A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies
Horstman, A. ; Nougalli Tonaco, I.A. ; Boutilier, K.A. ; Immink, R.G.H. - \ 2014
International Journal of Molecular Sciences 15 (2014). - ISSN 1661-6596 - p. 9628 - 9643.
bimolecular fluorescence complementation - bifc-based fret - living cells - ternary complexes - visualization - arabidopsis - assay - expression - fragments - cloning
Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.
Synergistic stiffening in double-fiber networks
Rombouts, W.H. ; Giesbers, M. ; Lent, J.W.M. van; Wolf, F.A. de; Gucht, J. van der - \ 2014
Biomacromolecules 15 (2014)4. - ISSN 1525-7797 - p. 1233 - 1239.
gelation properties - living cells - hydrogels - gels - copolymers - dipeptides - strength
Many biological materials are composite structures, interpenetrating networks of different types of fibers. The composite nature of such networks leads to superior mechanical properties, but the origin of this mechanical synergism is still poorly understood. Here we study soft composite networks, made by mixing two self-assembling fiber-forming components. We find that the elastic moduli of the composite networks significantly exceed the sum of the moduli of the two individual networks. This mechanical enhancement is in agreement with recent simulations, where it was attributed to a suppression of non-affine deformation modes in the most rigid fiber network due to the reaction forces in the softer network. The increase in affinity also causes a loss of strain hardening and an increase in the critical stress and stain at which the network fails.
Development of FRET biosensors for mammalian and plant systems
Hamers, D.S. ; Voorst Vader, L. van; Borst, J.W. ; Goedhart, J. - \ 2014
Protoplasma 251 (2014)2. - ISSN 0033-183X - p. 333 - 347.
yellow fluorescent proteins - photoactivated localization microscopy - resonance energy-transfer - living cells - mass-spectrometry - dynamic-range - time - ca2+ - resolution - indicators
Genetically encoded biosensors are increasingly used in visualising signalling processes in different organisms. Sensors based on green fluorescent protein technology are providing a great opportunity for using Förster resonance energy transfer (FRET) as a tool that allows for monitoring dynamic processes in living cells. The development of these FRET biosensors requires careful selection of fluorophores, substrates and recognition domains. In this review, we will discuss recent developments, strategies to create and optimise FRET biosensors and applications of FRET-based biosensors for use in the two major eukaryotic kingdoms and elaborate on different methods for FRET detection.
FRET-FLIM applications in plant systems
Bücherl, C.A. ; Bader, A.N. ; Westphal, A.H. ; Laptenok, S. ; Borst, J.W. - \ 2014
Protoplasma 251 (2014)2. - ISSN 0033-183X - p. 383 - 394.
lifetime imaging microscopy - resonance energy-transfer - fluorescence correlation spectroscopy - protein-protein interactions - living cells - subcellular resolution - molecular-interactions - spatial-resolution - fusion-proteins - global analysis
A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.
A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space
Fritz, R.D. ; Letzelter, M. ; Reimann, A. ; Martin, K. ; Fusco, L. ; Ritsma, L. ; Ponsioen, B. ; Fluri, E. ; Schulte-Merker, S. ; Rheenen, J. ; Pertz, O. - \ 2013
Science Signaling 6 (2013)285. - ISSN 1945-0877
cyan fluorescent protein - rho-family gtpases - dynamic-range - living cells - in-vivo - activation - kinase - cdc42 - indicators - reporters
Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal–regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.
Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology
Ershov, D.S. ; Cohen Stuart, M.A. ; Gucht, J. van der - \ 2012
Soft Matter 8 (2012). - ISSN 1744-683X - p. 5896 - 5903.
particle-tracking microrheology - cytoskeletal protein networks - micropipette aspiration - myosin-filaments - molecular motors - polymer networks - living cells - cortex - microscopy - membranes
There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids lining the surface of an oil droplet using biotin–streptavidin bonds. In this way we can form a thin actin network that can be visualized and studied by confocal microscopy. Our approach allows incorporation of different actin-binding and motor proteins into this 2D network and characterization of their effect on its mechanical properties in a quantitative way. To study the viscoelasticity of the network, we use passive particle tracking microrheology, which allows storage and loss moduli to be extracted from the mean square displacement of tracer particles. We show that adding cross-linkers to the cortex increases its elasticity by several orders of magnitude and addition of myosin in the presence of ATP results in a strong and rapid stiffening of the network. This approach opens up a variety of possibilities to study viscoelastic properties of the actin cortex in vitro, allowing incorporation of any protein of interest into the system.
Food-grade submicrometer particles from salts prepared using ethanol-in-oil mixtures
Paques, J.P. ; Linden, E. van der; Sagis, L.M.C. ; Rijn, C.J.M. van - \ 2012
Journal of Agricultural and Food Chemistry 60 (2012)34. - ISSN 0021-8561 - p. 8501 - 8509.
metal nanoparticles - living cells - evaporating solution - growth-kinetics - k2so4 crystals - vitamin-a - calcium - iron - microemulsions - stability
A simple method for preparing food-grade particles in the submicrometer range of ethanol soluble salts using ethanol-in-oil (E/O) mixtures is described. Salts CaCl2·2H2O and MgCl2·6H2O were dissolved in ethanol that subsequently was mixed with a medium-chain triglyceride oil phase. It was found that type and concentration of salt have a significant influence on the miscibility of ethanol and oil phase and on the stability of E/O mixtures. The ethanol phase was evaporated from the mixture at elevated temperatures, and salt particles with dimensions in the submicrometer range (6?400 nm) remained suspended in the oil phase. It was found that the concentration of salt and volume fraction of ethanol in MCT oil have a significant influence on the size distribution of salt particles. The size of CaCl2 and MgCl2 submicrometer particles was ascertained by scanning electron microscopy and dynamic light scattering.
Disentangling picosecond events that complicate the quantative use of the calcium sensor YC3.60
Laptenok, S. ; Stokkum, I.H.M. van; Borst, J.W. ; Oort, B.F. van; Visser, A.J.W.G. ; Amerongen, H. van - \ 2012
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 116 (2012)9. - ISSN 1520-6106 - p. 3013 - 3020.
cyan fluorescent protein - resonance energy-transfer - time-resolved spectra - living cells - fret - photoconversion - ca2+ - yfp - spectroscopy - indicators
Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.
Positive control of cell division: FtsZ is recruited by SsgB during sporulation of Streptomyces
Willemse, J. ; Borst, J.W. ; Waal, E. de; Bisseling, T. ; Wezel, G.P. van - \ 2011
Genes and Development 25 (2011)1. - ISSN 0890-9369 - p. 89 - 99.
escherichia-coli - coelicolor a3(2) - protein ftsz - assembly dynamics - bacillus-subtilis - crystal-structure - ring structure - living cells - growth - gene
In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Förster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle
Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
Visser, A.J.W.G. ; Laptenok, S. ; Visser, N.V. ; Hoek, A. van; Birch, D.J.S. ; Brochon, J.C. ; Borst, J.W. - \ 2010
European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 241 - 253.
lifetime imaging microscopy - maximum-entropy method - energy-transfer - living cells - polarized fluorescence - flavin fluorescence - lipoamide dehydrogenase - glutathione-reductase - dynamics - photoconversion
Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive
Membrane chemical stability and seed longevity
Golovina, E.A. ; Hoekstra, F.A. ; As, H. van - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 657 - 668.
nitroxide spin labels - phase-behavior - infrared-spectroscopy - molecular mobility - lipid-peroxidation - doxyl-stearates - viability loss - living cells - reduction - metabolism
Here, we investigate the relationships between the chemical stability of the membrane surface and seed longevity. Dry embryos of long-lived tomato and short-lived onion seeds were labeled with 5-doxyl-stearic acid (5-DS). Temperature-induced loss of the electron spin resonance signal caused by chemical conversion of 5-DS to nonparamagnetic species was used to characterize the membrane surface chemical stability. No difference was found between temperature plots of 5-DS signal intensity in dry onion and tomato below 345 K. Above this temperature, the 5-DS signal remained unchanged in tomato embryos and irreversibly disappeared in onion seeds. The role of the physical state and chemical status of the membrane environment in the chemical stability of membrane surfaces was estimated for model systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dried alone or in the presence of trehalose or glucose. Fourier transform infrared spectroscopy was used to follow temperature-induced structural changes in dry POPC. Spin-label technique was used to relate the chemical stability of 5-DS with the dynamic properties of the bilayer and 5-DS motion behavior. In all the models, the decrease in 5-DS signal intensity was always observed above T m for the membrane surface. The 5-DS signal was irreversibly lost at high temperature when dry POPC was embedded in a glucose matrix. The loss of 5-DS signal was moderate when POPC was dried alone or in the presence of trehalose. Comparison of model and in vivo data shows that the differences in longevity between onion and tomato seeds are caused by differences in the chemical status of the membrane surface rather than the degree of its immobilization
Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy
Borst, J.W. ; Laptenok, S. ; Westphal, A.H. ; Kühnemuth, R. ; Hornen, H. ; Visser, N.V. ; Kalinin, S. ; Aker, J.C.M. ; Hoek, A. van; Seidel, C.A.M. ; Visser, A.J.W.G. - \ 2008
Biophysical Journal 95 (2008). - ISSN 0006-3495 - p. 5399 - 5411.
resonance energy-transfer - living cells - segmental flexibility - reference convolution - transport-properties - molecular-dynamics - protein - microscopy - anisotropy - diffusion
Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding
Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteriod insensitive 1 receptor oligomerization
Hink, M.A. ; Shah, K. ; Russinova, E.T. ; Vries, S.C. de; Visser, A.J.W.G. - \ 2008
Biophysical Journal 94 (2008). - ISSN 0006-3495 - p. 1052 - 1062.
cross-correlation spectroscopy - protein-protein interactions - photon-counting histogram - live cells - signal-transduction - imaging microscopy - fusion proteins - plasma-membrane - energy-transfer - living cells
Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.
Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy
Koopman, W.J.H. ; Hink, M.A. ; Verkaart, S. ; Visch, H.J. ; Smeitink, J.A.M. ; Willems, Phgm - \ 2007
Biochimica et Biophysica Acta. B, Bioenergetics 1767 (2007)7. - ISSN 0005-2728 - p. 940 - 947.
correlation microscopy - transmitting cables - liver mitochondria - mammalian-cells - atp production - living cells - dynamics - deficiency - microtubule - transport
We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYTP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When Cl activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility.
In vivo hexamerisation and characterisation of the Arabidopsis thaliana AAA ATPase complex using FRET-FLIM and FCS.
Aker, J.C.M. ; Hesselink, R. ; Engel, R. ; Borst, J.W. ; Visser, A.J.W.G. ; Vries, S.C. de - \ 2007
Plant Physiology 145 (2007)2. - ISSN 0032-0889 - p. 339 - 350.
conformational-changes - living cells - protein - p97 - p97/vcp - receptor - membrane - cycle - phosphorylation - degradation
The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes
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
Spectral characterization of Dictyostelium autofluorescence.
Engel, R. ; Haastert, P.J.M. van; Visser, A.J.W.G. - \ 2006
Microscopy Research and Technique 69 (2006)3. - ISSN 1059-910X - p. 168 - 174.
fluorescence correlation spectroscopy - fluctuation spectroscopy - correlation microscopy - signaling events - fusion proteins - monomeric red - leading-edge - living cells - chemotaxis - dynamics
Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from ~500 to 650 nm with a maximum at ~510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, prebleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium
The Phosducin-Like Protein PhLP1 Is Essential for Gßgamma Dimer Formation in Dictyostelium discoideum
Knol, J. ; Engel, R. ; Blaauw, M. ; Visser, A.J.W.G. ; Haastert, P.J.M. van - \ 2005
Molecular and Cellular Biology 25 (2005)18. - ISSN 0270-7306 - p. 8393 - 8400.
fluorescence correlation spectroscopy - cytosolic chaperonin cct - wd-repeat - crystal-structure - molecular chaperones - subunit interactions - folding machine - fusion proteins - living cells - complex
Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering G beta gamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged G beta (GFP-G beta) and GFP-G gamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-G gamma occurs in wild-type cells but not in phlpI(-) and g beta(-) cells. Moreover, Goy dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-G gamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and g beta(-) cells. Collectively, our data strongly suggest the absence of G beta gamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Go and G gamma into a functional G beta gamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.
Time-resolved emission upon two-photon excitation of bis-N-carbazolyl-distyrylbenzene: mapping of water molecule distribution in the mouse brain
Vaganova, E. ; Yitzchaik, S. ; Sigalov, M. ; Borst, J.W. ; Visser, A.J.W.G. ; Ovadia, H. ; Khodorkovsky, V. - \ 2005
New Journal of Chemistry 29 (2005)8. - ISSN 1144-0546 - p. 1044 - 1048.
lifetime imaging microscopy - resonance energy-transfer - fluorescence-lifetime - living cells - proteins - glucose - spectroscopy - resolution - probes - flim
We present a method of mapping the water molecule distribution in mouse brain tissues using injected bis-N-carbazolyl-distyrylbenzene and the FLIM technique. The fluorescence lifetime of this two-photon absorbing chromophore diminishes when the amount of water in the surrounding area increases. The fluorescence lifetime of the injected in vivo chromophore strongly depends on the content of water in different areas. Thus, lifetimes of 900 ± 50 ps in the hippocampus (extracellular fluid), 520 ± 50 ps in the lateral ventricle (choroid plexus, cerebrospinal fluid), and 400-150 ps in blood vessels were observed. Moreover, the fluorescence lifetime distribution undergoes drastic changes when mice are deprived of water. Statistical analysis of the investigated samples showed that upon water deprivation water content decreased at the border of the hippocampus/lateral ventricle areas and increased in blood vessels
Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins
Visser, N.V. ; Borst, J.W. ; Hink, M.A. ; Hoek, A. van; Visser, A.J.W.G. - \ 2005
Biophysical Chemistry 116 (2005)3. - ISSN 0301-4622 - p. 207 - 212.
anisotropy decays - living cells - dynamics - spectroscopy - lifetimes - single
Visible fluorescent proteins from Aequorea victoria contain next to the fluorophoric group a single tryptophan residue. Both molecules form a single donor-acceptor pair for resonance energy transfer (RET) within the protein. Time-resolved fluorescence experiments using tryptophan excitation have shown that RET is manifested by a distinct growing in of acceptor fluorescence at a rate characteristic for this process. In addition, time-resolved fluorescence anisotropy measurements under the same excitation-emission conditions showed a correlation time that is similar to the time constant of the same RET process with the additional benefit of gaining information on the relative orientation of the corresponding transition dipoles.