Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    We will mail you new results for this query: keywords==fluorescence microscopy
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Single-enzyme analysis in a droplet-based micro- and nanofluidic system
Arayanarakool, R. ; Shui, L. ; Kengen, S.W.M. ; Berg, A. van den; Eijkel, J.C. - \ 2013
Lab on a Chip 13 (2013)10. - ISSN 1473-0197 - p. 1955 - 1962.
fluorescence microscopy - pyrococcus-furiosus - beta-glucosidase - propyl gallate - amplification - purification - evolution - kinetics - assays
The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtoliter carriers, achieving high product concentrations from single-molecule encapsulation. The tiny droplets (f ~ 2.5-3 µm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. The method presented here allows to follow large numbers of individual droplets over time. The instrumental requirements are furthermore modest, since the small droplet size allows to use of standard microscope and standard Pyrex glass chips as well as the use of relatively high enzyme concentrations (nM range) for single molecule encapsulation
Detection of illegal use of antibiotics in poultry by fluorescence microscopy
Rijke, E. de; Samson, D. ; Groot, M.J. ; Raamsdonk, L.W.D. van; Nielen, M.W.F. - \ 2013
antibiotica - pluimvee - fluorescentiemicroscopie - detectie - scheenbeen - vleeskuikens - oxytetracycline - antibiotics - poultry - fluorescence microscopy - detection - tibia - broilers
The possibility to use fluorescence microscopy of the antibiotic oxytetracyclin (OTC) in cross-sections of tibia bone is studied to distinguish between untreated broiler chicken and several treatment strategies.
Computational modeling of the BRI1-receptor system
Esse, G.W. van; Harter, K. ; Vries, S.C. de - \ 2013
Plant, Cell & Environment 36 (2013)9. - ISSN 0140-7791 - p. 1728 - 1737.
brassinosteroid signal-transduction - plasma-membrane - plant-growth - arabidopsis-thaliana - auxin transport - root-growth - fluorescence microscopy - gsk3-like kinases - egf receptors - protein
Computational models are useful tools to help understand signalling pathways in plant cells. A systems biology approach where models and experimental data are combined can provide experimentally verifiable predictions and novel insights. The brassinosteroid insensitive 1 (BRI1) receptor is one of the best-understood receptor systems in Arabidopsis with clearly described ligands, mutants and associated phenotypes. Therefore, BRI1-mediated signalling is attractive for mathematical modelling approaches to understand and interpret the spatial and temporal dynamics of signal transduction cascades in planta. To establish such a model, quantitative data sets incorporating local protein concentration, binding affinity and phosphorylation state of the different pathway components are essential. Computational modelling is increasingly employed in studies of plant growth and development. In this section, we have focused on the use of quantitative imaging of fluorescently labelled proteins as an entry point in modelling studies
The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites
Ribeiro, D.M.O.G. ; Jung, M. ; Moling, S. ; Borst, J.W. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2013
The Plant Cell 25 (2013)9. - ISSN 1040-4651 - p. 3602 - 3614.
virus nucleocapsid protein - strand rna viruses - tobacco by-2 cells - punta toro virus - movement protein - uukuniemi virus - intracellular-transport - fluorescence microscopy - golgi localization - secretory pathway
In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES
Visualizing brassinosteroid receptor hetero-oligomers in Arabidopsis roots
Bücherl, C.A. - \ 2013
University. Promotor(en): Sacco de Vries, co-promotor(en): Jan Willem Borst. - S.l. : s.n. - ISBN 9789461736543 - 195
brassinosteroïden - biochemische receptoren - arabidopsis - wortels - beeldanalyse - signaalpeptide - signaaltransductie - fluorescentiemicroscopie - brassinosteroids - biochemical receptors - roots - image analysis - signal peptide - signal transduction - fluorescence microscopy

Living matter is continuously challenged by the dynamics of its environment and intrinsic fluctuations. In the course of evolution, cells have developed mechanisms to detect and adapt to environmental and endogenous cues by the use of a wide array of receptors (Afzal et al., 2008). These receptors perceive specific signals, which, in turn, initiate a sequence of molecular events within the cells that convert signal perception into an adequate physiological response. Collectively, these processes of signal perception, signal transmission and cell adaptation represent so-called signal transduction pathways.

For the perception of signals such as hormones or pathogens cells are equipped with receptors that are often located at the cell surface. In plants, many of these receptors belong to the class of leucine-rich repeat receptor-like kinases (LRR-RLKs) (Shiu and Bleecker, 2001). They comprise an extracellular LRR domain for ligand binding, a transmembrane domain, which anchors them within the plasma membrane (PM) of their host cells, and an intracellular kinase domain for transducing the event of ligand binding into the cell interior. One of the best-described plant LRR-RLKs is the Brassinosteroid insensitive 1 (BRI1) receptor. Since the discovery in 1997 (Li and Chory, 1997) its mode of action has been studied extensively and has resulted in the elucidation of a complete set of molecular components constituting the brassinoteroid (BR) signal transduction pathway (Clouse, 2011).

BRs, the ligands of BRI1, are a group of polyhydroxy lactones that are structurally similar to animal steroid hormones (Grove et al., 1979). This class of phytohormones regulates several aspects of plant growth and development (Kutschera and Wang, 2012). During the last decade it has been shown that BRI1 indeed perceives BRs at the PM (Kinoshita et al., 2005), however, initiation of BR signal transduction requires interaction of BRI1 with other, non-ligand binding receptors (Nam and Li, 2002; Wang et al., 2008; Gou et al., 2012). These coreceptors belong to the family of Somatic embryogenesis receptor-like kinases (SERKs) and have a related structural architecture to BRI1, but with a smaller extracellular domain. Three members of this protein family are involved in BR signaling: SERK1, SERK3 (also known as BAK1 for BRI1-associated kinase 1), and SERK4 (also known as BKK1 for BAK1-like kinase 1). Besides their role as coreceptors of BRI1, the SERKs have also been implicated in various other signaling processes like somatic embryogenesis, male fertility, cell death regulation and plant immunity (Chinchilla et al., 2009).

In the first Chapter of this thesis, the BR signaling pathway was introduced in further detail and it was highlighted how genetic and biochemical approaches attributed to the identification of cellular components that link signal perception of BRs at the PM to BR dependent transcriptional regulation in the nucleus. Based on these findings a model for BRI1-mediated signal transduction was established, which often serves as a paradigm for plant PM receptor signaling. Even though the molecular determinants of BR signaling have been revealed, full mechanistic detail is still missing. The aim of this thesis was to describe BRI1-mediated signal transduction and the respective role of SERK3, the main coreceptor of BR signaling (Albrecht et al., 2008), at (sub)cellular level in Arabidopsis roots. For this purpose different fluorescence imaging techniques were applied, which allowed investigating the spatiotemporal localization and interaction dynamics of BRI1 and SERK3 in their natural environment.

One of the main microscopic methods applied throughout this thesis was fluorescence lifetime imaging microscopy (FLIM). Most imaging approaches, like confocal microscopy, only rely on fluorescence intensities as read-outs. However, the fluorescence lifetime τ is an additional parameter of fluorescence microscopy. This parameter is sensitive to the local environment of fluorescent probes and therefore can be exploited to illuminate cellular processes in live cells and tissues. In Chapter 2, the theoretical background of FLIM was introduced and it was illustrated how this technique can be used to reveal protein-protein interactions in Arabidopsis mesophyll protoplasts based on Förster resonance energy transfer (FRET). Next to a protocol for protoplast isolation and transient transfection, we provided a tutorial for analyzing time-resolved fluorescence intensity images using the software package SPCImage (Becker & Hickl). By determining the fluorescence lifetimes of a FRET donor fluorophore in the absence and the presence of a FRET acceptor chromophore physical interaction between the fluorescently tagged proteins of interest can be deduced. If the two proteins of interest, and thus the conjugated fluorophores, reside in close proximity FRET can occur and will result in a decrease of donor fluorescence lifetime. Besides the applicability to live cells and organisms, another important advantage of FRET-FLIM is the possibility to spatially resolve protein interactions within the two-dimensional imaging plane.

In Chapter 3, this technique was applied to live Arabidopsis roots. In our attempt to visualize the molecular events upon initiation of BR signaling, we performed FRET-FLIM on a double transgenic plant line expressing BRI1-GFP (Friedrichsen et al., 2000) and SERK3-mCherry. In accord with the current model of BR signal transduction (Jaillais et al., 2011a), a time-dependent and ligand-induced hetero-oligomerization between BRI1 and SERK3 was observed, similar to previous reports using coimmunoprecipitation (Wang et al., 2005; 2008; Albrecht et al., 2012). In addition, the spatially resolved FLIM images enabled us to localize these BRI1-SERK3 receptor complexes to restricted areas within the PM of live epidermal root cells, a cell file known to exhibit active BR signaling (Hacham et al., 2011). Application of brefeldin A (BFA), a fungal toxin reported to inhibit recycling (Nebenführ et al., 2002), allowed the visualization of intracellular receptor oligomers, which were most likely endocytosed from the PM. In contrast to the established BRI1 signaling model, FRET-FLIM revealed that a substantial amount of the BRI1-SERK3 hetero-oligomers was preformed. Constitutive receptor oligomerization is a well-established concept in animal signaling research (Gadella and Jovin, 1995; Martin-Fernandez et al., 2002; Issafras et al., 2002; Van Craenenbroeck et al., 2011), however in the plant field only a single study reported similar findings (Shimizu et al., 2010).

Besides the physical interaction between BRI1 and SERK3, also their localization and colocalization patterns were investigated (Chapter 3). As expected, most of the fluorescently tagged receptors localized to the PM. The intracellular fraction of BRI1-GFP mainly resided in punctate endosomal structures as documented previously (Geldner et al., 2007; Viotti et al., 2010; Irani et al., 2012). Similar endomembrane compartments were also observed for SERK3-mCherry, though to a lesser extent. In contrast to BRI1, for SERK3 an additional intracellular compartment was elucidated, the tonoplast. A further difference in the localization patterns of BRI1 and SERK3 was revealed when BFA was applied. Whereas BRI1-GFP strongly accumulated in BFA bodies, SERK3-GFP was only marginally affected, which hints at a differential endocytic pathway for both receptors. Although BRI1 and SERK3 showed distinct localization patterns, the two fluorescently tagged proteins also overlapped to some degree. Comparative colocalization analysis revealed that both the PM and the intracellular overlap between both LRR-RLKs is responsive to the BR signaling status. Application of brassinolide (BL), an endogenous BRI1 ligand, as well as BFA, which was demonstrated to elevate BR signaling activity (Geldner et al., 2007; Irani et al., 2012), resulted in an increased number of colocalizing BRI1 and SERK3 proteins. Thus FRET-FLIM and confocal imaging based colocalization analysis indicated that activation of the BR signaling system is accompanied by spatially distinct association of the two signal transduction inducing receptors BRI1 and SERK3.

As just illustrated, fluorescence microscopy is a valuable tool for investigating signal transduction processes in the natural environment of the executing molecular components. Unfortunately, a major drawback of the various techniques is that often only qualitative read-outs are obtained. Therefore we examined (Chapter 4) two different quantitative colocalization approaches in their ability to discriminate varying colocalizing proteinpopulations. First, the cytosolic colocalization of BRI1-GFP with the endosomal markerproteins ARA6 and ARA7 was investigated. Both tested and freely available ImageJ plugins Coloc2 and PSC Colocalization (French et al., 2008) revealed that BRI1-GFP preferentially localized to ARA7-mRFP labeled endosomal compartments. This finding was confirmed by manual counting of the respective endosomal structures and verified the reliability of the two quantitative colocalization methods. A biological explanation of the obtained result is given by the identity of the labeled endomembrane compartments. ARA7 localizes to both early endosomes (EEs), which enable recycling to the PM, and late endosomes (LEs; also known as multivesicular bodies [MVBs]), which are determined for vacuolar fusion. In contrast, ARA6 labels mainly LEs/MVBs. Thus both markers overlap to a certain degree during the maturation of LE but still have distinct localization patterns (Ueda et al., 2004; Ebine et al., 2011). Since BRI1 undergoes constitutive recycling (Geldner et al., 2007), our finding of preferential colocalization between BRI1 and ARA7 is plausible. In addition, similar observations were recently also reported for Flagellin sensing 2 (FLS2), an LRR-RLK involved in plant immunity, which also exhibits constitutive recycling (Beck et al., 2012).

After establishing the applicability of both colocalization approaches, we also intended to confirm our previous observation of increased BRI1 and SERK3 colocalization in response to BFA obtained with the Coloc2 plugin (Chapter 3). The application of PSC Colocalization indeed confirmed our initial colocalization results. The elevated colocalization of BRI1 and SERK3 upon drug treatment mostly like reflects the PM-stabilizing effect of BFA (Irani et al., 2012), which may also account for SERK3, since both Manders’ colocalization coefficients were increased. Nonetheless, a difficulty of quantitative colocalization analysis is the interpretation of colocalization coefficients obtained for individual images. However, they enable to assess image data sets, recorded under the same imaging conditions, in a comparative manner and that way allows drawing quantitative conclusions (Dunn et al., 2011). Colocalization analysis is not the only approach that suffers from qualitative read-outs and interpretations. The same accounts for FRET-FLIM studies. In particular the observation of preformed BRI1-SERK3 hetero-oligomers triggered our interest in developing a quantitative FLIM analysis procedure, which would be able to resolve ligand-independent and ligand-induced receptor complex populations. The details of our approach, which is based on time-correlated single photon count (TCSPC) measurements, were described in Chapter 4. Using this novel FLIM analysis procedure enabled us to estimate the different populations of BRI1 and SERK3 complexes. Upon BL stimulation around 10% of PM-located BRI1-GFP receptors were in complex with SERK3-mCherry. This finding is in line with recently reported data based on an in silico modeling approach (van Esse et al., 2012) and semi-quantitative coimmunoprecipitation (Albrecht et al., 2012), which suggested that active BR signal transduction involves between 1-10% of BRI1 receptors. Unfortunately, there are no quantitative data available for constitutive BRI1-SERK3 hetero-oligomers, even though their existence was proposed (Wang et al., 2005). Based on our imaging approach and analysis procedure we estimate that approximately 70% of PM BRI1-SERK3 heterooligomers are preformed. Finding such a considerable amount of preformed BRI1-SERK3 receptor complexes in the PM of root epidermal cells was intriguing since it contradicts the current view on BR signaling, which assumes a strictly ligand-dependent association of the two LRR-RLKs (Jaillais et al., 2011a). This posed the question when or where these preformed complexes are established. To address this point we investigated in Chapter 5 which cellular compartments harbor individual BRI1 and SERK3 receptors, and in which organelles these two receptors colocalize. Comparative colocalization analysis in live Arabidopsis roots revealed that both LRR-RLKs follow the traditional secretory and retrograde transport routes. These observations confirmed and extended previous findings for BRI1 using live cell (Friedrichsen et al., 2000; Geldner et al., 2007; Viotti et al., 2010; Irani et al., 2012) and electron microscopy (Viotti et al., 2010). For SERK3, to date only localization to EEs was suggested (Russinova et al., 2004).

Using the transient expression system of Arabidopsis protoplasts we could moreover show that both receptors also colocalize in the various endomembrane compartments of anterograde and retrograde trafficking. However, using electron microscopy a striking difference between their localization in retrograde endosomal compartments was elucidated. Whereas BRI1 was previously shown to reside at the membranes of the enclosed vesicles (Viotti et al., 2010), SERK3 was visualized at the limiting membrane of prevacuolar compartments (PVCs). This finding also explains, why SERK3, but not BRI1, was observed at the tonoplast (Chapter 3). Fusion of MVBs with the vacuole results in the release of BRI1 along with the inner MVB vesicles into the vacuolar lumen. PVC-localized SERK3 instead is incorporated into the tonoplast after membrane fusion. Collectively, the colocalization analysis of BRI1 and SERK3 with respect to endomembrane compartments revealed that subpopulations of both LRR-RLKs probably follow the same route to the PM, but that after endocytosis from the PM, possibly during the maturation of TGN/EEs to LEs/MVBs, a separation occurs. Still, these findings do not answer where or when BRI1-SERK3 hetero-oligomers are established. For that reason we applied FRET-FLIM on the subcellular compartment, in which BRI1 and SERK3 colocalized for the first time, the endoplasmic reticulum (ER). Similar to our observations at the PM of root epidermal cells (Chapter 3), most of the ER membrane did not show BRI1-SERK3 receptor complexes. Still, in restricted ER membrane regions strongly reduced donor fluorescence lifetimes were observed, indicating that BRI1-SERK3 hetero-oligomers are established already in the ER before entering the anterograde trafficking pathway. Finally, using a heat-shock inducible plant system we could confirm the establishment of BRI1-SERK3 hetero-oligomers shortly after biogenesis on their way to the PM. Thus, the observed preformed receptor complexes in the PM of root epidermal cells (Chapter 3) mostly likely originated from the ER and were inserted via targeted transport into the PM, the site where they fulfill their function as BR signaling units.

Primary photosynthetic processes: from supercomplex to leaf
Broess, K. - \ 2009
University. Promotor(en): Herbert van Amerongen. - [S.l.] : S.n. - ISBN 9789085852988 - 124
fotosynthese - fluorescentie - fluorescentiemicroscopie - spectroscopie - membranen - chloroplasten - fotosysteem ii - planten - photosynthesis - fluorescence - fluorescence microscopy - spectroscopy - membranes - chloroplasts - photosystem ii - plants
This thesis describes fluorescence spectroscopy experiments on photosynthetic complexes that cover the primary photosynthetic processes, from the absorption of light by photosynthetic pigments to a charge separation (CS) in the reaction center (RC). Fluorescence spectroscopy is a useful tool in photosynthetic particles, because the latter are densely packed with fluorescence pigments like chlorophylls (Chl). The fluorescence of each pigment is affected by its environment and provide information about structure and dynamics of the photosynthetic complexes. In this thesis time-resolved fluorescence of Chl molecules is used for studying the ultrafast kinetics in membrane particles of photosystem II (PSII) (chapter 2, 3 and 4). In chapter 5 fluorescence lifetime imaging microscopy (FLIM) of is applied to study entire chloroplasts, either in the leaf or in isolated chloroplast form. The advantage of FLIM is that the interactions of the fluorescence pigments in both photosystems can be spatially resolved up to a resolution of 0.5 x 0.5 x 2 µm to indentify and quantify photosynthetic processes in their natural environment.

Excitation energy transfer and charge separation in PSII membranes (chapter 2,3 and 4)

In this thesis time-resolved fluorescence measurements of PSII containing membranes, the so called BBY particles, are performed in low-light conditions with open reaction centers. The BBY particles do not contain photosystem I (PSI) or stroma lamellae, but do support electron transfer and carry out oxygen evolution with high activity and are comparable with the grana in vivo. The fluorescence decay kinetics of the BBY particles are faster than observed in previous studies and also faster than observed for PSII in chloroplasts and thylakoid preparations. The average lifetime is 150 ps, which, together with previous annihilation experiments on light-harvesting complex II (LHCII) suggests that excitation migration from the antenna complexes contributes significantly to the overall charge separation time. This is in disagreement with the commonly applied exciton / radical-pair-equilibrium (ERPE) model that assumes that excitation energy diffusion through the antenna to the RC is much faster than the overall charge-separation time.
A simple coarse-grained method is proposed, based on the supramolecular organization of PSII and LHCII in grana membranes (C2S2M2). The proposed modelling procedure for BBY particles is only approximate and many different combinations of excitation migration time and the charge separation time can explain the observed fluorescence kinetics. However it is clear that charge transfer should be rather fast and is accompanied with a large drop in free energy.
In chapter 3, the fluorescence kinetics of BBY particles with open RCs are compared after preferential excitation at 420 and 484 nm, which causes a difference in the initial excited-state populations of the inner and outer antenna system. The fluorescence decay is somewhat slower upon preferential excitation of chlorophyll (Chl) b, which is exclusively present in the outer antenna. Using the coarse-grained model it was possible to fit the 420 and 484 nm results simultaneously with a two-step electron transfer model and four parameters: the hopping rate between the protein-pigment complexes, the CS rate, the drop in free energy upon primary charge separation and a secondary charge separation rate. The conclusion is that the average migration time contributes ~25% to the overall trapping time. The hopping time obtained in chapter 3 is significantly faster than might be expected based on studies on trimeric and aggregated LHCII and it is concluded that excitation energy transfer in PSII follows specific pathways that require an optimized organization of the antenna complexes with respect to each other. Analysis of the composition of the BBY particles indicates that the size of the light-harvesting system in PSII is smaller than commonly found for PSII in chloroplasts and explains why the fluorescence lifetimes are smaller for the BBY’s.
In chapter 4, four different PSII supercomplex preparations were studied. The main difference between these supercomplexes concerns the size of the outer antenna. The average lifetime of the supercomplexes becomes longer upon increasing the antenna size. The results indicate that the rate constants obtained from the coarse-grained method for BBY preparations, which is based on the supercomplex composition C2S2M2, should be slightly faster (~10%) as predicted in chapter 3. The observation that the average lifetime of the supercomplexes is relatively slow compared to what one might expect based on the measurements on BBY particles, and this will require further future studies.

Photosynthesis in plant leaves (Chapter 5)

With the use of femtosecond two-photon excitation TPE at 860 nm it appears to be possible to measure fluorescence lifetimes throughout the entire leaves of Arabidopsis thaliana and Alocasia wentii. It turns out that the excitation intensity can be kept sufficiently low to avoid artifacts due to singlet-singlet and singlet-triplet annihilation, while the reaction centers can be kept in the open state during the measurements. The average fluorescence lifetimes obtained for individual chloroplasts of Arabidopsis thaliana and Alocasia wentii in the open and closed state, are approximately ~250 ps and ~1.5 ns, respectively. The maximum fluorescence state correspond to a state in which all reaction centers are closed. The kinetics are very similar to those obtained for chloroplasts in vitro with the FLIM setup and to in vivo results reported in literature. No variations between chloroplasts are observed when scanning throughout the leaves of Arabidopsis thaliana and Alocasia wentii. Within individual chloroplasts some variation is detected for the relative contributions of PSI and PSII to the fluorescence. The results open up the possibility to use FLIM for the in vivo study of the primary processes of photosynthesis at the level of single chloroplasts under all kinds of (stress) conditions.

General conclusions

This thesis gives new insight of the kinetic processes in PSII membranes. With the use of a coarse-grained method that provides an easy way to incorporate existing knowledge and models for individual complexes, valuable conclusions can be drawn about the excitation energy transfer and the CS which hopefully contributes to an improvement of the knowledge about PSII functioning. In general it was shown that a large drop in free energy is needed in PSII membranes for all simulations with the coarse-grained method.
The presented results on the kinetics of chloroplasts obtained in vitro and in vitro are very similar and verify that conclusions drawn from isolated chloroplasts can be extrapolated to photosynthetic processes in their natural environment.

The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants
Lacorte, C.C. ; Ribeiro, S.G. ; Lohuis, H. ; Goldbach, R.W. ; Prins, M.W. - \ 2007
Protein Expression and Purification 55 (2007)1. - ISSN 1046-5928 - p. 17 - 22.
transgenic plants - fluorescence microscopy - nucleocapsid protein - beta-glucuronidase - mosaic-virus - expression - system - gene - tospovirus - resistance
Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.
Tomato spotted wilt virus Gc and N proteins interact in vivo
Snippe, M. ; Borst, J.W. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2007
Virology 357 (2007)2. - ISSN 0042-6822 - p. 115 - 123.
nucleocapsid protein - fluorescence microscopy - mammalian-cells - matrix protein - living cells - fever virus - membrane - glycoproteins - localization - microtubules
Tomato spotted wilt virus (TSWV) virions consist of a nucleocapsid core surrounded by a membrane containing glycoproteins Gn and Gc. To unravel the protein interactions involved in the membrane acquisition of RNPs, TSWV nucleocapsid protein (N), Gn and Gc were expressed and analyzed in BHK21 cells. Upon coexpression of Gn, Gc and N, a partial colocalization of N with both glycoproteins was observed in the Golgi region. In contrast, upon coexpression of Gc and N in the absence of Gn, both proteins colocalized to a distinct non-Golgi perinuclear region. Using FLIM and FRET, interaction was demonstrated between N and Gc, but not between N and Gn, and was only observed in the region where both proteins accumulated. The genuine character of N¿Gc interaction was confirmed by its presence in purified virus and RNP preparations. The results are discussed in view of TSWV particle assembly taking place at the Golgi complex
Tomato spotted wilt virus particle assembly : studying the role of the structural proteins in vivo
Snippe, M. - \ 2006
University. Promotor(en): R.W. Goldbach, co-promotor(en): Richard Kormelink. - [S.l. ] : S.n. - ISBN 9085043263 - 128 p.
solanum lycopersicum - tomaten - tomatenbronsvlekkenvirus - tospovirus - viruseiwitten - virale regulatoire eiwitten - glycoproteïnen - fluorescentiemicroscopie - genexpressieanalyse - tomatoes - tomato spotted wilt virus - viral proteins - viral regulatory proteins - glycoproteins - fluorescence microscopy - genomics
New insights in cellular biochemistry owing to the use of fluorescence microscopy
Borst, J.W. - \ 2006
University. Promotor(en): Ton Visser. - [S.l. ] : S.n. - ISBN 908504474X - 132 p.
fluorescentiemicroscopie - redoxreacties - lipidenperoxidatie - arabidopsis - fluorescence microscopy - redox reactions - lipid peroxidation
Towards in vivo imaging of early Rhizobium Nod factor responses
Krogt, G.N.M. van der - \ 2006
University. Promotor(en): Ton Bisseling. - [S.l.] : S.n. - ISBN 9085043719 - 111
rhizobium - rhizobium rhizogenes - wortelharen - wortelknolletjes - fluorescentiemicroscopie - transformatie - moleculaire biologie - celbiologie - root hairs - root nodules - fluorescence microscopy - transformation - molecular biology - cellular biology
Germination inhibitors of fungal spores: identification and mode of action
Chitarra, G.S. - \ 2003
University. Promotor(en): Frans Rombouts, co-promotor(en): Tjakko Abee; J. Dijksterhuis. - [S.l.] : S.n. - ISBN 9058089142 - 111 p.
schimmelsporen - kiemremmers - fusarium culmorum - penicillium - fluorescentiemicroscopie - antimycotica - fungal spores - germination inhibitors - fluorescence microscopy - antifungal agents
Flow cytometry, fluorescent probes, and flashing bacteria
Bunthof, C.J. - \ 2002
University. Promotor(en): F.M. Rombouts; Tjakko Abee; P. Breeuwer. - S.l. : S.n. - ISBN 9789058086327 - 159
doorstroomcytometrie - fluorescentiemicroscopie - melkzuurbacteriën - voedselmicrobiologie - lysis - zuivelonderzoek - flow cytometry - fluorescence microscopy - lactic acid bacteria - food microbiology - dairy research
<font size="3"><p> </p><p><hr/></p><p>Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, <em>Lactococcus lactis</em> , <em>Lactobacillus plantarum</em> , cheese, milk, probiotic</p><p> </p><p>In food industry there is a perceived need for rapid methods for detection and viability assessment of microbes. Fluorescent staining and flow cytometry provide excellent tools for microbial analysis. This thesis describes fluorescent techniques for assessment of the physiological state of lactic acid bacteria.</p><p>Lysis of lactic acid bacteria plays a crucial role in cheese manufacturing. It is generally considered that lysis results in leakage of intracellular enzymes in the cheese curd and, thus, plays an important role in ripening and flavor formation. <em>Bac</em> Light (Molecular Probes) was applied for monitoring the lysis process of <em>Lactococcus lactis</em> MG1363 in a buffered suspension with high osmolarity to mimic cheese conditions. The <em>Bac</em> Light kit combines the nucleic acid dyes propidium iodide (PI) and SYTO 9. PI is commonly used to determine membrane integrity based on dye exclusion. When used in combination with the permeant SYTO 9, membrane-damaged cells are stained by PI (red) while the intact cells are stained by SYTO 9 (green). Lysis was induced with mutanolysin and followed in time using fluorescence microscopy and flow cytometry. Also, enzyme assays and plate counts were performed. The results demonstrated a transient permeable cell status that has a significant role in the lysis process. Furthermore, permeable cells were demonstrated in ripening cheese with confocal scanning laser microcopy and <em>Bac</em> Light.</p><p>Viability assessment by conventional plate counting requires long incubation times and provides limited information. Flow cytometric assessment of the viability of lactic acid bacteria was investigated and compared with plate counts. The esterase substrate carboxyfluorescein diacetate (cFDA) and the impermeant nucleic acid dyes PI and TOTO-1 were tested using exponential phase at 70°C heat-killed cultures of a <em>Lactococcus</em> , a <em>Streptococcus</em> , three <em>Lactobacillus</em> , two <em>Leuconostoc</em> , an <em>Enterococcus</em> , and a <em>Pediococcus</em> species. The combination of cFDA and TOTO-1 gave the best results. The intact and membrane-damaged subpopulations were distinguished well. Sorting and plating showed that cFDA stained the culturable and TOTO-1 the nonculturable cells. The assay was applied to cultures exposed to deconjugated bile salts or to hydrochloric acid and results corresponded well with plate counts.</p><p>Subsequently, flow cytometry with cFDA and TOTO-1 staining was applied to <em>Lactobacillus plantarum</em> WCFS 1 suspended in milk. To facilitate flow cytometry clearing of the milk was required. A procedure based on a milk clearing solution was optimized to increase the signal-to-noise-ratio and flow cytometry enumerations were accurate to a lower limit of 10 <sup>5</SUP>cells/ml.</p><p>Finally, the novel assay was applied to starter cultures for cheese and yogurt and to the probiotic products Yakult, Mona Vifit, and Orthiflorplus. Flow cytometry in combination with plate counts revealed three populations: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed between the tested products.</p><p>In conclusion, the development of flow cytometry for bacteria is an important asset for microbiological research. The rapid novel methods described in this thesis provide possibilities for examination of fermentation processes and food products.
Imaging of oxidative stress in plant cells by quantitative fluorescence microscopy and spectroscopy
Borst, J.W. ; Uskova, M.A. ; Visser, N.V. ; Visser, A.J.W.G. - \ 2002
In: Fluorescence Spectroscopy, Imaging and Probes, New Tools in Chemical, Physical and Life Sciences / Kraayenhof, R., Visser, A.J.W.G., Gerritsen, H.C., Heidelberg : Springer Verlag - p. 337 - 348.
fluorescentiemicroscopie - fluorescentie-emissiespectroscopie - cellen - planten - stress - fluorescence microscopy - fluorescence emission spectroscopy - cells - plants
Probing nod factor perception in legumes by fluorescence microspectroscopy
Goedhart, J. - \ 2001
University. Promotor(en): Ton Bisseling; T.W.J. Gadella Jr.. - S.l. : S.n. - ISBN 9789058084811 - 142
peulgewassen - rhizobium - stikstof - groei - symbiose - wortels - wortelharen - wortelknolletjes - membranen - n-acetylglucosamine - cytologie - fluorescentiemicroscopie - moleculaire biologie - legumes - nitrogen - growth - symbiosis - roots - root hairs - root nodules - membranes - cytology - fluorescence microscopy - molecular biology
<p>Plants of the family of legumes are capable of forming a symbiosis with Rhizobium bacteria. These Gram-negative bacteria invade the root system of a host legume and fix nitrogen in a specialized organ, the so-called root nodule. In exchange for sugars, the bacteria convert atmospheric nitrogen to ammonia which can be used by the plant. This remarkable alliance allows the plant to grow independently from nitrogen sources provided by the soil. Examples of leguminous plants are clover, pea, and soybean.</p><p>The symbiosis is initiated by a molecular dialogue. The plant produces flavonoid compounds which are recognized by the bacterial NodD protein. The signaling pathway which is activated leads to the synthesis and secretion of lipo-chitooligosaccharides which are also called Nod factors. The production of Nod factors by the Rhizobium bacteria is an essential step for accomplishing symbiosis and also determines host specificity. The general structure of Nod factors comprises a chitin backbone of three to five b-1,4-linked N-acetylglucosamine units. A fatty acid of 16-20 carbon atoms is N-linked to the terminal non-reducing sugar residue. The exact molecular structure can comprise different acyl chains and a variety of decorations on the chitin backbone depending on the Rhizobium species.</p><p>After successful recognition of the bacteria by the legume, a remarkable morphogenic process takes place, which is known as root hair curling. The root hair curls around the Rhizobium colony by which the bacteria are entrapped within the so-called shepherd's crook. Subsequently, the rhizobia enter the root hair through an infection thread, starting from the center of the curl. Via the infection thread several cell layers are crossed after which the bacteria are released in nodule primordium cells, where they differentiate into bacteroids that fix nitrogen.</p><p>Nod factors in the absence of bacteria, either purified from Rhizobium cultures or chemically synthesized can elicit a wide variety of responses on a compatible legume host. When Nod factors are applied to roots, the earliest visible response takes place in root hairs. Root hairs are single tip-growing cells that develop from the epidermis of a root and grow perpendicular from the longitudinal axis of the root. Generally, root hairs that are terminating growth are susceptible to Nod factors and respond by swelling of the tip of the root hairs, followed by the re-initiation of tip growth in a random direction. This typical Nod factor response is referred to as root hair deformation and can be observed with a microscope 2-3 hours after addition of Nod factors.</p><p>The perception of Nod factors by the plant, and the downstream signaling cascades that are activated are major research topics in the Rhizobium-legume interaction. The low concentration (down to 10-12 M) at which Nod factors can still induce root hair deformation and the dependence of the bioactivity on specific decorations of the Nod factor suggest that these molecules are perceived by receptors at the root hair. However, to date no such receptors are characterized. Moreover, it is far from clear where Nod factor recognition by root hairs takes place. Therefore an approach was taken in which fluorescent Nod factor derivatives are synthesized, allowing to probe the ligand binding sites on legume root hairs.</p><p>The research described in this thesis focuses on the quantification, characterization and perception by legumes of Nod factors. In order to detect Nod factors at physiologically relevant concentrations sensitive techniques are required. A number of fluorescence spectroscopy and microscopy based techniques can be used to study fluorescent derivatives of signaling molecules. In chapter 1, the use of fluorescence microspectroscopic techniques available in the laboratory are discussed. Examples how these techniques can be used for the study of root hairs and other living cells are described.</p><p>In chapter 2, two methods to quantify purified Nod factors are described. An enzymatic step which is crucial for the first method was analyzed in detail. The second method was optimized and validated using fluorescent and radiolabeled Nod factor derivatives. The chapter describes in detail how the two optimized methods can be used for quantifying Nod factors as well as potential pitfalls.</p><p>In chapter 3, the spectral properties of three novel fluorescent Nod factor derivatives are described. It is checked whether these fluorescent Nod factors can still elicit root hair deformation on Vicia sativa roots. The properties of the amphiphilic signaling molecules were characterized in vitro in the absence and presence of micelles and model membrane systems using fluorescence spectroscopy. Time-correlated single photon counting fluorescence spectroscopy was used to measure rotational mobility of the fluorophore. These experiments are complemented with fluorescence correlation spectroscopy to examine diffusional mobility of the Nod factors. A lipid transfer assay was used to measure the rate of intermembrane transfer and intramembrane flip-flop of Nod factors.</p><p>In chapter 4, a detailed study is reported describing the sites at which the fluorescent Nod factors accumulate. Fluorescence microscopy is used to examine the location of fluorescent Nod factors on root hairs during the initial perception and during root hair deformation. Subsequently, the diffusional mobility of the fluorescent Nod factors is measured in vivo using fluorescence correlation microscopy (FCM), allowing quantification of molecular mobility and concentration of fluorescent Nod factors in living root hairs at a molecular level. This study is continued in chapter 5 in which also novel sulfated fluorescent Nod factors are used and characterized, enabling a direct comparison between sulfated and non-sulfated Nod factors on a host and non-host legume. Also, the origin of the molecular mobility of the Nod factors is studied in more detail.</p><p>In chapter 6 a novel approach towards manipulating phospholipid second messengers in single cells with spatiotemporal control is presented. The synthesis of a fluorescent and caged derivative, NPE-phosphatidic acid, which releases phosphatidic acid upon exposure to UV is described. The release of phosphatidic acid from the caged compound is studied in vitro and in vivo. The use of photoreleasable phosphatidic acid for studying phospholipid signaling in vivo is evaluated.</p><p>Chapter 7 summarizes the conclusions that can be drawn from the results described in this thesis. The implications for Nod factor secretion by the bacterium and subsequent perception by legume root hairs are discussed. Based on the results presented in this thesis, it is tempting to speculate that spatial restriction of signaling molecules in plants is achieved by immobilization in the cell wall. Subseqent perception of Nod factor takes place either in the plasma membrane or within the cell wall as is illustrated by two proposed modes of perception. The results of this thesis are discussed with respect to these two models.</p>
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