Research
Research interest
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)
Multi-color FFS
High-resolution microscopy (PALM/STORM)
Fluorescent proteins
Signal transduction pathways
Fluorescence fluctuation spectroscopy (FFS)
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 observationvolumethan 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.
recommended literature:
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).
Multi-color FFS
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.
recommended literature:
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).
High-resolution microscopy (PALM/STORM)
It is well known that there is a spatial limit to which light can focus: approximately half of the wavelength of the light you are using. But this is not a true barrier, because this diffraction limit is only true in the far-field and localization precision can be increased with many photons and careful analysis. The image of a point source on a microscope detector is called the point-spread function (PSF), which is limited by diffraction to be approximately half the wavelength of the light. But it is possible to simply fit that PSF with a Gaussian to locate the center of the PSF, and thus the location of the fluorophore with a much higher accuracy (compare the 'standard' LSM image <left> with the PALM image <right>).
Betzig et al. (see image above from Science) developed photo-activated localization microscopy (PALM) while Zhuang and co-workers used a similar technique called stochastic optical reconstruction microscopy (STORM). In both techniques samples filled with many dark fluorophores are imaged. The dyes can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, well separated molecules "turn on". Then Gaussians are fit to their PSFs in order to localize the centre of the particle. After the few bright molecules photobleach (sometimes actively by using another differently colored excitation source), the next flash of the photoactivating light activates random fluorophores again and the PSFs are fit of these different molecules. This process is repeated many times, building up an image. Because the molecules were switched on-and-off (and thus localized) at different times, the 'resolution' of the final image can be much higher than that limited by diffraction. The current limitation of these techniques is that it can take on the order of hours to collect enough photons per molecule.
recommended literature:
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642 (2006).
Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793 (2006).
Fluorescent proteins
The gene encoding green fluorescent protein (GFP) was isolated from the jellyfish Aequorea victoria, initiating a revolution in cell biology. It has been demonstrated that the GFP does not requireaco-factorto become fluorescent and that it can be expressedin any type of organism. Nowadays, GFP is probably the mostpopular fluorescent probe to study proteins in living cells and has almost completely replaced the fluorescent labeling of proteins by organic fluorophores. The advantage of being a genetically encoded probe usually outweighs the disadvantage of its molecular size.
In past years many labs have been involved in expanding the color palette of the available FPs, now ranging from blue to red. In addition many variants have been generated with improved photophysical properties, having a higher brightness and a lower sensitivity to pH and photobleaching. Our laboratory has created variants of cyan and yellow FPs that have different fluorescence lifetimes, making it possible to discriminate multiple dyes, having a similar emission spectrum, by using only one detector. Recently the group of photoswitchable FPs has been expanded. These FPs can (irreversibly) change color upon illumination or be turned on-and-off reversibly by applying two different excitation wavelengths. These fluorophores are ideally suited to track molecules in the cell and localize proteins with a high resolution (see PALM/STORM)
recommended literature:
Shaner NC, Patterson GH, Davidson MW. Advances in fluorescent protein technology. J Cell Sci. 15, 4247 (2007).
Kremers GJ, Goedhart J, Van Munster EB, Gadella Jr. TWJ. Cyan and yellow superfluorescent proteins with improved brightness, protein folding and FRET Förster radius. Biochemistry 45, 6570 (2006).
Signal transduction pathways: Molecular interactions
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.
recommended literature:
Dohlman HG and Thorner, JW. Regulation of G-protein-initiated signal transduction in yeast: Paradigms and principles. Annu. Rev Biochem. 70, 703 (2001).
Fluorescence spectroscopy
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.
Webpage van Leeuwenhoek Centre for Advanced Microscopy (CAM)