Fluorescence fluctuation spectroscopy applied to living plant cells

Keywords: Fluorescence correlation spectroscopy, photon counting histogram, intracellular, plant, AtSERK1 In order to survive organisms have to be capable to adjust theirselves to changes in the environment. Cells, the building blocks of an organism react to these changes by sending signal molecules (for example hormones). Important biological processes like cell-division, -growth, -differentiation and - death are started after receipt of specific signal molecule that will trigger and activate other signal molecules. During evolution a complex network of signal cascades has been developed. Errors in this network may lead to disorders, diseases or even death. To study the molecules involved in this network the method, that has been used in this thesis, is both very selective as sensitive: fluorescence. After excitation using light of a specific color, a fluorescent molecule will emit light of a different color. Since most molecules are hardly fluorescent their selves, fluorescent groups can be coupled to the molecule of interest in order to distinguish it from the other molecules. The research described in this thesis is devoted to the application of two novel fluorescence techniques fluorescence correlation spectroscopy (FCS) and photon counting histogram analysis (PCH) to intracellular plant research. Both techniques retrieve information from the fluctuations in the fluorescence intensity that can be observed in a small volume element. The high sensitivity allows measurements under equilibrium conditions at the single-molecule detection level. Moreover, FCS-analysis can retrieve a large number of parameters, describing the time-dependent decay of the fluctuations, such as the local particle concentration, mobility of the fluorescent particles and rate constants of fast reversible reactions like triplet kinetics and protonation of the chromophore. Hence, it has been recognized that FCS has a high potential to monitor the behaviour of fluorescently labeled biomolecules in living cells at physiological relevant concentrations. Therefore, FCS could be a valuable tool in cell biology, especially in the study of molecular interactions. The behaviour of fluorescently labeled molecules was first studied in model biochemical systems, before applying FCS to the complex intracellular environment. The binding of a enhanced GFP labeled single chain antibody fragment (scFv-GFP) to its antigen, lipopolysacharide, present in the outer cell membrane of Ralstonia solanacearum bacteria clearly demonstrates the ability of FCS to distinguish particles on basis of the difference in diffusion coefficient. The diffusion coefficients of micelles loaded with a fluorescent phospholipid have been determined by FCS as well as by dynamic light scattering (DLS). The measurements of the micelle hydrodynamic volume using both techniques have been demonstrated to be equivalent. However, the concentration sensitivity of FCS is orders of magnitude higher than that of DLS and is, moreover, able to determine the extremely low critical micelle concentration by measuring the diffusion time as function of detergent concentration. Intracellular experiments may be complicated by the presence of autofluorescent molecules. Plant cells may contain molecules like chlorophyll, localized in the chloroplast, and pigment molecules in the vacuole that will not only fluoresce rather strongly but have broad absorption bands as well, able to absorb the fluorescence light emitted by other fluorophores. Hence, either experiments have been carried out in intracellular regions of cowpea protoplasts lacking these compartments or cell types were selected that originate from root tissue and therefore lack many pigment molecules that are responsible for intense autofluorescence. In order to achieve high signal to noise ratios (SNR) without disturbance of autofluorescence, dye depletion or cellular damage, the optimum emission wavelength of the fluorophores should lie between 560 and 610 nm and the excitation intensities in the visible range (458-514 nm) should not exceed 10 kW.cm -2. The diffusion rate of synthetic fluorophores in the cytoplasm and nucleus of the plant cells was retarded by a factor 2 to 4 as compared to buffer due to the viscous intracellular environment. Moreover, FCS was able to distinguish between different types of motion: The diffusion of dyes such as rhodamine green and Cy5 did show a clear deviation from normal Brownian motion in the cytoplasm and the curves could only be fitted according to a multi-component diffusion model or an anomalous diffusion model, indicating the restricted movement of the fluorophores. One reason for this behaviour is the non-specific interactions with (large) cellular structures. The diffusion of fluorescent lipid analogues in the plant plasma membrane showed a clear deviation from Brownian motion as well, most likely caused by the presence of inhomogeneities in the plasma membrane such as microdomains. Green fluorescent protein (GFP) and the large number of variants available have changed microscopic research in (plant) cell biology completely. The possibility to genetically fuse the fluorescent protein moiety to the protein of interest have replaced the laborious and sometimes inefficient methods of protein labeling, purification and microinjection. On basis of their photophysical characteristics EGFP is the best fluorophore to select for single channel fluctuation spectroscopy experiments and ECFP-EYFP is the best pair to be used in dual-color based methods such as fluorescence resonance energy transfer (FRET) or dual-color fluorescence cross-correlation microscopy (FCCM). To test the applicability of FRET-FCM using ECFP and EYFP as donor-acceptor pair, fusion proteins of ECFP and EYFP having a linker of 8 or 25 alanine residues (CA 8 Y and CA 25 Y, respectively) were created. Fluorescence lifetime measurements and spectral imaging microscopy yielded FRET-efficiencies of 25% for CA 8 Y and only 8% for CA 25 Y both in vitro and in vivo . FRET-FCS measurements by autocorrelating the sensitised emission of EYFP suffered from very low fluorescence count rates. Prolonged measurement times and precise bleed-through correction were required to visualize the presence of FRET in the CA 8 Y protein. FRET measurements within the plant cells were not successful most likely due to severe scattering of the fluorescence in the plant cells, which further reduced the detected fluorescence intensity. In order to detect molecular interactions FRET can be used but this technique requires that the acceptor and donor molecules are in close proximity of each other. However, dual-color cross-correlation microscopy (FCCM) does not require a small distance between two fluorophores since the technique has been based upon the coincidence of intensity fluctuations in two different detection channels. This difference can be illustrated with CA 25 Y. Although some FRET occurred (E = 8%) as have been retrieved from fluorescence lifetime and spectral analysis, autocorrelation of the sensitised acceptor intensity traces did yield a FRET-FCM curve. However, using FCCM the cross-correlation curve for the CA 25 Y protein was clearly present within 2 minutes. This implies that in studies involving interactions between molecules within large complexes or across the plasma-membrane, where FRET studies might not be possible due to the relatively long distances between the molecules, dual-color FCCM is an attractive alternative. To study the oligomerization state and mobility of Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1), a transmembrane protein involved in the embryogenesis of plant cells, AtSERK1 - cDNA was fused to that of ECFP or EYFP and transiently expressed in cowpea protoplasts. PCH analysis showed that 16% of the total amount of AtSERK1 fusion protein in the plasma membrane is present in dimerised form, while no evidence was found for higher oligomeric complexes. By FCCM measurements it was shown that both the monomeric as the dimerised form of fluorescent AtSERK1 diffuse in the plasma membrane according to a two-dimensional Brownian diffusion model. Although it has been shown that fluorescent lipid molecules can be restricted in their motion along the plasma membrane of cowpea protoplasts, no indication for anomalous diffusion was found for the AtSERK1 fusion proteins. In conclusion the application of fluorescence fluctuation techniques in living plant cells is an important asset for cell biological research. The approaches described in this thesis provide possibilities to study the dynamics of molecules at physiological relevant concentrations inside the living cell in a sensitive and selective way.