptenok1, S. Krumova2, R. Koehorst2, J.W. Borst2, A. van Hoek2, Z. Gombos3, H. van Amerongen2 1Vrije Universiteit Amsterdam, WAGENINGEN, Netherlands, 2Wageningen University, WAGENINGEN, Netherlands, 3Institute of Plant Biology, SZEGED, Hungary In order to understand the origin of the the fluorescence in FLIM experiments upon two-photon excitation streak-camera measurements were performed at two excitation wavelengths - 590 nm light was used to predominantly excite phycobilisomes, whereas 400 nm light is absorbed mainly by chlorophyll. The resolved lifetimes of the different components were compared to the ones observed in the FLIM experiments. It appears that in WT cells mainly the phycobilisomes are excited by the 860 nm two-photon excitation since the observed lifetimes and their respective amplitudes are very similar to the ones resolved by streak camera measurements upon 590 nm excitation and detected in the region 663-738 nm (the detection region for FLIM experiments).

P.089 Cost and colour of photosynthesis M.A. Marosvolgyi, H.J. van Gorkom Leiden Institute of Physics, LEIDEN, Netherlands The question why plants are green has been revisited in several articles recently. A common theme in the discussions is to explain why photosynthesis appears to absorb less of the available green sunlight than expected. This expectation is shown to be wrong, because it fails to take the energy cost of the photosynthetic apparatus into account. Depending on that cost, the red absorption band of the chlorophylls may be closely optimized to provide maximum growth power. If that is the case, the same optimization predicts no absorption at other wavelengths. The strong absorption of blue light by the same chlorophylls and by carotenoids is not selected because of its contribution to photosynthetic light-harvesting.


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P.090 Effect of hydrophobic mismatch and lipid composition on the mobility of bilayer spanning proteins and lipids S. Ramadurai1, A. Holt2, V. Krasnikov1, J. Killian2, B. Poolman1 1University of Groningen, GRONINGEN, Netherlands 2Utrecht University, UTRECHT, Netherlands Saffman-Delbrck developed a continuum hydrodynamic model for lateral and rotational diffusion of proteins in which lateral diffusion is strongly dependent on thickness (h) and viscosity () of the lipid membrane. We employ the model proteins GltT and LacY to study the lateral diffusion in varying bilayer thickness and phospholipid composition in the membrane. Proteins were purified, fluorescently labelled and reconstituted in LUVs made from varying acyl chain length and different phospholipid composition. FCS measurements were performed using a dual-color laser scanning confocal microscope. The diffusion coefficient of lipids is consistent with SD model, while the proteins show an 'anomalous' thickness dependence

P.091 Electrochemical sensing using nanofluidic devices P.S. Singh1, E.D. Goluch1, M.A.G. Zevenbergen1, B.L. Wolfrum2, A.W.J. Tepper3, H.A. Heering3, G.W. Canters3, S.G. Lemay1 1Delft University of Technology, DELFT, Netherlands 2Forschungszentrum Julich GmbH, JULICH, Germany 3Leiden University, LEIDEN, Netherlands We aim to develop new single-molecule techniques based on coupling electrical detection and nanofluidics. We fabricate solid-state devices consisting of a 50-nm high solution-filled cavity bounded by two parallel electrodes in which electrochemically-active molecules transfer charge between the two suitably biased electrodes. Redox cycling is employed, where each molecule contributes multiple electrons to the measured current by repeatedly traveling between electrodes, thus rendering single-molecule detection possible. These versatile devices allow us to examine rapid electron-transfer kinetics, investigate the adsorption dynamics of molecules, study enzyme kinetics, and extend sensing capabilities to the single-molecule level.

P.092 Self-assembly of triskelions into closed polyhedral cages W.K. den Otter, M.R. Renes, W.J. Briels University of Twente, ENSCHEDE, Netherlands The self-assembly of rigid three-legged particles into closed polyhedral cages is investigated by patchy particle simulations. A four-site anisotropic interaction potential is developed to make pairs of legs bind in an anti-parallel fashion, to form the edges of the lattice. The model's propensity to grow buckyballs of particular sizes is largely determined by the pucker of the rigid unit, while the strong preference for specific polyhedral structures appears innate to this class of cages. Nature explores a similar building unit, the clathrin triskelion, to regulate vesicle formation at the cell membrane during endocytosis.


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P.093 Probing the reactivity of nitrite reductase via dynamic protein-protein interaction J.M. Nusrat Jahan1, A.M. Elmalk1, H.A. Heering2, G.W. Canters1, T.J. Aartsma1 1Leiden Institute of Physics, LEIDEN, Netherlands 2Leiden Institute of Chemistry, LEIDEN, Netherlands Interaction between copper containing blue nitrite reductase (NiR) and a redox partner protein amicyanin (Ami) has been exploited to monitor the catalytic activity of NiR by electrochemistry and fluorescence. Electron transfer between the redox partner proteins via a dynamic and transient interaction of NiR-Ami leads to a conversion of nitrite to nitrous oxide. Thus activity of NiR i.e was investigated by analyzing the catalytic cyclic voltammograms exhibited by a spatially ordered hydrophobin coated gold with immobilized partner protein (Ami). The catalytic event was also detected from the readout signature of labeled amicyanin based on Frster resonance energy transfer from the fluorescent label to amicyanin.

P.094 Molecular dynamics simulations of polystyrene surfaces for biomedical applications S.A. Muntean, A.V. Lyulin, M. Kemper, L.J. van IJzendoorn, M.W.J. Prins Eindhoven University of Technology, EINDHOVEN, Netherlands The sensitivity of biosensors that rely on immunoassays is largely determined by the chemical reactivity and conformation of immobilized proteins at the sensor surface. The goal of this research project is to obtain a detailed understanding of the interaction between proteins and modi ed polymer substrate materials by a combination of experiments and MD calculations. In this poster we report on the morphological properties of oxidized polystyrene surfaces obtained from molecular dynamics simulations. The properties of the unmodified hydrophobic polystyrene are compared with those of the hydrophilic oxidized polystyrene surface. The results are compared with experimental data obtained by XPS and AFM.

P.095 Direct measurement of forces on protein-coated DNA molecules in solid-state nanopores A.R. Hall, S. van Dorp, S.G. Lemay, C. Dekker TU Delft, DELFT, Netherlands Solid-state nanopores are an emerging system for single-molecule biophysics wherein an electric field is used to drive charged biomolecules into a nano-scale aperture in a thin membrane. Here, we present measurements in which bead-tethered molecules are delivered to a pore with optical tweezers and captured statically. The force is then measured through bead displacement. We study DNA molecules coated with the protein RecA, which have greater size and charge than bare DNA. We find that forces acting on RecA-DNA are larger than those acting on bare DNA and can be described by an electrophoretic/osmotic model. We also study the conductance changes caused by the RecA-DNA inside a nanopore and find results consistent with the molecular charge.


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P.096 Multiple FRET studies reveal molecular features of the off-pathway folding intermediate of apoflavodoxin S. Lindhoud, A.H. Westphal, C.P.M. van Mierlo Wageningen University and Research Centre, WAGENINGEN, Netherlands FRET is used in our laboratory to study the folding behaviour of apoflavodoxin. For this purpose, a donor dye label is always attached to cysteine at position 69 and an acceptor label is coupled to another cysteine, which resides either on position 1, 100, 131 or 178 of the 179-residue protein. FRET measurements of apoflavodoxin folding show that the conformation of the off-pathway intermediate differs dramatically from the conformation of the native protein. Most inter-dye distances are shorter in the off-pathway intermediate as compared to their distances in native apoflavodoxin.

P.097 In vitro measurements of transport across a single biomimetic nuclear pore complex S.W. Kowalczyk1, L. Kapinos2, R.Y.H. Lim2, C. Dekker1 1Kavli Institute of NanoScience, DELFT, Netherlands 2Biozentrum, BASEL, Switzerland Transports of macromolecules (proteins, RNA) between the nucleus and the cytoplasm occurs through large protein complexes known as nuclear pore complexes (NPC) - the 'gatekeepers' of the nucleus - which selectively allows some cargos to pass but others not. We have made biomimetic nuclear pores, combining solid-state nanopores with key nuclear pore proteins rich in FG-repeat domains, with the aim to test the fundamental mechanism of the NPC. We will present electrophysiological single-molecule data of transport of transporter molecules like Importin-Beta (ImpB) across such a 'minimalist' NPC. We find that ImpB takes ~20 times longer to translocate through a biomimetic nuclear pore than through a bare nanopore.

P.098 Using GFRD to study pattern formation due to the interplay of active and passive transport T.R. Sokolowski, L.P. Bossen, T.W. Miedema, P.R. ten Wolde AMOLF, AMSTERDAM, Netherlands Cells exploit the interplay of active transport along cytoskeletal tracks and cytosolic passive diffusion to establish a wide range of spatial patterns of functional proteins, mRNA and specialized organelles. Such systems are not well-stirred, so standard simulation techniques can be very expensive while coarse-graining may be inappropriate. Green's function reaction dynamics (GFRD) is an exact event-driven chemical simulation scheme based on analytical solutions of the Smoluchowski equation. Its efficiency is significantly higher as compared to common Brownian dynamics schemes. Based on GFRD we develop a framework which allows for a spatio-temporal stochastic simulation of both active and passive transport in different geometries.


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P.099 DNA nanomechanics: Analyzing DNA-protein tension N.B. Becker1, R. Everaers2 1AMOLF, AMSTERDAM, Netherlands 2ENS, LYON, France Cellular DNA is strongly distorted by proteins, for example by histones, transcription factors, and restriction enzymes. In all these cases, mechanical DNA deformation affects binding rates and thus biological function. We present an analysis that allows to characterize the local tensions in DNA-protein complexes, taking only DNA conformation as input. The method requires high-resolution structural data (often available from crystallography or NMR structure determination), and relies on the rigid base-pair elastic model of DNA, parameterized by experiments and simulations.

P.100 A novel assay to study Rad51 dynamics on ssDNA in real time M.A.M. Franker1, A. Candelli1, M. Modesti2, G. Wuite1, E.J.G. Peterman1 1Vrije Universiteit Amsterdam, AMSTELVEEN, Netherlands 2CNRS, MARSEILLE, France Recombinase protein Rad51 is essential for homologous recombination in Eukaryotes. Dynamic association and dissociation of the Rad51 nucleoprotein filament are crucial for proper strand exchange. However, little is known about the mechanochemistry of these processes, in particular on single-stranded DNA. Here, we present a novel single-molecule assay using a combination of single-molecule fluorescence and optical trapping to study the dynamics of Rad51 on ssDNA in real time. Using this assay we have seen multiple Rad51 nucleations appearing on ssDNA after incubating for 30 seconds in the presence of Ca2+. Buffer containing Mg2+ triggered complete dissociation of the filament within minutes.

P.101 Photosystem I of chlamydomonas reinhardtII B. Drop, M. Birungi, E.J. Boekema, R. Croce University of Groningen/GBB, GRONINGEN, Netherlands Photosystem I (PSI) of Chlamydomonas reinhardtiiis a large chlorophyll-binding proteins complex located in thylakoid membranes. The exact composition of the complex and its supramolecular organization are however not known. The aim of this project is to get functional and structural information about PSI complexes of C.reinhardtii cells growing under different environmental conditions. Isolated PSI complexes were characterized using absorption, low and room temperature fluorescence spectroscopy (steady state and time-resolved), biochemical techniques and electron microscopy.


Abstracts posters

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P.102 Characterizing polystyrene for studying protein interactions M. Kemper, L.J. van IJzendoorn, S.A. Muntean, A. Lyulin, M.W.J. Prins Technische Universiteit Eindhoven, EINDHOVEN, Netherlands The sensitivity of biosensors that rely on immunoassays is largely determined by the chemical reactivity and conformation of immobilized proteins at the sensor surface. The goal of this research project is to obtain a detailed understanding of the interaction between proteins and modified polymer substrate materials by a combination of experiments and MD calculations. We use the adsorption of myoglobin on modified polystyrene as a model system. In this poster we report on the chemical and morphological properties of oxidized polystyrene surfaces characterized with XPS and AFM. Oxidation by potassium permanganate and UV/ozone treatment are compared and the morphology of polystyrene surfaces is compared to results from MD calculations.

P.103 Microscopic evaluation of the role and dynamics of CD6 in T cell activation using microprinted surfaces M.B.M. Meddens1, A. Esseling-Ozdoba2, J. Witsenburg3, R. Brock3, A. Cambi2, C.G. Figdor2 1NCMLS, NIJMEGEN, Netherlands 2Department of Tumor Immunology, NCMLS, UMCN, NIJMEGEN, Netherlands 3Department of Biochemistry of Integrated Systems, NCMLS, UMCN, NIJMEGEN, Netherlands During T cell activation by antigen presenting cells an immunological synapse (IS) is formed. We investigate the role and dynamics of the costimulatory receptor CD6 in this process. Using microcontact printing, we mimick the IS on a glass substrate by making spots of antibodies that stimulate the T cell receptor (TCR) and CD6 in combination with other costimulatory receptors. T cells are allowed to interact with these spots. Using confocal microscopy and image quantification, we show that spots stimulating TCR and CD6 increase p-Tyr signaling compared to TCR stimulation alone. Furthermore, CD6 recruitment is independent of CD6 stimulation, suggesting a role for CD6 in a supra-molecular signaling complex involved in T cell activation.

P.104 Unraveling chaperonin activity of trigger factor at the single molecule level A. Mashaghi Tabari, P. Bechtluft, S.J. Tans AMOLF, AMSTERDAM, Netherlands Trigger factor (TF) is a ribosome associated chaperone. The exact molecular mechanism of action of this chaperone is not known. Previous studies suggest that TF prevents aggregation of denatured polypeptides and newly translated proteins. With the use of optical tweezers, we studied the effect of TF on the folding pathways of maltose binding protein (MBP). In order to investigate the role of TF in preventing aggregation of unfolded proteins, a tandem repeat of four MBPs was constructed. Our preliminary data suggests that in the presence of TF, unfolded MBP either refolds or makes a folding competent complex with the chaperone. TF prevents aggregation of unfolded tandem 4MBP repeat that otherwise misfolds into a tight aggregate.


Abstracts posters

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P.105 A synthetic regulatory cascade for quantitative studies M. Kogenaru, S. Tans AMOLF, AMSTERDAM, Netherlands Advances in the field of synthetic, molecular, and quantitative biology allows the construction of synthetic gene regulatory networks using model regulatory proteins and DNA regulatory regions, and also to measure their behavior quantitatively in vivo. We constructed a synthetic regulatory cascade that controls the expression of three different fluorescent reporters using the P_BAD /AraC, P_BAD/TetReCFP, P_LtetO-1 /LacI-mCherry, and P_TRC /eYFP systems. We also engineered an E. coli strain as a host for the joint functioning of these three regulatory proteins. Each of the components and the cascade as a whole is characterized by measuring the transfer function in vivo using fluorescent plate reader.

P.106 Towards a reconstituted SARS-coronavirus replication complex at single molecule level A.J.W. te Velthuis1, S. Hage2, S.H.W. van den Worm1, E.J. Snijder1, N.H. Dekker2 1Leiden University Medical Center, LEIDEN, Netherlands 2Delft University of Technology, DELFT, Netherlands The SARS-coronavirus and related nidoviruses encode a large set of proteins involved in viral RNA synthesis, including a primer-dependent RNA polymerase, a primase and a helicase. Currently, however, little detail is known about how they act together to synthesize the ~30 kb long viral RNA. Here we report a first step towards their mechanistic elucidation, using magnetic tweezers. The helicase was able to unwind dsDNA with a mean velocity of 30 bp/s, with stalling and rezipping events dependend on the template sequence. Such events were overcome by an increase of the ATP concentration or the applied force. Future experiments will be aimed at studying the helicase on tethered RNA hairpins as well as in concert with the polymerase.

P.107 Cooperation and cheating in bacterial metapopulations P. Galajda, R. Woolthuis, J. Mannik, F. Hol, C. Dekker, J. Keymer Delft University of Technology, DELFT, Netherlands The interests of an individual may be different from those of a community. To study this conflict in bacterial populations we used mixed cultures of wild type (cooperator) and a GASP mutant (cheater) strain E. coli bacteria in homogeneous (batch culture) and spatially structured (microfabricated chip) environments. We find that the characteristics of the competitive 'game' that these bacteria play largely depend on conditions such as cell densities, nutrient concentration and growth phase. Moreover, in spatially structured environments motility, aggregation and possibly cell-to-cell signaling give rise to an increased survival of cooperative strategies compared to well mixed cultures.


Abstracts posters

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P.108 SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that bind to EB1 and its homologues B. van der Vaart, A. Akhmanova, I. Grigoriev, S. Montenegro Gouveia, J. Demmers, N. Galjart Erasmus MC, ROTTERDAM, Netherlands One of the most conserved families of microtubule plus-end tracking proteins (+TIPs) are the End Binding (EB) proteins. Using EB1-GST pull downs combined with mass spectrometry we identified SLAIN1/2 as new EB partners. Both SLAINs are highly conserved and widely expressed proteins. SLAIN plus-end tracking is mediated by SLAIN C-terminus and is dependent on EB1. SLAINs also bind to other +TIPs such as CLASPs, CLIP-170 and p150glued. The C-terminus of SLAIN terminates with a tyrosine (Y) residue, similar to EBs and a-tubulin. As is the case for EBs, SLAIN Y residue is essential for binding to CLIP-170 CAP-Gly domains. Currently we are investigating SLAIN function using RNAi in cultured cells.

P.109 The biological relevance of photochemical parameters and microscopic rate constants of the light-sensor protein YtvA from Bacillus subtilis Y. Nakasone, M. Avila-Pérez, J. Hendriks, K.J. Hellingwerf University of Amsterdam, AMSTERDAM, Netherlands The bacterial blue light sensor YtvA is a multi-domain protein with an N-terminal LOV domain. This protein mediates the initiation of the light-induced stress response in B. subtilis. Its LOV domain shows an unusually slow dark recovery rate and a light-induced branching reaction. Here, we investigated the importance of these features on the biological characteristics of the stress response. The most striking of these is its very low-fluence response, which could be precisely reproduced by a mathematical model that incorporates the slow dark recovery rate and light-induced branching reaction of the photosensory protein. The relationship between this biological activity and the theoretical basis of this model will be discussed.

P.110 Aquaporin-2 binds LIP5: Insight into the structural interaction of a cargo protein to the ESCRT complex S.M. Nabuurs, M. Boone, H. Venselaar, J.H. Robben, G. Vriend, G.W. Vuister, P.M.T. Deen Nijmegen Centre for Molecular Life Sciences, NIJMEGEN, Netherlands Concentration of urine by renal principal cells is critically regulated by the insertion into and internalization of the Aquaporin-2 water channel in the apical membrane, but little is known about the regulation of this shuttling. Yeast two-hybrid and GST pull down assays showed that the MIT1 domain of the lysosome trafficking interacting protein 5 (LIP5) interacts with the proximal C-terminal tail of AQP2. LIP5 is a factor in the ESCRT-III complex, which is involved in internalization of membrane proteins. Molecular modelling confirmed the potential binding of MIT1 and the AQP2 peptide (L230-D243). NMR spectroscopy will gain structural insight into the first binding of a cargo protein to the ESCRT complex.


Abstracts posters

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P.111 Zero-mode waveguides: A powerful tool for single-molecule optical studies Z. Huang, S.P. Donkers, N.H. Dekker Kavli Institute of NanoScience, DELFT, Netherlands Single-molecule fluorescence studies of enzymes that incorporate fluorescently labeled substrate nucleotides typically operate at substrate concentrations well below their Km values. While this is inevitable in conventional fluorescence microscopy, the biological relevance of the insights gained into enzyme mechanism may be compromised. Zero-mode waveguides (ZMWs) provide an excellent solution to this problem. We report the nanofabrication of ZMWs, the surface treatment for controlled immobilization of biomolecules and background noise reduction. We also present the development of an assay to monitor in real time the incorporation of fluorescently-labeled nucleotides, which paves the way for the studies of nucleic acid polymerizing enzymes.

P.112 Evolution of antibiotic resistance in patchy ecosystems F. Hol, P.J. Galajda, J. Männik, C. Dekker, J.E. Keymer TU Delft, DELFT, Netherlands The spatial structure of an ecosystem plays a crucial role in the evolutionary dynamics of its supported populations. In an unstructured habitat, competitive exclusion prevents the coexistence of competing subpopulations leaving one sequentially evolving population; in a patchy habitat, a parallel evolving metapopulation will arise. Patchiness also enhances biodiversity through local saturation and ensuing induction of stress responses. Here, we present an assay using nanofabricated ecosystems to study the relationship between habitat topological structure and the evolutionary dynamics of the inhabiting bacteria. We compare adaptation to the presence of antibiotics in habitats with different degrees of patchiness and landscape connectivity

P.113 LEDGF/p75 switches from a dynamic to a tight chromatin interaction upon binding to HIV-1 integrase J.H. Hendrix, Y. Engelborghs, Z. Debyser KU Leuven, LEUVEN, Belgium Human transcriptional co-activator LEDGF/p75 is hijacked by HIV-1 integrase (IN) during the replication of HIV. Little is still known about the molecular complex of these two proteins in the living cell. In this work we first studied the cellular chromatin interaction of eGFP-tagged LEDGF/p75 with tunable-focus fluorescence correlation spectroscopy (TF-FCS) and show that LEDGF/p75 is in equilibrium between a free Brownian motion and a very slow movement on the chromatin. Being dependent on the size of the laser focus, this slow movement represents a continuous association-dissociation-reassociation process that is governed by diffusion.


Abstracts posters

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P.114 Electrical contacting of redox enzymes through DNA-directed self-assembly A.W.J.W. Tepper Leiden University, LEIDEN, Netherlands The direct electrochemical contacting of redox enzymes to solid electrode supports offers great potential for biosensing applications, but finding appropriate immobilization methods has proven difficult. I will discuss a new strategy based on the DNA-directed immobilization (DDI) of redox-protein components and show that nitrite reductase (NiR) can be electrochemically contacted by immobilizing it onto a gold electrode together with its natural electron-exchange partner pseudoazurin (Paz). The DNA tethered to the electrode surface acts as flexible place-holder for the Paz and NiR, allowing Paz to shuttle electrons between the electrode and the NiR enzyme.

P.115 Epigenetic gene regulation of the eukaryotic genome: A systems biology approach using synthetic cell systems P.J. Verschure1, L.C.M. Anink1, D.G.E. Piebes1, M.C. Brink1, A. Schwabe2, F.J. Bruggeman2 1University of Amsterdam, AMSTERDAM, Netherlands 2Centre for Mathematics and Computer Sciences, AMSTERDAM, Netherlands We analyzed the role of epigenetic regulatory proteins involved in gene silencing establishing higher order chromatin structures using advanced-microscopy-techniques, via: (i) interference with epigenetic regulatory proteins using a dominant negative approach and (ii) in vivo targeting of lac-repressor tagged epigenetic regulatory proteins to an amplified lac-operator site containing chromosomal domain. We created synthetic epigenetic cell systems allowing to change the chromatin state and measure the causal effect on epigenetic gene control using a systems biology approach. This research opens an unexplored field of research with great potential for medicine.

P.116 A multifocal 2-photon fluorescence setup for single-molecule tracking of chromatin dynamics in living cells B. van den Broek, T.H. Oosterkamp, S.J.T. van Noort Leiden Institute of Physics, LEIDEN, Netherlands The organization of chromatin plays a key role in gene regulation and processing. To date, most data comes from in vitro experiments or FISH studies on fixed samples. We aim to study chromatin dynamics in living cells using fluorescent probes. For this purpose we have constructed a multifocal 2-photon fluorescence microscope setup that combines advantages of 2-photon microscopy with the speed and sensitivity of wide field detection. We show that 3D images of cells can be obtained within 2 seconds. We compare the suitability of fluorescent dyes, quantum dots and gold nanorods for 2-photon 3D tracking. In future studies, by labeling specific genes with PNA we plan to quantify structural changes of chromatin at the single-gene scale.


Abstracts posters

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P.117 Modular coupling in E.coli chemotaxis revealed by FRET with time-varying stimuli T.S. Shimizu1, Y.T. Tu2, H.C.B. Berg3 1AMOLF, AMSTERDAM, Netherlands 2IBM T.J. Watson Research Center, YORKTOWN HEIGHTS, NY, United States of America 3Harvard University, CAMBRIDGE, MA, United States of America We characterized the transfer functions of the Escherichia coli chemotaxis signaling pathway by fluorescence resonance energy transfer (FRET) measurements of intracellular signaling responses. Receptor sensitivity was characterized by step stimuli, the gradient sensitivity by exponential ramp stimuli, and the frequency response by exponential sine-wave stimuli. Feedback near steady state was found to be weak, consistent with strong fluctuations and slow recovery from small perturbations. Gradient sensitivity and frequency response both depended strongly on temperature. Our results demonstrate how dynamic input-output measurements can serve as powerful tools in deciphering cell signaling mechanisms.


Abstracts posters

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List of participants

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name affiliation lunch 1 dinner room lunch 2 Anna Akhmanova EUR X X X X Namik Akkilic LEI X X X X José Alvarado AMOLF X X X X Herbert van Amerongen WUR X X X X Alessio Andreoni LEI X X X X Zeinab Anvarian UMC-U X X X X Philipp Bechtluft AMOLF X X X X Nils Becker AMOLF X X X X Martin Bennink UT X X X X Paul van Bergen en Henegouwen UU X X Andreas Biebricher VU X X X X Kerstin Blank RU X X X X Johan den Blanken TUD X X X X Egbert Boekema RuG X Peter Bolhuis UvA X X X Sylvestre Bonnet UU X X X X Peter Bosch UT X X X Sarah Boulineau AMOLF X X Vincent Breukels RU X X X X Chase Broedersz VU X X X X Bram van den Broek LEI X X X X Onno Broekmans VU X X X X Wybren Jan Buma UvA X X X Ruth Buning LEI X X X X Arend van Buul RU X X X X Stephane Calmat VU X X X X Alessandra Cambi RU X X X Andrea Candelli VU X X X X Vivek src Chand UT X X X X Debasish Chaudhuri AMOLF X X X X Fan-Tso Chien LEI X X X X Yi-Da Chung RU X X X X Victor Claessen RU X X X X Mireille Claessens UT X X X X Jeroen Cornelissen UT X X X X Michele Cristovao EMC X X X X Roberta Croce RuG X X X X Remus Dame LEI X X X X Moumita Das VU X X X Jan De Sonneville LEI X X X X Iwijn De Vlaminck TUD X X Eva Deinum AMOLF X X X Dirk Roelof Dekker UT X X X X Linda Dekker VU X X X X Jan Dekker VU X X X X Cees Dekker TUD X X X X Nynke Dekker TUD X X X X Martin Depken VU X X X X Heidi Dietrich TUD X X X X Bartlomiej Drop RuG X X X X Weina Du UvA X X X X Natalya Dudkina RuG X X X X



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name affiliation lunch 1 dinner room lunch 2 Esther van Duijn UU X X Michel Duits UT X X X X Michel Ebskamp Genetwister X X X X Rianne van Eerd FOM-bureau X X X X Abdalmohsen Elmalk LEI X X X Marc Emanuel LEI X X X X Anne-Mie Emons WUR X X X X Maryana Escalante UT X X X Geraldine Farge VU X X X X Susanne Fenz LEI X X X X Mariella Franker VU X X X X Peter Galajda TUD X X X X Swapna Ganapathy TUD X X X X Vanni Gasbarri KULeuven X X X X Brian Gentry AMOLF X X X X Nora Goosen LEI X X Ilya Grigorev EMC X X X X Rienk van Grondelle VU X X X X Huub de Groot LEI X X X X Peter Gross VU X X X X Marloes ten Haaff UT X X X X Adam Hall TUD X X X X Nima Hamedani Radja LEI X X X X Liesbeth Hartsuiker UT X X X X Thijn van der Heijden LEI X X X X Iddo Heller VU X X X X Freek van Hemert LEI X X X X Jelle Hendrix KULeuven X X X X Jennifer Herek UT X X X X Nicolaas Hermans EMC X X X X Hans Heus RU X X X X Silvia Hodorogea LEI X X X X Tjalle Hoekstra VU X X X X Felix Hol TUD X X X X Frank Holstege UMC X X X X Thomas Holthausen EMC X X X X Laurent Holtzer LEI X X X X Hannie van der Honing WUR X X X X Jacob Hoogenboom TUD X X X X Michiel van den Hout TUD X X X X Zhuangxiong Huang TUD X X X X Martina Huber LEI X X X X Timon Idema LEI X X X X Ienas Idriss VUMC X X X X Leo van IJzendoorn TU/e X X Matthias Irmscher TU/e X X X Monique Jacobs RU X X X Tessa Jager TUD X X X X Marcel Janson WUR X X X X Xander Janssen TU/e X X X Albertas Janulevicius TUD X X X X Arthur de Jong TU/e X X



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name affiliation lunch 1 dinner room lunch 2 Djurre de Jong RuG X X X X Aniek Jongerius AMOLF X X X X Svenja-Marei Kalisch AMOLF X X X X Olya Kalmykowa LEI X X X Muhammad Kamran LEI X X X X Lukas Kapitein EMC X X X X Alexej Kedrov RuG X X X X Sandra de Keijzer RU X X X X Marijn Kemper TU/e X X X John Kennis VU X X X Juan Keymer TUD X X X X Henk Kieft WUR X Antoinette Killian UU X X X X Eri Kinoshita EMC X X X X Malgorzata Kluba KULeuven X X X X Rob Koehorst WUR X X X Gijsje Koenderink AMOLF X X X X Manjunatha Kogenaru AMOLF X X X X Albert Konijnenberg RU X X X X Stefan Kowalczyk TUD X X X X Yvonne Kraan UT X X X X Vincent Krudde TUD X X X X Tjaart Kruger VU X X X X Maxim Kuil LEI X X X X Ilja Kusters RuG X X X X Liedewij Laan AMOLF X X X X Zdenek Lansky WUR X X X X Sergey Laptenok VU X X X X Niels Laurens VU X X X X Peter Jan Laverman TUD X X X Milena Lazova AMOLF X X X X Joyce Lebbink EMC X X X X Tony Lee Univ. Southampton X X X X Kirsten van Leijenhorst-Groener UT X X X X Serge Lemay UT X X X Ruifang Li LEI X X X Jelmer Lindeboom WUR X X X X Simon Lindhoud WUR X X X X Marijn van Loenhout TUD X X X X Cesar Lopez RuG X X X X Remy Loris VU Brussel X X X X Petra van Luling FOM-bureau X X X X Alexey Lyulin TU/e X X X X Yujie Ma UT X Fred MacKintosh VU X Tomas Magalhaes TUD X X X X Jaan Männik TUD X X X X Marcell Marosvolgyi LEI X X X X Alireza Mashaghi Tabari AMOLF X X X X Marjolein Meddens RU X X X X Carlo van Mierlo WUR X X X X Mohamed Miqyass LEI X X X



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name affiliation lunch 1 dinner room lunch 2 Geri Moolenaar LEI X X X X Mohammad Mahdi Motazacker AMC X X X X Emilia Motoasca TU/e X Bela Mulder AMOLF X X X X Teun Munnik UvA X X X X Andrea Muntean TU/e X X X Sanne Nabuurs RU X X X X Yusuke Nakasone UvA X X X X Jeanette Nguyen AMOLF X X X X Jana Nieder FUBerlin X X X X Bernard Nienhuis UvA X X X X John van Noort LEI X X X X Maria Noutsou UMC-U X X X X Sanghamitra Nusrat Jahan LEI X X X X Maarten van Oene TUD X X X Kim van Ommering TU/e X Jos Oomens Rijnhuizen X X X X Bart van Oort VU X X X X Anne van Oosten TU/e X X X Wouter den Otter UT X X X X Rik Paesen Philips X Anjali Pandit LEI X X X Francesca Passarini RuG X X X X Francesco Pedaci TUD X X Xavier Periole RuG X X X X Erwin Peterman VU X X X X Anna Pezzarossa LEI X X X X Indriati Pfeiffer EMC X X Izabela Piechocka AMOLF X X X X Bram Prevo VU X X X X Menno Prins TU/e X X Peter Prinsen LEI X X X X Vishnu Vardhan Pully UT X X X X Sivaramakrishnan Ramadurai RuG X X X Britta Redlich Rijnhuizen X X X X Alexander van Reenen TU/e X Daniela Rhodes MRC X X X X Emile de Rijk VU X X X X Maylon Rojer TUD X X X Elisabet Romero VU X X X X Wiet de Ronde AMOLF X X X X Bart van Rooijen UT X X X X Wouter Roos VU X X Theo Saat NWO X X X X Marian Salumbides-Baclayon VU X X X X Jante Salverda LEI X X Humberto Sanchez EUR X X X X Max Schlager EMC X X X X Thomas Schmidt LEI X X X X Marieke Schor UvA X X X X Sarah Schreurs KULeuven X X X Anne Schwabe CWI X X X



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name affiliation lunch 1 dinner room lunch 2 Andrew Scott TUD X X X X Ine Segers-Nolten UT X X X X Stefan Semrau LEI X X X X Maryam Shabestari LEI X X X X Paige Shaklee AMOLF X X X X Shashank Shekhar UT X X X X Thomas Shimizu AMOLF X X X X Pradyumna Singh TUD X X X X Gary Skinner TUD X X X X Marieke Snijder-van As UT X X X X Marina Soares e Silva AMOLF X X X X Thomas Sokolowski AMOLF X X X X Andreas Stahl VU X X X Oscar Stassen TUD X X X Bjorn Stuhrmann AMOLF X X X X Vinod Subramaniam UT X X X X Kim Sweers UT X X X X Olga Sytina AMOLF X X X X Leandro Tabares LEI X X X X François Taddei INSERM X X X X Sander Tans AMOLF X X X X Mariliis Tark UvA X X X X Juliane Teapal WUR X X X X Armand Tepper LEI X X X X Aniket Thete TU Ilmenau X X X X Lijin Tian WUR X X X X Marije aan den Toorn WUR X X X X Filipe Tostevin AMOLF X X X X Feng-Ching Tsai AMOLF X X X X Cicerone Tudor UT X X X X Babet van der Vaart EMC X X X X Willem Vanderlinden KULeuven X X X X Aartjan te Velthuis LUMC X Cassandra Verheul LEI X X X X Pernette Verschure UvA X X X X Iztok Vidic EMC X X X X Ana Vila Verde AMOLF X X X X Rifka Vlijm TUD X X X X Silvia Völker LEI X X X X Jarno Voortman UU X X Marjon de Vos AMOLF X Jocelyne Vreede UvA X X X X Joke van Vugt RU X X X X Koen Wagner LEI X X X X Anna Wegner TUD X X X X Adrie Westphal WUR X X X X Mirte Westra VU X X X X Emilie Wientjes RuG X X X X Matthew Wiggin TUD X X X X Martijn de Wild AMOLF X X X X Siet van den Wildenberg VU X X X X Kathelijne Wintraecken WUR X X X X



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name affiliation lunch 1 dinner room lunch 2 Pieter Rein ten Wolde AMOLF X X X X Chien-Ching Wu UT X X X X Gijs Wuite VU X X X X Claire Wyman EMC X X X X Ted Xia LEI X X X X Ke Yang TU/e X X X Kah Wai Yau EMC X X X X Ka Lou Carol Yu EMC X X X X Thomas van Zanten IBEC X X X X Rosanne Zeiler UvA X X X X Marcel Zevenbergen TUD X X X X Ying Zhang WUR X X X X Lizhe Zhu UvA X X X X Xiaowei Zhuang Harvard X X X X Niels Zijlstra UT X X X X Loes van Zijp TU/e X X Jelena Zivkovic RU X X X X Franziska Zosel LEI X X X X Wouter van der Zwan EUR X X X X

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