Section of Molecular Cytology
van Leeuwenhoek Centre for Advanced Microscopy
Masters in Molecular Sciences, Wageningen University , The Netherlands
Doctorate with thesis "Fluorescence correlation spectroscopy applied to living plant cells", Wageningen University, The Netherlands
Manager van Leeuwenhoek for Advanced Microscopy (LCAM-FNWI), University of Amsterdam, The Netherlands
Assistant professor, Molecular Cytology, University of Amsterdam, The Netherlands
Postdoctoral fellow, Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany
Postdoctoral fellow, Cell Biology and Biophysics, European Molecular Biology Laboratory, Heidelberg, Germany
Postdoctoral fellow, MicroSpectroscopy Centre, Wageningen University, The Netherlands
Fluorescence fluctuation spectroscopy
Signal transduction pathways
One of the most intriguing challenges in life sciences is to understand how a complex mixture of molecular particles and structures can make up a living cell. Despite the immense number of studies still much is unknown about the molecular basis of numerous biological processes such as cell proliferation, differentiation, intra- and extra-cellular communication and apoptosis. To increase our understanding about the complexity of these processes in living cells, experimental data on the spatial-temporal organization is required. Fluorescence based techniques are ideal tools for this type of studies.
Fluorescence Fluctuation Spectroscopy (FFS) is a family of fluorescence techniques that is capable of detecting concentration, dynamics and interactions of fluorescent particles down to the single-molecule level and, if desired, in the living cell. We are applying, optimizing and expanding these techniques to describe signal transduction pathways quantitatively, like we have done for a part of the yeast pheromone signaling cascade. Thereto, the proteins of interest are genetically labeled with the various color- and lifetime variants of the green fluorescent protein (partly developed in our laboratory), integrated into the genome and studied by advanced fluorescence microscopes.
Below you will find a brief description of the 'keywords' in our research:
Fluorescence fluctuation spectroscopy (FFS)
Signal transduction pathways
Fluorescence fluctuation techniques like fluorescence correlation spectroscopy ( FCS ) and photon counting histogram ( PCH ) monitor concentrations and mobility-, binding- and conformational state dynamics of fluorescent molecules and their complexes in situ. Since FCS and PCH are single-molecule techniques, molecules f.e. fluorescently labelled proteins can be studied at the nanomolar level. For many proteins (especially those involved in signal transduction) this is the physiological relevant concentration in a living cell, thus no over-expression of the protein is required. For FCS and PCH the fluorescence intensity is monitored in the small observation volume of a confocal microscope (green), which is continuously illuminated (blue). A particle (red) with a given molecular brightness produces an intensity fluctuation as it passes the observation volume. Particles with a higher molecular brightness will result in stronger intensity fluctuations. Since small particles will diffuse more rapidly through the observation volume than large molecules, the duration of the fluorescence bursts contains information on the diffusion speed of the particles.
Both PCH and FCS analysis use the same experimental data, but each technique focuses on a different property of the signal. While FCS is a measure of the time-dependent decay of the fluorescence fluctuations yielding parameters like particle number, diffusion time and dark-state kinetics, PCH calculates the amplitude distribution of these fluctuations yielding the distribution of molecular brightness per particle (Chen et al., 1999). When no fluorescence quenching occurs this distribution provides a direct readout of the oligomerization state of the particle. The same principle can be applied to a stack of fluorescent images using the family of Image Correlation Spectroscopy (ICS) techniques.
Schwille & Haustein. Fluorescence Correlation Spectroscopy: An introduction to its concepts and applications, www.biophysics.org/education/schwille.pdf
Chen Y, Müller JD, So PT, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J. 77 , 553 (1999).
Still, the resolving power of FCS to distinguish particles of different molecular size is limited. Therefore, dual-color fluorescence cross-correlation spectroscopy ( FCCS ) has been developed by Schwille et al. (1997). Here two spectrally different fluorescent groups, e.g. green and red emitting dyes, are used to label each of the interacting partners. Each dye is excited and detected by separate light sources and detectors. Molecular interactions can be studied by following the coincidence of the fluorescence fluctuations in the two detectors. The amplitude and decay rate of the cross-correlation curve correspond to the number and dynamics of those complexes that carry both fluorescent dyes. Recentlythe repertoire of fluctuation microscopies has been extended by introduction of fluorescence lifetime correlation spectroscopy ( FLCS ) and pulsed-interleaved excitation FCCS ( PIE-FCCS ). These new additions enable to distinguish dyes with similar emission spectra but different fluorescent lifetimes, and eliminate spectral cross-talk artifacts, which is especially useful for multiparameter characterization of biomolecular processes.
Bacia K, Kim SA, Schwille P. Fluorescence cross-correlation spectroscopy in living cells. Nat. Methods 3 , 83 (2006).
Lamb DC, Müller BK, Bräuchle C. Enhancing the Sensitivity of Fluorescence Correlation Spectroscopy by Using Time-Correlated Single Photon Counting. Curr. Pharm. Biotech. 6 , 405 (2005).
Cells can communicate with their environmentvia receptors at the plasma membrane and recognize signals from the extracellular environment. The "G-protein coupled receptors" (GPCRs) comprise the largest family of receptors. Heterotrimeric G-proteins are intracellular partners of the GPCRs. Upon activation the Gα·GDP/Gβγ heterotrimers promote GDP release and GTP binding. Both the Gα-GTP and Gβγ dimer are capable of activating downstream effectors. These effectors include adenylate cyclase, phospholipase C-beta, PI 3-kinase and RhoGEF. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation - a cycle accelerated by 'regulators of G-protein signaling' (RGS proteins). To regulate this signaling a complex network has been evolved (see left, snapshot from Qiagen) where molecular interactions are crucial to pass the signal.
Another signaling pathway of interest is the response of the yeast Saccharomyces cerevisiae to mating pheromone, that is mediated by a MAPK signalingcascade. The binding of the α-factor at the GPCR Ste 2 activates a trimeric G-protein. The released Gβγ-dimer interacts with the PAK kinase Ste20 and the scaffold protein Ste5. In conjunction with bound Ste4, a conformational change of Ste5 is generated that results in Ste5 oligomerization and the activation of Ste11 (also known as MAPKKK) via Cdc42-activated Ste20. Requirements for Ste7 (MAPKK) and Fus3 (MAPK) phosphorylation include: the membrane recruitment of Ste5 through cryptic lipid-binding domains; the interaction of Ste5 with Gβγ; and the self-interaction of Ste5. Fus3 phosphorylation of its Thr 180 and Tyr 182 residues in the activation loop generates the active form of Fus3, Fus3PP. The nuclear pool of Fus3PP regulates a series of transcriptional regulators that modulate mating-specific gene expression.
Dohlman HG and Thorner, JW. Regulation of G-protein-initiated signal transduction in yeast: Paradigms and principles. Annu. Rev Biochem. 70, 703 (2001).
Besides the techniques described above, we are also using other advanced fluorescence techniques like fluorescence lifetime imaging microscopy ( FLIM ), fluorescence recovery after photobleaching ( FRAP ), total internal reflection microscopy ( TIRF ), spectral imaging( SPIM ) using controled light exposure microscopy ( CLEM ) and confocal imaging ( CLSM ) to monitor and characterize our proteins of interest. These experiments are performed in the van Leeuwenhoek Centre for Advanced Microscopy (LCAM). LCAM has a complete range of microscopes, image analysis and data storage equipment to facilitate scientific research. The staff of LCAM has the expertise, skills and experience to give professional support and coaching to research groups within and outside SILS. Since LCAM is part of our department of Molecular Cytology, facilities for molecular biological work, biochemical analysis and cell culturing are available as well.
There are always possibilities for students to
do an internship in our group.
Please enquire for specific projects currently available (see link below).
Depending on the length of the practical training period (minimum 4 months), and the
interest of the student, one has the possibility to work on several different disciplines,
including molecular biological andbiochemical work, cell culturing, microscopy
(development), fluorescent protein optimalization and data analysis (development).
Bio-organische chemie, Biochemie &
Moleculaire Microscopie van de Levende Cel &Ziekteprocessen (BW04K)
Molecular Structure in Biology (004LS)
Medical Biochemistry and Molecular Biology (BMS005)
Advanced micrscopy (XXXX)
Monthly confocal user course LCAM
contact Erik Manders (+ 6225) or Ronald Breedijk (+ 6211)
Confocal light microscopy (May 2010)
In the footsteps of Antoni van Leeuwenhoek (Nov 2009)
FEBS advanced course
Microspectroscopy: Monitoring Molecular Interactions
inLiving Cells, Wageningen, Netherlands (Sept 08)
B-Basic course Visualization of Cellular Processes , Groningen, Netherlands(Oct 08)
EMBO Practical Course on Fluorescene (Cross-) Correlation Spectroscopy
(FCS/FCCS) for Cell Biology Applications , Heidelberg, Germany (May 09)
EMBO course on light microscopy in living cells , Lisbon, Portugal (Jun 09)
Bravissimo network workshop: Microspectroscopy: Monitoring Cellular Biochemistry
in vivo , Wageningen, The Netherlands (Jun 09)
2nd European short course Time-Resolved Microscopy and Correlation Spectroscopy ,
Berlin, Germany (Feb 2010)
IEEE International Symposium on Biomedical Imaging , Rotterdam,
The Netherlands (Apr 2010)
FEBS advanced course Microspectroscopy: Probing Protein Dynamics and
Interactions in Living Cells , Wageningen, Netherlands (Sept 2010)
33. Klarenbeek J.B., Goedhart, J., Hink, M.A., Gadella,
T.W.J. and Jalink, K. A mTurquoise-based cAMP sensor for both
FLIM and ratiometric read-out has improved dynamic range. PLoS
ONE 6(4): e19170 (2011).
32. Goedhart, J., L. van Weeren, M.A. Hink , N.O.E Vischer , K. Jalink and Th.W.J. Gadella Jr. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nature Methods 7, 137 (2010).
31. Bellou, S., M.A. Hink, E. Bagli, E. Panopoulou, P.I.H. Bastiaens, C. Murphy and T. Fotsis. VEGF auto-regulates its proliferative and migratory ERK1/2 and p38 MAPK cascades by enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells. Am. J. Physiol. Cell. Physiol. 297, 1477 (2009).
30. Willems, P.H.G.M., H.G. Swarts, M.A. Hink and W.J.H. Koopman. The use of fluorescence correlation spectroscopy to probe mitochondrial mobility and intramatrix protein diffusion. Methods Enzymol. 456, 287 (2009).
29. Koopman, W.J.H. , F. Distelmaier, M.A. Hink, S. Verkaart, M. Wijers, J. Fransen, J.A.M. Smeitink and P.H.G.M. Willems. Inherited complex I deficiency is associatedwith faster protein diffusion in the matrix of moving mitochondria. Am. J. Physiol. Cell. Physiol. 294, 1124 (2008).
28. Hink, M.A., K. Shah, E. Russinova, S.C. de Vries and A.J.W.G. Visser. Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization. Biophys. J. 94, 1052 (2008).
27. Maeder, C.I.*, M.A. Hink*, A. Kinkhabwala, R. Mayr,
P.I.H. Bastiaens & M. Knop. Spatial regulation of Fus3 MAP
kinase activity through a reaction-diffusion mechanism in yeast
pheromone signalling Nat. Cell Biol. 9, 1319 (2007).
(* authors contributed equally)
26. Koopman, W.J.H., M.A. Hink, S. Verkaart, F. de Lange, J.A.M. Smeitink and P.H.G.H.M. Willems. Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. BBA Bioenergetics 1767, 940 (2007).
25. Sutter M., S. Oliveira, N.N. Sanders, B. Lucas, A. van Hoek, M.A.Hink, A.J.W.G. Visser, S.C. De Smedt, W.E. Hennink, W. Jiskoot. Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye nile red. J. Fluoresc. 17, 181 (2007).
24. Zorrilla, S., M.A. Hink, A.J.W.G. Visser and M. Pilar Lillo. Translational and rotational motions of proteins in a protein crowded environment. Biophys. Chem. 125, 298 (2007).
23. Slootweg, E.J., H.J. Keller, M.A. Hink, J.W. Borst, J. Bakker and A. Schots. Fluorescent T7 display phages obtained by translational frameshift. Nuc. Acids Res. 34, e137 (2006).
22. Lavalette, D., M.A. Hink, M. Tourbez, C. Tetreau and A.J.W.G. Visser. Proteins as micro viscosimeters: Brownian motion revisited. Eur. Biophys. J. 35, 517 (2006).
21. Veldhuis, G., M. Hink, V. Krasnikov, G. van den Bogaart, J. Hoeboer, A.J.W.G.Visser, J. Broos and B. Poolman. The oligomeric state and stability of the mannitol transporter from Escherichia coli, EnzymeIImtl: a fluorescence correlation spectroscopy study. Protein Sc. 15, 1977 (2006).
20. Westphal, A.H., A. Matorin, M.A. Hink, J.W. Borst, W.J.H. van Berkel and A.J.W.G. Visser. Real-time single flavoenzyme dynamics studied with fluorescence correlation spectroscopy of p hydroxybenzoate hydroxylase. J. Biol. Chem. 281, 11074 (2006).
19. Visser, N.V., J.W. Borst, M.A. Hink, A. van Hoek and A.J.W.G. Visser. Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophys. Chem. 116, 207 (2005).
18. Skakun, V.V.*, M.A. Hink*, A.V. Digris*, R. Engel,
E.G. Novikov, V.V. Apanasovich and A.J.W.G. Visser. Global
analysis of fluorescence fluctuation data. Eur. Biophys. J. 34,
(* authors contributed equally)
17. Borst, J.W., M.A. Hink, A. van Hoek and A.J.W.G. Visser. Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins. J. Fluoresc. 15, 153 (2005).
16. Ruchira, M.A. Hink, L. Bosgraaf, P.J.M. van Haastert and A.J.W.G.Visser. Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy. J. Biol. Chem. 279, 10013 (2004).
15. Becker,W., A. Bergmann, M.A. Hink, K. König, K. Benndorf and C. Biskup. Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58 (2004).
14. Hink, M.A., N.V. Visser,J.W. Borst, A. van Hoek and A.J.W.G. Visser. Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster resonance energy transfer (FRET) studies. J. Fluoresc. 13, 185 (2003).
13. Hink,M.A., J.W. Borst and A.J.W.G. Visser. Fluorescence correlation spectroscopy of GFP fusion proteins in living plant cells. Methods Enzymol. 361, 93 (2003).
12. Hink, M.A., T. Bisseling and A.J.W.G. Visser. Imaging protein-protein interactions in living cells. Plant Mol. Biol. 50, 871 (2002).
11. Visser, N.V., M.A. Hink, J.W. Borst, G.N.M. van der Krogt and A.J.W.G. Visser. Circular dichroism spectroscopy of fluorescent proteins. FEBS Lett. 521, 31 (2002).
10. Van den Berg, P.A.W., J. Widengren, M.A. Hink, R. Rigler and A.J.W.G. Visser. Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects. Spectrochim. Acta A 57, 2135 (2001).
9. Uskova, M.A., J.W. Borst, M.A. Hink, A. van Hoek, A. Schots, N.L. Klyachko and A.J.W.G. Visser. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles. Biophys. Chem. 87, 73 (2001).
8. Hink, M.A., R.A. Griep, J.W. Borst, A. van Hoek, M.H.M. Eppink, A. Schots and A.J.W.G. Visser. Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. J. Biol. Chem. 275, 17556 (2000).
7. Goedhart, J., M.A. Hink, A.J.W.G. Visser, T. Bisseling and T.W.J. Gadella. In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls. Plant J. 21, 109 (2000).
6. Visser, A.J.W.G. and M.A. Hink. New perspectives of fluorescence correlation spectroscopy. J. Fluoresc. 9, 81 (1999).
5. Visser, N.V., M.A. Hink, A. van Hoek and A.J.W.G. Visser. Comparison between fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy as illustrated with a fluorescent dextran. J. Fluoresc. 9, 251 (1999).
4. Goedhart, J., H. Rohrig, M.A. Hink, A. van Hoek, A.J.W.G. Visser, T. Bisseling and T.W.J. Gadella,Jr. Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur. Biochemistry 38, 10898 (1999).
3. Koopman,W.J.H., M.A. Hink, A.J.W.G. Visser, E.W. Roubos and B.G. Jenks. Evidence that Ca2+-waves in Xenopus melanotropes depend on calcium-induced calcium release: a fluorescence correlation microscopy and linescanning study. Cell Calcium 26, 59 (1999).
2. Hink, M.A., A. van Hoek and A.J.W.G. Visser. Dynamics of phospholipid molecules in micelles: Characterization with fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy. Langmuir 15, 992 (1999).
1. Brock, R., M.A. Hink and T.M. Jovin. Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys. J. 75, 2547 (1998).
8. Hink, M.A., S.C. de Vries and A.J.W.G. Visser. Fluorescence fluctuation analysis of receptor kinase dimerization. In Methods in Molecular Biology, Humana Press, (2010) in press
7. Kwaaitaal, M.A.C.J., M. Schor, M.A. Hink, A.J.W.G. Visser and S.C. de Vries. Applying fluorescence correlation spectroscopy and fluorescence recovery after photo bleaching to study receptor kinase mobility in planta. In Methods in Molecular Biology, Humana Press, (2010) in press
6. Borst, J.W., I. Nougalli-Tonaco, M.A.Hink,A. van Hoek, R.G.H. Immink and A.J.W.G. Visser. Protein-proteininteractions invivo: Use ofbiosensors based on FRET. In Reviews in Fluorescence. C.D. Geddes and J.R. Lakowicz (eds.) Kluwer Academic/Plenum Publishers,New York , 341-355 (2006).
5. Rodrigo F. M. de Almeida, M.A. Hink,J.W. Borst, A.J.W.G.Visser and M.Prieto. Lipid domains and rafts studied by time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy. In Biochemistry & Biophysics of Lipids. A. Pramanik (ed.) Kerala, pp. 31-62 (2006).
4. Borst, J.W., M.A. Hink, A. van Hoek and A.J.W.G. Visser. Multiphoton microspectroscopy in living plant cells. In Multiphoton Microscopy in the Biomedical Sciences III,. A.Periasamy and P.T.So (eds.) Proc. Spie 4963, pp. 231-238 (2003).
3. Hink, M.A. Fluorescence correlation spectroscopy applied to living plant cells. Thesis. Wageningen University . pp. 1-138 (2002).
2. Visser, A.J.W.G., P.A.W. vanden Berg, M.A. Hink, V.N.Petushkov. Fluorescence correlation spectroscopy of flavins and flavoproteins. In Fluorescence Correlation Spectroscopy. Theory and Applications. E. Elson and R. Rigler (eds.) Springer-Verlag , Berlin , pp. 9-24 (2001).
1. Hink, M.A. and A.J.W.G. Visser. Characterization of membrane mimetic systems with fluorescence correlation spectroscopy. In Applied Fluorescence in Chemistry, Biology and Medicine.W. Rettig, B. Strehmel, S. Schrader and H. Seifert (eds.) Springer-Verlag Berlin, pp. 101-118 (1998).