Microtrabecular Lattice Research Articles

Polyethylene glycol (PEG) embedding and subsequent de‐embedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy

AbstractA simple and reliable method to make resinless sections for electron microscopy was recently developed by using polyethylene glycol (PEG) as a transient embedding media. In this paper the practical procedure of this PEG method is described in detail. Normal ultrastructure of several types of in‐situ cells in resinless sections is demonstrated. The cytoplasmic matrix of all in‐situ cells examined is revealed to consist of the microtrabecular lattice. A result from application of this technique to immuno‐electron microscopy is also illustrated. This method is shown to have potential in overcoming the problem of intracellular penetration of macromolecular antibodies. Several artifacts caused by failures in specimen preparations are displayed. The real or artifactual nature of the microtrabecula is briefly discussed.

Observations préliminaires sur l'organisation du cytosquelette des cellules végétales. Mise en évidence d'un “réseau microtrabéculaire”

Electron microscopic observations of particularly favourable sections of pericycle and vascular parenchyma cells fixed with glutaraldéhyde have revealed the existence of a network of exceedingly fine filaments in the cytoplasmic ground substance of plant cells. This highly intricated structure to which polysomes seem to be attached interconnects the cytoskeletal fibers and the various other cellular components. It corresponds evidently to the "microtrabecular lattice" described in animal cells. Moreover, this report points out that microfilaments, which are 6- to 8-nm putative actin filaments, contract with microtubules or sheets of agranular reticulum, to form privileged close relationships whose functional significance is discussed.

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

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.

Electron microscopy of resin-free sections of plant cells

A technique for producing resin-free sections of plant roots for observation with the high voltage electron microscope is described. The resulting images have high contrast and reveal a three-dimensional filamentous network in the cytoplasm and nucleoplasm. This network is briefly compared with the microtrabecular lattice found in animal cells.

Evidence for the possible presence of a microtrabecular lattice in plant cells

Tangential longitudinal sections of the differentiating xylem of species ofBrugmansia, Eucalyptus andPinus and the basal meristem of leaves ofSansevieria were examined after conventional fixation and embedding procedures, followed by removal of the embedding medium, and shadow-casting with platinum-palladium alloy. In the images obtained, in addition to microtubules and in some instances, other identifiable organelles, a fibrous or tubular reticulum could be identified which resembled the microtrabecular system previously reported as present in animal cells.

Evidence that cytoplasmic birefringence is a normal feature of rat adrenal chromaffin cells

The rat adrenal medulla was fixed by isobaric whole-body perfusion with glutaraldehyde. Specimens treated with anisotropic stains exhibited a diffuse cytoplasmic birefringence. Evidence is presented that birefringence is not only associated with "dark" cell formation, but may be a normal feature of fixed chromaffin cells. It is suggested that the birefringence is caused by the fixed contents of the chromaffin vesicles and/or the microtrabecular lattice recently described in rat chromaffin cells.

The Distribution of Water in the Cytoplasm

It has become apparent in recent years that the cytoplasmic matrix (cytomatrix) is structured. If the evidence available for this is correctly interpreted, we should put aside the concept that the majority of the enzymes which catalyze metabolic reactions are in solution in a soluble phase of the cytoplasm, also called the cytosol. This evidence, derived in large part from high voltage micrographs of whole cultured cells, says that the cytomatrix is a three-dimensional mesliwork of fine strands measuring 5-20 nm in diameter and 50-200 nm in length. By its existence, the microtrabecular lattice transforms the cytosol into a two-phase system, one protein-rich and the other water-rich. It is the purpose of this paper to report a few observations on the latter of these two, i.e. the water-rich phase.

Hormonally induced changes in the cytoskeleton of human thyroid cells in culture.

Serially cultivated thyroid follicular cells are not active in hormone synthesis but retain a thyrotropin-responsive adenylate cyclase. The exposure of such cells to thyrotropin leads to an increase in the concentration of intracellular cAMP and a drastic change in morphology including a total cytoplasmic arborization. The present communication describes these changes at the cytoskeletal level using a cell line derived from a human functioning thyroid adenoma. Phase contrast microscopy showed that the cytoplasmic arborization was preceded by a total disappearance of stress fibers, visible within 20 min of exposure. Small marginal membrane ruffles could also be seen. These morphological changes could also be induced by the addition of dibutyryl cAMP. The action of both thyrotropin and dibutyryl cAMP was potentiated by theophylline. High voltage electron microscopy of whole mounted cells confirmed the loss of stress fibers (microfilament bundles). In addition, thyrotropin treatment led to an uneven redistribution of the cytoplasmic ground substance and to changes in the organization of the microtrabecular lattice. Stereo images demonstrated numerous minute surface ruffles. The thyrotropin-induced arborization was reversible even in the presence of thyrotropin. After 24 h of treatment, cells had flattened and then contained very straight and condensed microfilament bundles. The results thus demonstrate that thyrotropin induces a disintegration of microfilament bundles in human, partially dedifferentiated, follicular cells and that this effect to all appearances is caused by cAMP, the second messenger in thyrotropin action. The relation of this event in partially dedifferentiated cells to the effect of thyrotropin in the intact thyroid gland is unclear. The fact that several other cultured hormone-responsive cells round up or become arborized in conjunction with an increase in cAMP levels implies that cAMP may be a major factor in the disassembly of microfilament bundles in these cells.

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.

The microtrabecular lattice of the adrenal medulla revealed by polyethylene glycol embedding and stereo electron microscopy

The microtrabecular lattice of the adrenal medulla revealed by polyethylene glycol embedding and stereo electron microscopy

Interrelationships between Water and Cell Metabolism in Artemia Cysts. IX. Evidence for Organization of Soluble Cytoplasmic Enzymes

A large fraction of the intermediary metabolism of eukaryotic cells occurs in the cytoplasm, and much of that has traditionally been considered to occur in what has been variously called the cytosol, cytoplasmic ground substance, hyaloplasm, or soluble phase of the cell (Anderson and Green 1967; Schneider 1976). These soluble metabolic pathways are generally believed to take place in solution within the cytoplasm, their operation and regulation occurring by random collisions of the participants of the pathway, governed by simple mass-action principles. According to this view, such pathways exist in that volume of the cytoplasm not occupied by the very considerable amount of architecture that is present, including the latest addition to an already crowded cytoplasm, the microtrabecular lattice (MTL) (Buckley and Porter 1975; Wolosewick and Porter 1979). For convenience I refer to that volume as the aqueous cytoplasm and use the term soluble enzyme to refer to those enzymes...

Epithelial cell motility: the effect of 2-deoxyglucose on cell migration, ATP production, and the structure of the cytoplasmic ground substance in lamellipodia of epithelial cells in culture.

Using a line of epithelial cells (SCCA5) derived from a spontaneous rat carcinoma, the glucose analogue 2-deoxyglucose (2DG) has been shown by time-lapse cinemicrography to produce a cessation of motility by 1 hour that can be reversed by replacement of the 2DG, and does not occur in equivalent media with or without glucose or in 2DG-containing media with added pyruvate and citrate. The effect on the cells at the edge of an epithelial island is to prevent the formation of new lamellipodia and produce a progressive retraction and condensation of lamellipodia already present. This effect of 2DG on motility corresponds with a significant reduction in the level of ATP that is partially restored after 30 minutes in the recovery incubation. Only a slight reduction in protein synthesis occurs in the presence of 2DG. The external morphology and the cytoplasmic ground substance of the cells were studied by scanning electron microscopy and high voltage electron microscopy respectively. It was found that after incubation in 2DG for 1 hour the outline of the free edges of the cells was distorted resulting in redistribution of microvilli, condensation of cytoplasm into strands, and irregular projections from the edges of residual lamellipodia. The structure of the cytoplasmic ground substance in lamellipodia from cells incubated in 2DG for 3 hours was distinctly different from that in cells incubated for 3 hours in 2DG then recovered for 25 minutes, or in cells incubated in glucose-containing medium for 3 hours. In the 2DG-treated cells the lattice-like structure evident in critical-point-dried cells was condensed into short thick strands that terminated in bulbous ends, whereas in cells recovered for 25 minutes the lattice material was elongated and tapering and the interlattice space relatively expanded. The results obtained support the concept of modulation occurring in the structure of the microtrabecular lattice component of the cytoplasmic ground substance coincident with alterations in cell function and metabolic state.

Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: Anaphase B but not anaphase a requires ATP

Permeabilized PtK 1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs, ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase A does not require exogenous ATP and is unaffected by these inhibitors. These results are consistent with “dynein-like” ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.

An electrochemical interpretation of metabolism

FEBS LettersVolume 134, Issue 2 p. 133-138 HypothesisFree Access An electrochemical interpretation of metabolism Michael N. Berry, Michael N. Berry Department of Clinical Biochemistry, Flinders Medical Centre, Bedford Park, SA 5042, AustraliaSearch for more papers by this author Michael N. Berry, Michael N. Berry Department of Clinical Biochemistry, Flinders Medical Centre, Bedford Park, SA 5042, AustraliaSearch for more papers by this author First published: November 16, 1981 https://doi.org/10.1016/0014-5793(81)80585-6Citations: 27AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume134, Issue2November 16, 1981Pages 133-138 ReferencesRelatedInformation

Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.

Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

Chapter 4 Stereomicroscopy of Whole Cells

Publisher Summary This chapter describes the different aspects of the stereomicroscopy of whole cells. The applications of stereomicroscopy of whole cells are expanding rapidly and will take on greater significance when more high-voltage microscopes become available. The correlative information made available by this approach will help to define precisely the influence that changes in cytoskeletal components can exert on the external cell surface morphology. This approach has already been successfully applied to follow virus propagation, maturation, and release from host cell systems. The same methods will also be useful in analyzing the ultrastructural basis for endocytosis, especially where it involves selective uptake by clathrin-coated pits and vesicles. The capping process in cells and the involvement of the subsurface lattice also lend themselves to this approach. Cell responses to a number of drugs such as cytochalasin B and Taxol offer attractive opportunities for observing fine structural changes associated with the gross form changes that the treated cells displays. The stereo micrographs depict for the cytoplasmic ground substance the same microtrabecular lattice as is found in images of the whole cell. The dimensions and the volume ratios of trabeculae to intertrabecular space are different, but that is to be expected, as differentiated cells normally contain less H2O.

Role of the microtrabecular lattice in axoplasmic transport

High voltage electron microscopy (HVEM) stereo images of whole mounted cells have revealed that the cytoplasmic ground substance comprises a three-dimensional lattice of slender strands from 4 to 10 nm dia. (termed microtrabeculae). The lattice, in effect, forms an integral cytoskeletal component that cross-links other filamentous elements to function in one fashion or another as a framework for the maintenance of cell anisometry. In Neuroblastoma, the lattice emerges from the subplasmalemmal surfaces as one of the main cytoskeletal elements involved in growth cone formation and axonal elongation. Most importantly, the microtrabeculae cross-link axonal microtubules in parallel arrays to form linear channels that direct the bidirectional flow of particles from the cell soma to the growth cone. The microtrabeculae (3–7 nm dia.) also seem to interconnect particles (lipid, lysosomal, vesicular, mitochondrial) with microtubules to facilitate their translocation. In time lapse studies, the particles in nearby channels are seen to move simultaneously by linear saltations in opposite directions, and often to reverse their direction of motion in subsequent saltations. This suggests that microtrabeculae behave as nonstatic structures capable of constantly changing, and displaying localized contractions and extensions which result in particle transport through the lattice. Further, the apparently non-ordered process of particle motion suggests that highly localized cellular control mechanisms exist to regulate lattice structure and function within individual microtubule channels. We have investigated how changes in divalent cation ratios ( Ca 2+Mg 2+) affect both lattice organizational properties and the saltation of particles along microtubule channels. Based on these studies, possible mechanisms controlling the behavior of nerve cells in general are discussed.

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). I. Energy requirements

Erythrophores isolated from the scales of the marine teleost, Holocentrus ascensionis (Osbeck), are capable of rapidly aggregating or dispersing numerous red pigment granules within their cytoplasm by translocating them along radial paths delineated by bundles of radially oriented microtubules. Pigment translocation is accompanied by transformations in the morphology of the cytoplasmic matrix, or microtrabecular lattice (MTL), in which the pigment granules are suspended. It appears that the MTL as a whole contracts toward the cell center during aggregation, carrying the pigment granules inward along with it, and is restructured during dispersion, using the radial microtubules as guides. We examined the energy requirements of pigment migration and the accompanying MTL transformations. Cellular ATP was depleted using the specific metabolic inhibitors 2,4 dinitrophenol, NaCN and oligomycin. All three of these drugs, which inhibit oxidative phosphorylation by different mechanisms, prevent both pigment dispersion and MTL transformation to dispersed morphology, while aggregation is unaffected. Inhibitor-treated cells recover normal pigment movements and MTL morphology when inhibitor is washed out of the cells with fresh medium. Potential energy apparently is stored in the MTL by some ATP-dependent process during dispersion and is converted to kinetic energy during aggregation. The results of this study strengthen the hypothesis that the MTL, working in concert with the radial microtubules, is the vehicle for pigment translocation in the erythrophore system.

The Cytoplasmic “Microtrabecular Lattice” - Reality or Artifact

In vertebrate cells microtubules, 10 nm “intermediate” filaments and 6 nm actin filaments are generally accepted as the major units of the so-called “cytoskeleton.” Recently an additional organelle has been proposed as a component of the cytoskeleton, the “microtrabecular lattice” (1). This network of filaments 4-10 nm thick is said to form the ground substance in which other organelles are embedded. Its structure resembles spongy bone, forming a three-dimensional network of “trabeculae” which characteristically vary in thickness as if plastically deformed by stress. This appearance is not seen in sections of embedded cells but is found in micrographs of whole mounts of cells dried by the critical point procedure.