The aim of these studies is the quantitative investigation of protein-protein interactions in the AP1 system in

vivo. First results of FCS measurements show an exchange in the nucleus of the proteins Fos-CFP and Jun-YFP

in the stably mono-transfected HeLa-Cells. This is also shown by fitting the bleaching curves measured in the

nucleus with an appropriate model. We obtained dissociation times between 10 and 20 seconds in the nucleus. In

the autocorrelation function a free and an obstructed component of diffusion are shown. For further studies

doubly transfected cells with both proteins, Fos-CFP and Jun-YFP, were prepared. These cells will now be

characterized with FCCS to investigate the protein-protein interactions. In order to obtain the dissociation rates

of the complex in the cell nucleus bleaching curves will be recorded on these cell lines. We also overexpressed

and purified Jun-YFP and Fos-CFP for in vitro studies

Baudendistel, N. (Nina)

Knoch, T.A. (Tobias)

Langowski, J. (Jörg)

Müller, G. (Gabriele)

Wachsmuth, M. (Malte)

Waldeck, W. (Waldemar)

Weidemann, T. (Thomas)

Erasmus University Digital Repository

In Vivo Characterization of Protein-Protein Interactions in the AP1 System with Fluorescence Correlation Spectroscopy (FCS)AP1 System FCSandFCCS- group of transcription activator proteins- subunits: c-Fos, FosB, c-Jun, JunB, JunD, FraI, FraII-major components are c-Fos and c-JunNina Baudendistel Tobias A. Knoch, Gabriele Müller, Malte Wachsmuth, Thomas Weidemann, Waldemar Waldeck, Jörg Langowski Deutsches Krebsforschungszentrum, Abteilung Biophysik der Makromoleküle, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germanye-mail: nb@dkfz.defluorescentparticleobjectivtrajectorylaser focusA laser is coupled into a microscope, illuminating a sub-femtoliter volume (Fig. 8a).Fluorescent molecules cross  this focus by Brownian motion.Their fluorescence light is collected by the same optics and detected by confocally arranged detectors.Single molecules can be detected due to the high sensivity of the detectors (avalanche photodiods).Experiments1. Construction of fusion proteinsStuI  HIND III C                 reg.                     region         CFP      YFP SV40polyAf1 oriPf1oriSV40  oriPSV40   ori Kan  &Neorr       HSV    TKpoly Ap UCoripSV AP1-XFP-HisHindIIIHindIIIAgeIBsrGINotIM        C             SHis6c-Jun-YFP c-Fos-CFPTwo color FCCSdiffusion timecon-centrationτ [ms]G(τ)1G(τ) =F(0) F(τ)F2 –Signal fluctuations induced by molecules diffusing across the focus are recorded over time and the autocorrelation function (ACF) of the signal is calculated (Fig. 8b).Fitting an appropriate model to the autocorrelation function results in:concentrations, diffusion coefficients and other physical parameters of the different fluorescent species in the system (Fig. 8c).In Fluorescence Cross-Correlation Spec-troscopy (FCCS) two fluorophores with distinct spectra are detected simultaneously in two channels and their signals are cross-correlated (Fig. 9a+b).Only particles which carry both fluorophores contribute to the cross correlation signal.FCCS will enable us to measure for the first time protein-DNA and protein-protein interactions  in living cells.203040500 1 2 4 5 6τ[kHz]F(t)t [s]2 400.031 10 100.01G 12(τ)010.01 1 10 2203040500 1 2 4 5 6F(0) F(τ)Fτt [s][kHz]F(t)τ[ms]Fig. 9b fluctuation of the intensity in both channelsFig. 9c Cross-Correlation ConclusionsFig.5 the leucine zipper is formed as a coiled coil- dimerization of AP1 is required for DNA binding before transcription- AP1 binds to the TPA responsive element in the promoter region- AP1 is controlled by phosphorylation and ubiquitinationTransactivationDNA-BindingDimerizationXFPc-JunJunBc-FosJunDFraIFraIIFosBTRE TATA Boxc-Junc-FosAP1TPA responsiveelementATG+1transcription startDNARNA-polymerase IIFig.6 3D image of the AP1-DNA complexRNAAll proteins have a basic region leucine zipper (bZIP) for dimerizationProtein domainsDimerization and DNA-bindingThe aim of these studies is the quantitative investigation of protein-protein interactions in the AP1 system in vivo.First results of FCS measurements show an exchange in the nucleus of the proteins Fos-CFP and Jun-YFP in the stably monotransfected HeLa-Cells. This is also shown by fitting the bleaching curves measured in the nucleus with an appropiate model. We obtained dissociation times between 10 and 20 seconds in the nucleus.In the autocorrelation function a free and an obstructed component of diffusion are shown.For further studies doubly transfected cells with both proteins, Fos-CFP and Jun-YFP, were prepared. These cells will now be characterized with FCCS to investigate the protein-protein interactions. In order to obtain the dissociation rates of the complex in the cell nucleus bleaching curves will be recorded on these cell lines.We also overexpressed and purified Jun-YFP and Fos-CFP for in vitro studies.0 2-2 log(t/1ms)0.10.20G(t)Fig. 4 FCS in the nucleus of Jun-YFP cells: a fast (μs) and a slow component (ms) of diffusion are obtained in the ACF corresponding to free and obstructed diffusion. A fast exchange in the nucleus is observed.  Concentrations of the free component are 50-100 nM. Transactivation DNA-Binding DimerizationOne color FCS12Fig. 7Fig. 8a Fig. 9aFig. 1 plasmid map of pSV-AP1-XFP-HisFig. 3 Photobleaching in the nucleus of Fos-CFP cells: in the nucleus an equilibrium between the freely diffusing and the bound component of AP1 exists. By means of bleaching curves and an appropriate fit model we can calculate the dissociation rates k   in the nucleus, if the fraction of the immobilized component is known. We obtain retention periods between 10 and 20 seconds corresponding to the exchange between the free and the bound component. diss51015202530350 5 10 15 20 25 30Z04_02b00s.ascCount Ratet [s]CountRateRegion around an AP1-regulated promoterFig. 8b Fig. 8cslowcomponentfastcomponenta b cd e fFig. 2 Vektor pSV-AP1-XFP in HeLa cells stably transfected. The main fluorescence is in the nucleus (a+d). FCS measurement in the nucleus (b+e). The cells are bleached uniformly (c+f).2. Expression of fusion protein and FCS measurements3. Resultsconcen-tration50-100 nMc-Fosc-JunFraIIn Vivo Characterization of Protein-Protein Interactions in the AP1 System with Fluorescence Correlation Spectroscopy (FCS) Baudendistel, N., Knoch, T. A., Müller, G., Wachsmuth, M., Weidemann, T.,           Waldeck, W. & Langowski, J. 5th Graduate Students Meeting of the German Cancer Research Center (DKFZ), Germany, May 2002.  Abstract   The aim of these studies is the quantitative investigation of protein-protein interactions in the AP1 system in vivo. First results of FCS measurements show an exchange in the nucleus of the proteins Fos-CFP and Jun-YFP in the stably mono-transfected HeLa-Cells. This is also shown by fitting the bleaching curves measured in the nucleus with an appropriate model. We obtained dissociation times between 10 and 20 seconds in the nucleus. In the autocorrelation function a free and an obstructed component of diffusion are shown. For further studies doubly transfected cells with both proteins, Fos-CFP and Jun-YFP, were prepared. These cells will now be characterized with FCCS to investigate the protein-protein interactions. In order to obtain the dissociation rates of the complex in the cell nucleus bleaching curves will be recorded on these cell lines. We also overexpressed and purified Jun-YFP and Fos-CFP for in vitro studies.  Keywords: Genome, genomics, genome organization, genome architecture, structural sequencing, architectural sequencing, systems genomics, coevolution, holistic genetics, genome mechanics, genome function, genetics, gene regulation, replication, transcription, repair, homologous recombination, simultaneous co-transfection, cell division, mitosis, metaphase, interphase, cell nucleus, nuclear structure, nuclear organization, chromatin density distribution, nuclear morphology, chromosome territories, subchromosomal domains, chromatin loop aggregates, chromatin rosettes, chromatin loops, chromatin fibre, chromatin density, persistence length, spatial distance measurement, histones, H1.0, H2A, H2B, H3, H4, mH2A1.2, DNA sequence, complete sequenced genomes, molecular transport, obstructed diffusion, anomalous diffusion, percolation, long-range correlations, fractal analysis, scaling analysis, exact yard-stick dimension, box-counting dimension, lacunarity dimension, local nuclear dimension, nuclear diffuseness, parallel super computing, grid computing, volunteer computing, Brownian Dynamics, Monte Carlo, fluorescence in situ hybridization, confocal laser scanning microscopy, fluorescence correlation spectroscopy, super resolution microscopy, spatial precision distance microscopy, auto-fluorescent proteins, CFP, GFP, YFP, DsRed, fusion protein, in vivo labelling.   Literature References  Knoch, T. A. Dreidimensionale Organisation von Chromosomen-Domänen in Simulation und Experiment. (Three-dimensional organization of chromosome domains in simulation and experiment.) Diploma Thesis, Faculty for Physics and Astronomy, Ruperto-Carola University, Heidelberg, Germany, 1998, and TAK Press, Tobias A. Knoch, Mannheim, Germany, ISBN 3-00-010685-5 and ISBN 978-3-00-010685-9 (soft cover, 2rd ed.), ISBN 3-00-035857-9 and ISBN 978-3-00-0358857-0 (hard cover, 2rd ed.), ISBN 3-00-035858-7, and ISBN 978-3-00-035858-6 (DVD, 2rd ed.), 1998. Knoch, T. A., Münkel, C. & Langowski, J. Three-dimensional organization of chromosome territories and the human cell nucleus - about the structure of a self replicating nano fabrication site. Foresight Institute - Article Archive, Foresight Institute, Palo Alto, CA, USA, http://www. foresight.org, 1- 6, 1998. Knoch, T. A., Münkel, C. & Langowski, J. Three-Dimensional Organization of Chromosome Territories and the Human Interphase Nucleus. High Performance Scientific Supercomputing, editor Wilfried Juling, Scientific Supercomputing Center (SSC) Karlsruhe, University of Karlsruhe (TH), 27- 29, 1999. Knoch, T. A., Münkel, C. & Langowski, J. Three-dimensional organization of chromosome territories in the human interphase nucleus. High Performance Computing in Science and Engineering 1999, editors Krause, E. & Jäger, W., High-Performance Computing Center (HLRS) Stuttgart, University of Stuttgart, Springer Berlin-Heidelberg-New York, ISBN 3-540-66504-8, 229-238, 2000. Bestvater, F., Knoch, T. A., Langowski, J. & Spiess, E. GFP-Walking: Artificial construct conversions caused by simultaneous cotransfection. BioTechniques 32(4), 844-854, 2002. Knoch, T. A. (editor), Backes, M., Baumgärtner, V., Eysel, G., Fehrenbach, H., Göker, M., Hampl, J., Hampl, U., Hartmann, D., Hitzelberger, H., Nambena, J., Rehberg, U., Schmidt, S., Weber, A., & Weidemann, T. Humanökologische Perspectiven Wechsel - Festschrift zu Ehren des 70. Geburtstags von Prof. Dr. Kurt Egger. Human Ecology Working Group, Ruperto-Carola University of Heidelberg, Heidelberg, Germany, 2002. Knoch, T. A. Approaching the three-dimensional organization of the human genome: structural-, scaling- and dynamic properties in the simulation of interphase chromosomes and cell nuclei, long- range correlations in complete genomes, in vivo quantification of the chromatin distribution, construct conversions in simultaneous co-transfections. Dissertation, Ruperto-Carola University, Heidelberg, Germany, and TAK†Press, Tobias A. Knoch, Mannheim, Germany, ISBN 3-00-009959-X and ISBN 978-3-00-009959-5 (soft cover, 3rd ed.), ISBN 3-00-009960-3 and ISBN 978-3-00-009960-1 (hard cover, 3rd ed.), ISBN 3-00-035856-9 and ISBN 978-3-00-010685-9 (DVD, 3rd ed.) 2002. Knoch, T. A. Towards a holistic understanding of the human genome by determination and integration of its sequential and three-dimensional organization. High Performance Computing in Science and Engineering 2003, editors Krause, E., Jäger, W. & Resch, M., High-Performance Computing Center (HLRS) Stuttgart, University of Stuttgart, Springer Berlin-Heidelberg-New York, ISBN 3- 540-40850-9, 421-440, 2003. Wachsmuth, M., Weidemann, T., Müller, G., Urs W. Hoffmann-Rohrer, Knoch, T. A., Waldeck, W. & Langowski, J. Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys. J. 84(5), 3353-3363, 2003. Weidemann, T., Wachsmuth, M., Knoch, T. A., Müller, G., Waldeck, W. & Langowski, J. Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. J. Mol. Biol. 334(2), 229-240, 2003. 

In vivo characterization of protein-protein interactions in the AP1 system with fluorescence correlation spectroscopy (FCS).

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