Localization of protein-protein interactions in live cells using confocal and spectral imaging FRET microscopy

Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy. The specific aims of the investigation of protein dynamics in live specimens dictate the selection of the microscope methodology. In this article confocal and spectral imaging methods to monitor the dimerization of alpha enhancer binding protein (C/EBPalpha) in the pituitary GHFT1-5 living cell nucleus have been described. Also outline are issues involved in protein imaging using light microscopy techniques and the advantages of lifetime imaging of protein-protein interactions.     

An improved no-moving-parts video-rate confocal microscope

Several years ago our research program developed a video-rate confocal microscope with no moving parts, based on synchronizing and aligning the scan of an image dissector tube (IDT) with the light returning from a microscope stage that has been acousto-optically scanned by a laser beam. Improvements on the original system have recently been completed. The laser power has been substantially increased and the laser scan is now brought into the Nikon Diaphot inverted microscope through the epi-illumination port. Aberrations in the scanned beam have been reduced by performing the beam shaping required by the acousto-optic deflectors using prisms instead of cylindrical lenses. The IDT is located at the side camera output port at the end of a simple, efficient light path. The new system is described in detail and results obtained using the microscope in reflection and fluorescence mode are presented.     

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.     

An improved cyan fluorescent protein variant useful for FRET

Many genetically encoded biosensors use F?rster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy

The crystallographic structures of functional fragments of ErbBs have provided excellent insights into the geometry of growth factor binding and receptor dimerization. By placing together receptor fragments to build structural models of entire receptors, we expect to understand how these enzymes are allosterically regulated; however, several predictions from these models are inconsistent with experimental evidence from cells. The opening of this gap underlines the need to investigate intact ErbBs by combining cellular and structural studies into a full picture.     

Dynamics of the formation of secondary reproductive structures in Streptomyces zone-forming colonies as determined from confocal microscopy data

The dynamics of the formation of secondary structures on the colony surface, so-called air mycelium concentric rings, was investigated by confocal microscopy and using Streptomyces zone-forming colonies as a model of a self-organizing system. The occurrence of luminescence in reproductive structures was registered, whereas in vegetative structures it was almost completely absent. It was shown that secondary reproductive structures begin to form within the thickness of substratum mycelium as branches or as an apical extension of vegetative gyphes, followed by the formation of numerous secondary branches filled with protospores.     

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.     

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.     

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

Summary The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.     

Bacterial community structure and colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy

) grown in natural forest soil in southern Germany was examined by fluorescence in situ hybridization (FISH) using fluorescent oligonucleotide probes, targeting phylogenetic relevant sequences of the 16S and 23S rRNA. Lactarius subdulcis, L. vellereus, L. rubrocinctus and Laccaria amethystina were found to be the prevalent fungi forming ectomycorrhizae with F. sylvatica. For FISH studies using confocal laser scanning microscopy, oligonucleotide probes labeled with carboxymethylindocyanine-succinimidyl ester allowed detection of associated bacteria, because the autofluorescence of ectomycorrhiza samples could be overcome in the infrared. Bacteria of the α-, β and γ-subclasses of the proteobacteriawere detected in high numbers on mantle surfaces, while members of other phylogenetically defined groups were found in smaller numbers. This contrasts with previous published results on the cultivation of mycorrhiza-associated bacteria. Hybridizing bacteria were also found within damaged cells of the hyphal mantle of L. rubrocinctus, as well as on emanating hyphae of L. amethystina. Using a newly developed extraction protocol for bacteria associated with ectomycorhizas, the two most common fungi on F. sylvatica, L. vellereus and L. subdulcis, were mostly associated with members of the α- and β-subclasses of the proteobacteria. The proportion of hybridizing bacteria varied between the two ectomycorrhizae, which were thus host to distinct populations of bacteria.     

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.     

Chromosome structure: improved immunolabeling for electron microscopy

To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIα (topoIIα) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIα and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIα and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIα tether chromatin to loops according to a scaffolding-type model.     

Three-dimensional structure of living chloroplasts as visualized by confocal scanning laser microscopy

Summary The newly developed confocal scanning laser microscope, together with image processing by computer, has been used to obtain three-dimensional information on the organization of grana in chloroplasts in living plant tissue. Chloroplasts are ideally suited for such studies because their pigments show bright autofluorescence. The high-resolution stereo images bridge a gap between classic light microscopy and electron microscopy. Our preliminary observations on several plant species resemble most the early observations of Strugger (1951: Die Strukturordnung im Chloroplasten. Ber Deutsch Bot Ges 64: 69–83) and suggest that the 3-D technique might well be suitable to solve discrepancies in the interpretation of classical light microscopic and electron microscopic observations.Abbreviations 3-D three dimensional - CSLM confocal scanning laser microscopy - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNA deoxyribonucleic acid     

Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy

In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 microm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.     

Imaging protein molecules using FRET and FLIM microscopy

F?rster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses. The widening application of FRET and FLIM has been driven by the availability of suitable fluorophores, increasingly sophisticated microscopy systems, methodologies to correct spectral bleed-through, and the ease with which FRET can be combined with other techniques. FRET and FLIM have recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity (e.g. clustering), in the study of conformational changes, in the analysis of binding sequences, and in applications such as high-throughput screening.     

3D structure of Syk kinase determined by single-particle electron microscopy

The cytoplasmic Syk kinase plays key roles in immune responses and comprises two N-terminal regulatory Src homology 2 (SH2) domains followed by a catalytic region. Atomic structures of these domains have only been solved in isolation. To gain insights into the three-dimensional structure of full-length Syk, we have used single-particle electron microscopy. Syk acquires a closed conformation resembling the inhibited structure of Zap-70, another member of the Syk family. Such configuration suggests an inhibition of the N-terminal domains on its catalytic activity. The phosphotyrosine binding pockets of both SH2 domains are not occluded and they could interact with other phosphoproteins.     

The first three-dimensional structure of phosphofructokinase from Saccharomyces cerevisiae determined by electron microscopy of single particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4alpha and 4beta subunits arranged in a dihedral point group symmetry D(2). The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0 degrees tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.