Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.     

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio‐temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore‐assisted light inactivation (CALI) of proteins, as well as for photo‐induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini‐antibodies or selective scaffold proteins and photo‐induced cytotoxicity of miniSOG, which is particularly promising for selective non‐invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

     

Engineering an affinity tag for genetically encoded cyclic peptides

Peptide expression libraries are valuable probes of cellular function. SICLOPPS technology merges the principal advantages of both genetic methods and small-molecule approaches in yielding superior library sizes of operationally stable, structurally well-defined entities with an established biological and medicinal record. Here, we describe development, application, and the first-generation library implementation of an expressed affinity tag for a library of cyclic peptides. A tripeptide streptavidin-binding motif (HPQ) proved to be compatible with presentation from a backbone cyclized template. A resulting peptide was employed as a sensitive indicator of peptide splicing, expression, and recovery as well as an affinity tag for one-step purification. Specific recognition of the tag by streptavidin was also critical for an analysis of intein mutants. Finally, the initially identified probe was used as a template for design of a streptavidin-responsive cyclic peptide library.     

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80?K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.     

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells

Background

Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P₂ in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane_. For controlled and reversible depletion of PtdIns(4,5)P₂, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.

Methods and Results

Expression and correct targeting of ECFP-PH-PLCδ1_, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P₂ in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K⁺ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P₂ was identified.

Conclusions

Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.     

Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms

Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

A genetically encoded, fluorescent indicator for cyclic AMP in living cells

Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.     

Convenient one-step purification and immobilization of lipase using a genetically encoded aldehyde tag

To avoid the unwanted and random covalent linkage between the cross-linker and enzyme's active site in covalent immobilization, a genetically encoded “aldehyde tag” was introduced into recombinant lipase and applied for the one-step purification and covalent immobilization of this enzyme. The effects of the immobilization time, temperature and the amount of enzyme were investigated, and the thermo-stability of immobilized lipase was also examined. The specific activity and the k_cat/K_m of the immobilized lipase using aldehyde tag (IL-AT) were 2.50 and 3.02 fold higher, respectively, than those of the traditionally immobilized lipase using glutaraldehyde (IL-GA). The newly immobilized lipase also presented better thermo-stability than the traditionally immobilized one. The results show that the recombinant enzyme could be conveniently immobilized without glutaraldehyde and that the enzyme's active site was well protected. This is a new immobilization method able to avoid glutaraldehyde or 2,4,6-trichloro-1,3,5-triazine as an activating agent. The greener method without hazardous chemicals for the one-step purification and immobilization of an enzyme using a genetically encoded “aldehyde tag” can be exploited for numerous other enzyme purification and immobilization applications.     

Subcellular motility: A correlated light and electron microscopic study using cultured cells

To assess the possible role of filaments in subcellular motility, particular cultured cells were studied by light and electron microscopy. Motile cell margins always contained meshworks of ~50 Å diam. filaments. Organelles moved within cytoplasm occupied by a meshwork of 50–100 Å filaments and microtubules. When cells were treated with cytochalasin B, movements of cell margins stopped, but organelle movements continued; electron microscopically, while subplasmalemmal filaments had disappeared, subcortical filaments and microtubules remained. When cells were treated with hypertonic medium, organelle movements ceased but marginal movements continued; electron microscopically, although cell margins contained normal filament arrays, few subcortical filaments remained. It is concluded that while cell margins are moved by a meshwork of filaments, organelle movement is mediated by a subcortical meshwork of filaments and microtubules.     

Quantitative imaging of intact cells and tissues by multi-dimensional confocal fluorescence microscopy

Confocal fluorescence microscopy enables visualisation and quantitation of fluorescent probes at high resolution deep within intact tissues, with minimal disturbance both of cell–cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix. We illustrate the principles of multiple-parameter 3-D (x,y,z) imaging using reconstruction of nuclear channels in mammalian cells. Repeated sampling in time generates 4-D (x,y,z,t) images which can be used to follow dynamic changes, such as blue-light-dependent chloroplast re-orientation, in intact tissues. Quantitative measurements from multi-dimensional images require calibration of the spatial dimensions of the image and the fluorescence intensity response. This must be determined throughout the volume, which must be sampled to correct for geometric distortion as well as photometric errors arising from the complete optical system, including the specimen. The effects of specimen calibration are illustrated for morphological analysis of stomatal closing responses to abscisic acid in Commelina from 4-D images. Calibrated 4-D imaging allows direct volume measurements and we have followed volume regulation of chondrocytes in cartilage explants during osmotic perturbation. In intact cartilage, unlike in isolated cells, the chondrocytes exhibit volume regulatory mechanisms. In other cases, the fluorescence intensity of the probe may be related to a physiological parameter of interest and changes in its distribution within the cell. Optical sectioning permits discrimination of signal in separate compartments within the cell and can be used to follow transport events between different organelles. We illustrate 3-D (x,y,t) measurements of vacuolar glutathione conjugate pump activity in intact roots of Arabidopsis by following the sequestration of a fluorescent conjugate between glutathione and monochlorobimane. Dynamic measurements of protein localisation are now possible following the introduction of chimeric fusion proteins with green fluorescent protein (GFP) from Aequoria victoria. We have analysed the disposition of heterochromatin in nuclei of living Schizosaccharomyces pombe cells expressing a chimeric construct between Swi6 and GFP. Heterochromatin dynamics can be followed throughout mitosis in 4-D (x,y,z,t) images. Statistical analysis of the fluorescence histograms from each nucleus over time provides quantitative support for aggregation and dispersion of Swi6-GFP clusters during mitosis, rather than dissociation of Swi6 from the heterochromatin. A wide range of single-wavelength and ratio probes are available for imaging different ion activities. We compare 3-D (x,y,t) measurements of ion activities made using single-wavelength (Fluo-3 for calcium) and ratio (BCECF for pH) measurements, using stomatal responses in Vicia faba to peptides from the auxin-binding protein of maize and tip growth in pollen tubes of Lilium longiflorum as examples. Ratioing techniques have many advantages for quantitative fluorescence measurements and we conclude with a discussion of techniques to develop ratioing of single-wavelength probes against alternative references, such as DNA, protein or cell wall material.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Aeroponics for the culture of organisms, tissues and cells

Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P₂ was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P₂ accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP₃ distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.     

A genetically encoded, high-signal-to-noise maltose sensor

We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.     

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy

Summary It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm³ can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50°C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.