Embedment-free Electron Microscopy Research Articles

A cytoplasmic gel network capable of mediating the conversion of chemical energy to mechanical work in diverse cell processes: A speculation

Enigmatic morphological features of the formation and fate of "dark" (hyper-basophilic, hyper-argyrophilic and hyper-electrondense) neurons suggest that the mechanical work causing their dramatic shrinkage (whole-cell ultrastructural compaction) is done by a previously "unknown" ultrastructural component residing in the spaces between their "known" (i.e. visible in the conventional transmission electron microscopy) ultrastructural constituents. Embedment-free section electron microscopy revealed in these spaces the existence of a continuous network of gel microdomains, which is embedded in a continuous network of fluid-filled lacunae. We gathered experimental facts suggesting that this gel network is capable of a volume-reducing phase-transition (an established physico-chemical phenomenon), which could be the motor of the whole-cell ultrastructural compaction. The present paper revisits our relevant observations and speculates how such a continuous whole-cell gel network can do both whole-cell and compartmentalized mechanical work.

What we have learned and will learn from cell ultrastructure in embedment‐free section electron microscopy

The limitations inherent in conventional electron microscopy (EM) using epoxy ultrathin sections for a clear recognition of biological entities having electron densities similar to or lower than that of epoxy resin have led to the development of embedment-free sectioning for EM. Embedment-free section EM is reliably performed using water-soluble polyethylene glycol (PEG) as a transient embedding medium, with subsequent de-embedment of PEG by immersion into water, followed by critical point-drying (CPD) of the embedment-free section. The present author has stressed that this approach clearly discloses structures whose contours and/or appearance are accordingly vague and/or fuzzy in conventional EM, but does not reveal any new structures. Based on embedment-free electron microscopy (PEG-EM), this article presents five major findings regarding strand- or microtrabecular lattices which have been clearly revealed to occur in the cytoplasmic matrix-an impossibility with conventional EM. These are (1) the appearance of lattices of different compactness in various cells and in intracellular domains of a given cell; (2) the faithful reproduction from an albumin solution in vitro of strand-lattices with correspondingly increasing compactness following increasing concentrations; (3) the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro; (4) the appearance of either greater or less lattice-compactness by hyper- or hypotonic pretreatments of cells; and (5) the appearance of certain intracellular proteins confined to the centripetal demilune-domain of centrifuged ganglion cells which is occupied with strand-lattices of a substantial compactness. From these findings, questions now arise as to the biological significance of the individual strand itself in the microtrabecular lattices in PEG-EM. In addition, it may be that the appearance of strand-lattices in a given biological domain represents the presence of soluble proteins; the lattice-compactness indicates the concentration of soluble proteins in the domain, and the aqueous cytoplasm is equivalent to the aqueous solution. Further, the appearance of two contiguous lattice domains exhibiting differing degrees of compactness in a given cell indicates that cytoplasmic proteins are solated in a domain with less compact lattices, whereas they are gelated in the other domain. These proposed interpretations need to be confirmed by further studies. If confirmed, the control mechanisms of the localization and movement of intracellular organelles could then be understood on the basis not only of information about the cytoskeletons but also of cell ultrastructure-related information on the concentration and sol-gel states of intracellular proteins. In addition, possible interpretations of the significance of strand-lattices in PEG-EM are also applicable to the nucleoplasm, especially extra-heterochromatin (euchromatin) areas. Finally, several potential uses/advantages of PEG-EM in the cell-ultrastructure have also been demonstrated, especially in three-dimensional reconstructions of nonmembranous structures including stereo-viewing using a pair of EM images with appropriate tilting as well as electron microscopic tomography.

Spatial Distribution of Actin and Tubulin in Human Sperm Nuclear Matrix‐intermediate Filament Whole Mounts—A New Paradigm

Sperm is a highly differentiated cell streamlined for fertilization. The function is thus heavily dependent on the cytoskeletal organization. Conventional methods limit the appreciation and correlation of this intricate cytoskeletal filament network in the context of an entire sperm. Our recent successful localization of nonmuscle myosin IIA on sperm nuclear matrix-intermediate filament (NM-IF) preparations from fertile men by embedment-free electron microscopy (EF-EM), prompted us to investigate the antigenic distribution of two major cytoskeletal proteins-actin and tubulin. The NM-IF preparations were subjected to a cocktail of buffered paraformaldehyde (2%) with a low concentration of glutaraldehyde (0.05%). These proteins were localized by indirect immunogold technique using EF-EM on sperm NM-IF whole mounts. Ultrastructure analysis revealed well preserved centrioles, outer dense fibers, axonemal filaments, and submitochondrial reticulum in the sperm NM-IF. Immunoreactive actin was localized along the length of the sperm whereas beta-tubulin was present in the axoneme alone. The spatial distribution of actin and tubulin in normal human sperm NM-IF reported here together with that of myosin on whole mount offers a powerful technique to understand sperm cytoskeletal supramolecular structure.

Embedment-free section electron microscopy

Because of potential hindrance of clear viewing in epoxy sections of biological entities having an electron density similar to and lower than that of epoxy resin, the author has stressed that the embedment-free section electron microscopy is necessary for re-examination and/or clarification of biological specimen structures, and that the embedment-free electron microscopy is reliably done by using water-soluble polyethylene glycol (PEG) as a transient embedding media and by critical point-drying of embedment-free sections after de-embedment of PEG by immersion of semithin sections into water. With the embedment-free electron microscopy, the author has presented five major findings: the appearance of microtrabecular lattices with different compactnesses in various cells and in intracellular domains of a given cell, the faithful reproduction of microtrabecula-like strand lattices in vitro with increasing compactnesses from artificial protein solutions at correspondingly increasing concentrations, the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro, the changeability in compactnesses of the microtrabecular lattices by hyper- or hypotonic shock treatments of cells, and the confined appearance of an intracellular protein in the centripetal demilune of centrifuged ganglion cells which is occupied with the microtrabecular lattices of a substantial compactness. From these findings, several conclusions are drawn: individual strands themselves of the microtrabeculae are meaningless, the appearance of microtrabeculae represents the presence of proteins at a certain concentration, and it is therefore likely that the aqueous cytoplasm is equivalent to the aqueous solution. In addition, it is possible that the appearance of two contiguous lattice domains exhibiting different compactnesses in a given cell may represent the occurrence of a contiguity of sol to gel states of cytoplasmic domains. It is thus proposed that the localization and movement of intracellular organelles are controlled not only by the cytoskeletons but also by the concentration and sol/gel states of intracellular proteins. In addition, several potential usefulness of the embedment-free electron microscopy has also been demonstrated.

Embedment-free electron microscopy in cell and tissue structure

Embedment-free electron microscopy in cell and tissue structure

A new look at the cellular scaffold by embedment‐free electron microscopy method

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.

Cytoplasmic matrix in embedment-free electron microscopy: non-molecular biological histology.

Embedment-free electron microscopy using polyethylene glycol as a transient embedment has revealed that slender strands, originally termed microtrabeculae and microtrabecular lattices, interconnect every organelle and conventional cytoskeletons as well as plasma membranes, resulting in the formation of 3-D meshworks in all portions of the cytoplasmic matrix of every cell. The microtrabeculae correspond well to the wispy components in the cytoplasmic matrix of conventionally epoxy-sectioned cell specimens that have been looked at but often neglected because of their poorly defined images due to the presence of embedding media having a substantial electron-scattering property. Because of the occurrence of similar meshworks in specimens that are supposed to be unstructured, such as the intramitochondrial matrix and blood plasma, together with the failure to detect any predictable changes of the microtrabecular lattices by experimental manipulation of cellular environments, it is inaccurate to conclude that all microtrabecular lattice represent structures equivalent to those in a living state of cells simply because of their clear appearance. Instead, three possible interpretations are newly proposed for the biological significance of the microtrabecular lattices. The first is that the appearance of lattices represents the presence of proteins, and that their approximate concentrations are speculated based on the compactness of the lattice. The second is that when an intracellular microdomain composed of more compact lattices is contiguous with another domain composed of looser lattices in a given cell, the former might represent the gelated state and the latter the solated state. Possible examples for these two interpretations are also proposed, possibly leading us to further elaborate the significance of microtrabecular lattices.

A novel embedment-free immunoelectron microscopy technique reveals association of apoptosis-regulating proteins with subcellular structures.

By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8.

The arrangement of microtrabecular network during chromatin migration process and immunolocalization of actin and myosin in pollen mother cells of David Lily

Abstract The pollen mother cells of David lily at synizesis of meiotic prophase I were studied using the light microscope and diethylene glycol distearate (DGD) embedment-free electron microscope. The results showed that there existed clear areas in front of and behind the chromatin masses undergoing migration via cytoplasmic channels (cytomixis) under light microscope. Observations of diethylene glycol distearate embedment free electron microscopy indicated that these clear areas were actually filled with microtrabecular components. Several major arrangement changes of microtrabecular network were observed as follows: When the nucleus moved aside from the center, an area filled with cytoplasmic microtrabecular network appeared behind. Later, when chromatin underwent cytomixis via cytoplasmic channels, another area filled with nuclear microtrabecular network appeared between nuclear envelope and the rear condensed chromatin, and some thick microtrabecular filaments linked the migrating chromatin masses in...

EXPRESSION OF BAX IN CELL NUCLEUS AFTER EXPERIMENTALLY INDUCED APOPTOSIS REVEALED BY IMMUNOGOLD AND EMBEDMENT-FREE ELECTRON MICROSCOPY

The unique combination of immunocytochemistry with embedment-free electron microscopy was applied for precise and specific localisation of BAX in the human colon adenocarcinoma COLO 205 cell line stimulated to undergo apoptosis by camptothecin (DNA topoisomerase I inhibitor). Camptothecin-induced apoptosis was associated with redistribution of BAX from cytosol to organelle membranes: mitochondria, Golgi apparatus, endoplasmic reticulum and via nuclear envelope pores to the nucleus, occurring within 60–180min of cell exposure to the drug. An increase in BAX immunoreactivity on fine filaments and the lamina-pore complex of the nuclear matrix was also observed. The increase in BAX expression in the nuclear area of camptothecin-treated COLO 205 cells was confirmed by quantitative analysis using laser scanning cytometry. The subcellular translocations of BAX preceded the appearance of any morphological symptoms of apoptosis.

Changes in the ultrastructure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation.

The cellular scaffold that comprises nuclear matrix and cytoskeleton provides mechanical support for the cell and plays a crucial role in motility, cellular signaling, regulation of gene transcription and DNA replication. In this study we examined the structure of cytoskeleton and nuclear matrix in the keratinocyte cell line HaCaT using a recently developed technique, embedment-free electron microscopy. With this method the three-dimensional structure of cellular scaffold is visualized in the cells extracted from soluble proteins and the chromatin. In actively proliferating cells the cytoskeleton appeared to consist of a continuous meshwork of 10--15 nm filaments with a smaller amount of thin (5 nm) and ultrathin (1--2 nm) filaments. In contrast to what could be expected from earlier immunofluorescence and electron microscopy studies, the cytoskeleton in HaCaT keratinocytes did not consist of superposed autonomous networks of different filaments but was a highly integrated, continuous structure filling whole cytoplasmic territory. Moreover, cytoskeletons of adjacent cells were in a direct physical contact. Nuclear matrix consisted of globular ribonucleoprotein aggregates attached to the meshwork of 20--40 nm filaments. Nuclear envelope was firmly fastened to the cytoskeleton. In keratinocytes induced to differentiation by calcium switch both the cytoskeleton and nuclear matrix were drastically rearranged and comprised a monomorphic, dense and regular meshwork of 10--15 nm filaments. Our data underscore the fact that in HaCaT keratinocyte monolayer in vitro, and probably also in the epidermis in vivo, the nuclear matrices and the cytoskeletons of adjacent cells seemed to form a continuous, highly ordered structure which is rapidly rearranged during cell differentiation. This feature may be crucial for the understanding of how the signal initiated by, e.g. mechanical forces generated through the cell--cell and cell--matrix interaction is transmitted to the nucleus producing ultimately changes in the pattern of gene expression.

STRUCTURAL ASSOCIATION OF BAX WITH NUCLEAR MATRIX AND CYTOMATRIX REVEALED BY EMBEDMENT-FREE IMMUNOGOLD ELECTRON MICROSCOPY

Bax is a cellular protein functioning as a promoter of apoptosis. It is ultrastructurally associated with mitochondrial membranes, where it participates in permeability transition pore formation. By employing embedment-free electron microscopy (EFEM), we present evidence that Bax is also associated with the nuclear matrix and cytomatrix of cultured human tumour cells (COLO 205, PA-1, U-373 MG). Extracted cellular scaffolds were probed with anti-Bax antibody using the immunogold electron microscopy technique. Bax immunoreactivity was found on 10–15nm intermediate filaments of karyo- and cytoskeleton, stretched between the nucleus, nuclear lamina and cell periphery. Bax immunoreactivity was preferentially localized to certain areas of filaments (spot-like). The target molecules for Bax binding in the cellular matrix and their physiological significance remain to be established.

Structure of cytomatrix and nuclear matrix revealed by embedment-free electron microscopy

Embedment-free electron microscopy (EFEM) is a new method which allows the visualisation of cytoskeleton in whole-mounted cells. In this study we employed EFEM to investigate the structure of cellular scaffolds in glioma C6 cell line. The cells were extracted with Triton X-100 that dissolves phospholipids in the membranes and removes most of cytoplasmic soluble proteins. The DNA and nuclear histones were removed with DNase I and high-salt buffer, respectively. The remaining cellular frameworks were temporary embedded in diethylene glycol distearate (DGD), sectioned and observed in transmission and scanning electron microscope after the removal of DGD. The predominant structure was the extensive meshwork of 10-20 nm filaments in the cytoplasm (cytomatrix) and 15-30 nm filaments in the nucleus (nuclear matrix). The 5 nm filaments, presumably corresponding to the actin filaments, were present in the cytomatrix, but not in the nuclear matrix. Moreover, the ultrathin (3 nm) filaments, connecting other cytoskeletal components were detected. Those are possibly identical with the previously described plectin filaments. For the first time we report the occurrence of ultrathin filaments in the nuclear matrix. Thus, in a addition to the well known cytoskeletal components (microtubules, intermediate filaments, actin microfilaments) EFEM showed a new type of filaments (the ultrathin filaments) in the cytomatrix and nuclear matrix. Further immunocytochemical studies are needed to determine the biochemical identity of the filaments observed in EFEM.

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracilis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix.

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with Mrs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the α-IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.

The nuclear lamina in male generative cells ofGinkgo biloba

Using selective extraction and diethylene glycol distearate embedment and embedment-free electron microscopy, we demonstrated nuclear lamina-like structures in sperm cells ofGinkgo biloba. A well-organized nuclear matrix network was also observed. Further studies were undertaken to determine whether or not lamin-like components exist in the pollen and sperm cells. Immunofluorescence staining using monoclonal antibodies against different animal lamins revealed lamins localized in the nuclear compartment of the sperm cells. Western blotting showed that in pollen grains there are two positive crossreaction bands at 66 kDa and 86 kDa, recognized by antibodies specific to animal lamins; in sperm cells there was only one, at 66 kDa. These results indicate that nuclear lamina containing both A-type and B-type lamins was present in male generative cells ofG. biloba. The data imply that plant lamins share some homology with animal lamins and may be conserved during evolution.

Rethinking cell structure.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.

On the real structure of the cytoplasmic matrix: learning from the embedment-free electron microscopy.

The recent debate on the nature of the cytoplasmic matrix is reviewed on the basis of results obtained by electron microscopy of embedment-free materials, i.e., the critical point-dried whole mount-cell method, the polyethylene glycol (PEG)-embedding and subsequent de-embedding section method, and the freeze-etching method. Fine structural images obtained by these methods are carefully evaluated and close correspondence with electron microscopy regardless of these methods is demonstrated. Especially, 'novel' filamentous structures--the microtrabecular strands and the cross-linkers--correspond well to each other; they are structures which have been included in epoxy sections by conventional methods, but have been obscured simply because of a similar property of electron scattering between the filamentous structures and epoxy resin. Although this correspondence seems to support the existence of the microtrabeculae, the electron microscopy of serum albumin, when processed by the PEG-method, also exhibits filamentous networks resembling the microtrabecular lattice. This, together with the finding on the centrifugation of in situ cells, strongly suggests a possibility that most, if not all, microtrabecular strands and cross-linkers in cells without pretreatment by detergents do not represent actual in situ structures.

Ultrastructural filament organization of hepatocyte cytoplasm revealed by embedment-free sections of rat liver.

The cytoplasmic matrix of rat liver cells was examined by embedment-free electron microscopy of polyethylene glycol (PEG) embedded tissue from which the PEG had been removed subsequently. Non-ionic detergent extraction of fresh blocks of tissue permitted the major cytoskeletal components to be visualized in relatively thick embedment-free sections. Immunolabelling confirmed the presence of the major cytoskeletal proteins (actin, cytokeratin and tubulin) in these sections. Hence, this method permits the examination of the ultrastructural organization of the cytoskeleton of the hepatocyte in blocks of fresh hepatic tissue.

Imaging cytoskeleton--mitochondrial membrane attachments by embedment-free electron microscopy of saponin-extracted cells.

Embedment-free electron microscopy images the cytoskeleton and nuclear matrix, which are very difficult to visualize in conventional electron micrographs. However, to be effective, cell structures must be depleted of soluble proteins, which otherwise shroud cell architecture. Nonionic detergents effect this extraction, releasing soluble proteins but also destroying all membranes. Saponin can permeabilize plasma membranes, releasing soluble proteins while preserving many cytoplasmic membranes. Stereoscopic electron microscopy of resinless sections shows the many connections of the cytoskeleton to mitochondrial membranes.