Embedment-free Electron Microscopy Research Articles

The cytoplasmic matrix of the adrenal chromaffin cells of rats under normal and stressed conditions.

In embedment-free electron microscopy with polyethylene glycol embedding and subsequent deembedding, the conventional cytoplasm of the chromaffin cells was revealed to consist of a three-dimensional lattice of microtrabeculae and gives the impression that the chromaffin granules are held in place by the lattice. After the restraint stress, a substantial number of chromaffin cells were almost free of granules, and the microtrabecular lattice was much more compact than that in cytoplasmic regions occupied with remaining granules or increased mitochondria. In immunocytochemistry, actin immunofluorescence was confined to the subplasmalemmal regions, while tubulin and tropomyosin immunofluorescence appeared throughout the entire cytoplasm of normal chromaffin cells. After the stress, the immunofluorescence for actin and tubulin increased in intensity, while that for tropomyosin decreased. Immunogold labelings for actin and tubulin were found mainly on the thinner subplasmalemmal microtrabeculae and the thicker perikaryal ones, respectively, while some were deposited in the form of small aggregates on portions of microtrabeculae. No specific association between the gold labelings for actin or tubulin and the chromaffin granules was found, even in the subplasmalemmal regions. A hypothetical interpretation was proposed in which a more compact lattice of the microtrabeculae in spatial association with a looser lattice represents a gelated state of the cytoplasm. The significance of the gel-sol transition of the cytoplasmic matrix in relation to the secretory mechanism was discussed.

Specialized cytoskeletal elements in mammalian eggs: Structural and biochemical evidence for their composition

Mammalian eggs and embryos contain an extensive detergent-resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet-like components by using embedment-free sections and freeze-fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS-polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trophectoderm.

THE ASSOCIATION OF VACCINIA VIRUS WITH INTERMEDIATE FILAMENT

The intermediate filament-lamina-nuclear matrix system (IF-lamina-Nm System) can be shown clearlyusing selective extraction, embedment-free electron microscopy and whole mount cell preparation. A del-icate intermediate filament network was demonstrated in vaccinia virus factory for the first time. It isa part of the cytoplasmic intermediate filament network. The components and substructures of vacciniavirus bind firmly to intermediate filaments. Both assembled and assembling vaccinia viruses are fixedin the intermediate filament network. Direct connections between viruses and intermediate filaments canbe observed. We propose that vaccinia virus assembly depends on intermediate filaments as support.

Resinless section electron microscopy of HeLa cell mitotic architecture.

The use of resinless sections extends embedment-free electron microscopy to the cytoskeleton of thick specimens. Here we examine HeLa cells rounded at mitosis. Extraction of mitotic HeLa cells with Triton X-100 removes lipids and soluble proteins, leaving the cytoskeletal framework and spindle apparatus. After fixation, the samples are embedded and sectioned, and the temporary embedding resin is removed for direct visualization in the electron microscope. The micrographs show that the cytoskeletal framework, chromosomes, spindle, and centrioles form an interconnected entity. The pericentriolar region, indistinct in conventional micrographs, appears composed of distinct fibers interconnecting the spindle microtubules and centriole. The resinless sections also reveal characteristic lacunae at late anaphase/early telophase. These probably result from reformation of the interphase cytoskeleton lagging reassembly of the nucleus.

New views of cell and tissue cytoarchitecture: embedment-free electron microscopy and biochemical analysis.

Our perception of the cell interior is undergoing a fundamental change. What once appeared to be formless plasms are now seen to contain highly organized structural scaffolds. Even the soluble proteins, those not a part of skeletal structures, are no longer considered free, but associated with the skeletal framework. The implications of these studies of cell structure are beginning to emerge. The end result will likely be a change in many of the paradigms of our science. As with most changes in paradigm, progress is halting and not without conflict.