Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'- dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.     

Stereo high voltage electron microscopy of melanophores : Matrix transformations during pigment movements and the effects of cold and colchicine

Pigment migration in isolated melanophores of the angelfish, Pterophyllum scalare, has been studied by high voltage electron microscopy. Cells were isolated from the scales by collagenase and allowed to spread on Formvar and carbon-coated gold grids. Melanophores were then fixed by glutaraldehyde and osmium tetroxide and critical-point dried for viewing of whole cells in a high voltage electron microscope (1000 kV). The three-dimensional organization of the cytoplasmic matrix was stereoscopically examined in different states of pigment distribution, as well as under cold and colchicine treatment. The most prominent matrix constituent is an extensive mesh of cytoplasmic filaments (microtrabeculae) 2–18 nm in diameter that make contact to microtubules, pigment granules, and mitochondria. Microtrabeculae undergo dramatic changes in structural appearance in association with different phases of pigment movements. Cells fixed in the process of pigment aggregation are characterized by thickened and beaded trabeculae which may form irregular clots. Part of this material trails behind centripetally moving melanosomes. In dispersing cells, microtrabeculae are straight and of relatively uniform thickness throughout their length and form a highly ordered three-dimensional lattice. Reconstruction of the mesh in part precedes the arrival of pigment granules.Under the influence of cold or colchicine treatment, microtrabeculae show a high degree of polymorphism, being beaded, branched, or flattened with globose ends. Rather formless heaps are found associated with the surface of pigment granules. Since, however, these treatments also remove microtubules, the other important component of the cytoplasmic frame, alterations in microtrabecular structure may simply be mediated through removal of this organelle. In an attempt to separate the effects on microtrabeculae and microtubules from one another, cells have been cold-treated for only 15 min, a procedure that leaves a considerable portion of microtubules intact. Also under these conditions, microtrabeculae are beaded or transformed to globose heaps and flattened sheets.The observations suggest an involvement of microtrabeculae in the process of granule movement. Centripetal melanosome migration thereby seems associated with a collapse of microtrabeculae which again are reconstructed during pigment dispersion. The cold and colchicine experiments indicate direct effects of these agents on the structure and possibly also the function of the trabecular mesh. The significance and possible chemical composition of microtrabeculae is discussed.     

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.     

Reexamination of the reality or artifacts of the microtrabeculae

The polyethylene glycol (PEG) method revealed that model systems such as erythrocytes and protein solutions, which are supposed to lack structured components, exhibit lattice structures not unlike the microtrabeculae. The compactness of the lattice was dependent on the concentration of proteins. The gelated state of gelatin exhibited lattices more compact than those of the solated state at any given concentration. Comparison of images by PEG and rapid-freezing, deep-etching replica methods showed no basic differences in the ultrastructure of the intestinal epithelial cell. This indicates that the PEG method, including chemical fixation, produces little, if any, disorganization of the cytoskeleton. All of the present findings suggest that cytoplasmic protein, nonstructure-bound or structure-forming, might be present in intact cells which could form microtrabecular structures when specimens are fixed by chemical fixatives without any extractions. Therefore, the microtrabeculae should generally be regarded as a simple marker for the presence of proteinaceous macromolecules. It is also suggested that the microtrabecular lattice, as a whole, might represent a gelated state in a given compartment when another, looser lattice is simultaneously present in the same compartment, i.e., within a single cell.     

Whatever happened to the ‘microtrabecular concept’?

Keith Porter culminated his stellar career as the founding father of biological electron microscopy by acquiring, in the late 1970s, a high-voltage electron microscope (HVEM). With this magnificent instrument he examined whole-mounts of cultured cells, and perceived within them a structured cytoplasmic matrix he named the "microtrabecular lattice". Over the next decade Porter published a series of studies, together with a team of outstanding young colleagues, which elaborated his broader "microtrabecular concept." This concept posited that microtrabeculae were real physical entities that represented the fundamental organization the cytoplasm, and that they were the physical basis of cytoplasmic motility and of cell-shape determination. The present review presents Porter's original images of microtrabeculae, after conversion to a more interpretable "digital-anaglyph" form, and discusses the rise and fall of the microtrabecular concept. Further, it explains how the HVEM images of microtrabeculae finally came to be considered as an artifact of the preparative methods Porter used to prepare whole cells for HVEM. Still, Keith's "microtrabecular concept" foretold of our current appreciation of the complexity and pervasiveness of the cytoskeleton, which has now been found by more modern methods of EM to actually be the fundamental organizing principle of the cytoplasmic matrix. During the impending eclipse of Porter's microtrabecular concept in the late 1980s, many of Keith's colleagues fondly described the cell as being filled, not with protoplasm, but with "Porterplasm." Despite the fact that Keith's view was clouded by the methods of his time, it would be fitting and apt to retain this name, still today, for the ordered matrix of cytoskeletal macromolecules that exists in the living cell. In the end, the story of what happened to Porter's microtrabecular concept should be an object lesson in scientific hubris that should humble and inform all of us in cell biology, even today--particularly when we begin to think that our most recent methods and observations are achieving "the last word".     

Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles

The three-dimensional organization and fine structure of cytoplasmic components within whole non-embedded bovine aortic endothelial cells were examined during their attachment and spreading in tissue culture. Cells were cultured directly on Formvar-coated gold grids, fixed in glutaraldehyde and osmium tetroxide, critical point dried and examined by transmission electron microscopy (TEM) using stereoscopic methods, and by scanning electron microscopy (SEM). Reorganization of cytoplasmic structures during cell spreading occurred in four sequential stages: (1) spreading of the plasma membrane and unstructured cytoplasmic matrix; (2) spreading of cytoplasmic fiber systems (microtubules, microfilament bundles and microtrabecular system); (3) alignment of microfilament bundles and formation of radial tracts of microtubules; and (4) centripetal movement of organelles along radial tracts. These stages observed by TEM correlated with progressive degrees of cell flattening as visualized by SEM. These studies demonstrate that a characteristic reorganization of intracellular fiber systems and organelles accompanies the spreading of endothelial cells in culture.     

IN SITU PRESERVATION OF THE TRANSVERSE FLAGELLUM OF PERIDINIUM CINCTUM (DINOPHYCEAE) FOR SCANNING ELECTRON MICROSCOPY1

Three-dimensional structure of the transverse flagellum was studied by means of scanning electron microscopy (SEM) in Peridinium cinctum (O.F.M.) Ehrenberg. Several fixation and dehydration procedures were compared. Cells fixed, rapidly in 1% osmium tetroxide and critical-point dried showed accurate preservation of structural features seen in living cells; in particular, both transverse and posterior flagella were retained in the furrows on the cell surface. Osmium-fixed and freeze-dried, samples were prone to cellular collapse and loss of flagella. Glutar-aldehyde-fixed and critical-point dried cells showed a layer of material covering the thecal plates, most conspicuously over the girdle region; flagella were absent. Stereo-pair micrographs illustrate the 3-dimensional configuration of the transverse flagellum. Modifications of previous structural models are proposed.     

High-voltage electron microscopy of whole,critical-point dried plant cells—fine cytoskeletal elements in the mossBryum tenuisetum

Summary Whole, critical-point dried cells of protonemata of the mossBryum tenuisetum have been examined in the high-voltage electron microscope at 1 MV. Fine cytoskeletal elements form a three-dimensional meshwork in the cytoplasm. This resembles, but has some differences from, the microtrabecular lattice seen in spread animal cells examined by the same technique.     

High voltage electron microscopy studies of axoplasmic transport in neurons: a possible regulatory role for divalent cations

Light and high voltage electron microscopy (HVEM) procedures have been employed to examine the processes regulating saltatory motion in neurons. Light microscope studies demonstrate that organelle transport occurs by rapid bidirectional saltations along linear pathways in cultured neuroblastoma cells. HVEM stereo images of axons reveal that microtubules (Mts) and organelles are suspended in a continuous latticework of fine microtrabecular filaments and that the Mts and lattice constitute a basic cytoskeletal structure mediating the motion of particles along axons. We propose that particle transport depends on dynamic properties of nonstatic microtrabecular lattice components. EXperiments were initiated to determine the effects of changes in divalent cation concentrations (Ca2+ and Mg2+) on: (a)the continuation of transport and (b) the corresponding structural properties of the microtrabecular lattice. We discovered that transport continues or is stimulated to a limited extent in cells exposed to small amounts of exogenously supplied Ca2+ and Mg2+ ions (less than 0.1 mM). Exposure of neurons to increased dosages of Ca2+ and Mg2+ (0.2-1.0 mM) stimulates transport for 2-4 min at 37 degrees C, but after a 5- to 20-min exposure the saltatory movements of organelles are observed gradually to become shorter in duration and rate particle motion ceases to occur. HVEM observations demonstrated that Ca2+ - and with the cessation of motion. Ca2+-containing solutions produced contractions of the microtrabecular filaments, whereas Mg2+-containing solutions had the opposing effect of stimulating an elongation and assembly (expansion) of microtrabeculae. On the basis of these observations we hypothesize that cycles of Ca2+/Mg2+-coupled contractions and expansions of the microtrabecular lattice probably regulate organelle motion in nerve cells.     

Scanning electron microscopical comparisons of insect virus occlusion bodies prepared by several techniques

Samples of baculoviruses, a cytoplasmic polyhedrosis virus, and an entomopoxvirus were prepared by several techniques for study in the scanning electron microscope. The techniques which gave satisfactory definition of surface ultrastructure were: (1) double fixation in glutaraldehyde and osmium tetroxide with critical point drying; and (2) double fixation as in (1) with a further postfixation by ligand-mediated osmium binding using thiocarbohydrazide as the ligand (OTO method) followed by critical point drying. The latter technique was superior to the former technique. Air drying of samples gave acceptable but inferior definition when compared with critical point-dried samples prepared by these techniques.     

The cytoplast: A unit structure in chromatophores

We followed the translocation of identifiable pigment granules in living erythrophores through normal aggregation and dispersion and observed that they always return in dispersion to the same location relative to the whole pigment complex. This is interpreted to mean that each granule occupies a fixed position within a unit structure, the cytoplast. This position is retained even though the cytoplast undergoes dramatic reversals in form from ellipsoid to spheroid and back again with each aggregation and dispersion. The major structural components of the cytoplast, besides pigment granules, are microtubules and microtrabeculae. The latter constitute an irregular lattice that is confluent with microtubules and contains the pigment granules. In aggregation, the microtrabeculae shorten and seemingly contribute to the contraction of the entire cytoplast plus pigment. In dispersion, the microtrabeculae elongate in an apparent restructuring of the ellipsoidal cytoplast. The microtubules, however, persist in the cell cortex and appear to give radial direction to the pigment motion.     

High voltage electron microscopy of cytoskeletal structures in whole plant cells

During the past decade, work on whole, critical-point dried animal cells has revealed a three-dimensional meshwork, the microtrabecular lattice or cytomatrix, which pervades the ground cytoplasm. This work was carried out on cells which could be spread out into thin layers on support films. Plant cells provide a more difficult problem since their rigid cell walls do not allow them to be spread into thin layers. Nevertheless high-voltage electron microscopy at up to 2.5 MeV permits examination of whole cells up to 30 μm thick, though both preparation and interpretation present problems. In algal cells flagellar roots and associated structures can be seen in three dimensions, while cells of mosses, ferns and lycopods show a cytomatrix of fine interconnecting filaments.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

A method for the chemical treatment of cells for the purpose of the subsequent study of the same cell culture preparation by light, scanning and transmission electron microscopy]

Cells cultured on transparent conductive substrates (glass coated with indium oxide) were fixed with aldehyde and osmium tetroxide and then treated with tannic acid, uranyl acetate and lead citrate. The same cell culture preparation could be sequentially studied by light microscopy (in water immersed condition), SEM (after dehydration and critical point drying) and TEM (after embedding in an epoxy resin). This method ensures the preservation of intact cell morphology, cell surface topography and intracellular structures. The treatments used render the cells conductivity and permit to carry out successfully SEM of uncoated cells cultured on conductive substrates. This method also provides a higher contrast of TEM images.     

The ultrastructure of Candida krusei

Various methods of chemical fixation and freeze-drying of Candida krusei were compared to determine the most appropriate method for the ultrastructural investigation of the thick walled organisms of this genus. Freeze-drying without chemical fixation was of little value because of insufficient variation in electron density. Potassium permanganate was able to penetrate the intact cell but failed to show cytoplasmic glycogen and lipid and some details of the cell wall. While normal glutaraldehyde, formaldehyde and osmium tetroxide treatment failed to permeate and preserve intracellular structures, several cycles of rapid freezing (–155°C) and thawing followed by glutaraldehyde fixation and osmium tetroxide post-fixation demonstrated the intracellular details of the majority of cells so treated.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe

In this study, fixation of attached glycocalyx on the interior surfaces of polyvinyl chloride distribution pipe remnants was compared with and without ruthenium red/osmium tetroxide and, in the final preparatory phase, with chemical dehydration and critical point drying. SEM examination of interior surface of the polyvinyl chloride pipe showed varying concentrations of adherent bacteria, depending on the preparatory technique used. It was concluded that using a combination of ruthenium red/osmium tetroxide and critical point drying is the optimum method for visually demonstrating aging biofilm on the interior surface of contaminated polyvinyl chloride pipe.     

ON THE FINE STRUCTURE AND COMPOSITION OF THE NUCLEAR ENVELOPE

Nuclei from nearly ripe eggs of Rana pipiens were isolated and cleaned in 0.1 M KCl. The whole nucleus was then digested to various degrees with ribonuclease or trypsin, followed by washing and fixation in either osmium tetroxide or potassium permanganate. The nuclear envelope was dissected off, placed on a grid, air dried, and compared with undigested controls in the electron microscope. Some envelopes were dehydrated, embedded in methacrylate, and sectioned. Annuli around "pores" are composed of a substance or substances, at least partially fibrillar, which is preserved by osmium but lost during permanganate fixation. Material within the "pores" is also preserved by osmium but partially lost after permanganate. No evidence of granules or tubules in the annuli was found in air dried mounts although a granular appearance could be seen in tangentially oriented thin sections. Thin sections of isolated envelopes give evidence of diffuse material within the "pores" as well as a more condensed diaphragm across their waists. In whole mounts of the envelope the total density within "pores" is relatively constant from "pore" to "pore." All material within "pores," including the condensed diaphragm, is removable by trypsin digestion. Wispy material from the "pore" structure projects into the nucleus and annular material extends into the cytoplasm. Both annular and diaphragm materials remain with the envelope when it is isolated and are thus considered a part of its structure, not merely evidences of material passing through. There is no evidence of ribonuclease-removable material in any part of the "pore" complex.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.