Microtrabecular Lattice Research Articles

Hydrogel formation by multivalent IDPs: A reincarnation of the microtrabecular lattice?

Based on high-voltage electron microscopic (HVEM) data of fixed cultured cells, an elaborate three-dimensional network of filaments, including and interconnecting other elements of the cytoskeleton, was observed in cells some half a century ago. Despite many attempts and comparative studies, this “microtrabecular lattice” (MTL) of the cytoplasmic ground substance could not be established as a genuine component of the eukaryotic cell, and is mostly considered today as a sample-preparation artifact of protein adherence and cross-linking to the cytoskeleton. Here we elaborate on the provocative idea that recent observations of hydrogel-forming phase transitions of repetitive regions of intrinsically disordered proteins (IDPs) bear resemblance in creation, organization and physical appearance to the MTL. We review this phenomenon in detail, and suggest that phase transitions of actin regulatory proteins, neurofilament side-arms and other proteins could generate non-uniform spatial distribution of cytoplasmic material in the vicinity of the cytoskeleton that might even give rise to fixation phenomena resembling the MTL. Whether such hydrogel formation by IDPs is a general physical phenomenon, will remain to be seen, nevertheless, the underlying organizational principle provokes novel experimental studies to uncover the ensuing higher-level regulation of cell physiology, in which the despised and long-forgotten concept of MTL might give some interesting leads.

Revisiting the microtrabecular lattice

The 'microtrabecular lattice' (MTL) that Keith Porter described in the 1970s and 1980s is reconsidered as a proposed fundamental cytoplasmic structure of eukaryotic cells. Although considered to be an artefact by most cell biologists of his time (and probably ours), the case is made that something like the MTL may well exist, but in a much more dynamic form than images from electron microscopy imply and convey.

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.

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.

On the fractal nature of cytoplasm

On the basis of a quantification of the fractal dimension, D, in micrographs of cytoskeleton components or microtrabecular lattice, we propose that the cellular cytoplasm can be described as a percolation cluster, a sort of ‘random fractal’. Our hypothesis deals with: (i) the existence of the percolation threshold — a remarkable property of percolation processes; and (ii) the reactivity increase — when enzymes, or targets, and substrates, or effectors, coexist in the same topological dimension.

Reconstruction of intracellular and extracellular networks from stereo IVEM images using computer graphic methods

High and intermediate voltage electron microscopes can be used to get good images of many types of thick biological specimens, including sections of embedded material up to several micrometers thick, whole cells, or isolated cellular components mounted on support films, stereoscopic pairs of images can be obtained by tilting the specimen between exposures, and this requires only relatively small tilt angles, often less than 15°. With a high tilt specimen holder, images covering a wider range of tilt angles can be obtained, increasing the amount of 3D information. Computer graphic methods for extracting quantitative 3D information from such images have been under development and use in this laboratory for several years.Many cellular structures take the form of networks. Examples that we study are transverse tubular networks in muscle, fibrin clots, and the microtrabecular lattice of the cytoplasm. We perform quantitative, 3D reconstruction and analysis of such networks from multiple stereoscopic image sets obtained using a JEOL 4000-EX TEM.

Does the introduction of a new player, the endoplasmic reticulum, create more or less confusion in understanding the mechanism(s) of pigmentary organelle translocations?

In 1925, Wilson listed, in his classic third edition of Cell in Development and Heredity, four theories for the morphological and physiological characteristics of cytoplasm; each theory provided some sort of explanation as to the mechanism(s) of organelle translocations. During the past twenty years, cell biologists have focused their attentions on the cell's cytoskeleton, microtrabecular lattice, and associated mechanochemical motors which drive organelles along cytoskeletal tracks. A number of cell types have been used to study organelle translocations, but chromatophores, pigment cells, from cold-blooded vertebrates have been one of the more popular models. This article reviews some of the research findings during the past twenty years, particularly those involving cytoplasmic elements: i.e, microfilaments, intermediate filaments, microtubules, and mechanochemical motors. In addition, it contrasts the proposed involvement of these elements in organelle translocations with the endoplasmic reticulum, a tubulovesicular organelle, which we recently demonstrated is responsible, through its elongation or retraction, for the translocations of carotenoid droplets in goldfish xanthophores and swordtail fish erythrophores. Here, the carotenoid droplets are not free in the cytoplasm and do not translocate via cytoskeletal tracks, but instead are attached to or are a part of the endoplasmic reticulum. On the other hand, carotenoid droplets of squirrel fish erythrophores are free in the cytoplasm and appear to translocate via microtubules. Finally, the rates of pigmentary organelle translocations are reviewed in light of the participation of the cytoskeletal elements with the endoplasmic reticulum.

Human fibroblast intracellular network prepared with digitonin for field emission scanning electron microscopy.

Results obtained by extracting human fibroblast cells for the study of cytoskeletal structures are compared. Cells grown in culture were treated with digitonin in contrast to other methods of detergent preparation using Triton and Saponin. The three dimensional intracellular network which resulted from digitonin treatment was found to be similar in appearance to the structures observed by high voltage transmission electron microscopy of untreated cells described as a microtrabecular lattice by Porter and Tucker (1981). Our results, obtained by high resolution, field emission scanning electron microscopy indicate that the microtrabecular lattice may indeed be one conformation of a dynamic cytoskeleton.

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.

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.

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase ( d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase ( d-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceralde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate: NAD + oxidoreductase (phosphorylating), EC 1.2.1.12), triosephosphate isomerase ( d-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase ( d-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho- d-glycerate 1-phosphotransferase, EC 2.7.2.3), enolase (2-phospho- d-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP: Pyruvate O 2- phosphotransferase , EC 2.7.1.40) and lactate dehydrogenase ( (S)- lactate : NAD + oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofruktokinase (ATP: d-fructose-6-phosphate 1-phosphortransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were obsereved. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.

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.

Diversity in the axonal transport of structural proteins: major differences between optic and spinal axons in the rat

Investigations of slow axonal transport reveal variation in both protein composition and the rate of movement. However, these studies involve a variety of nerve preparations in different species, and most lack the resolution needed to determine the kinetics of identified proteins. We have compared the axonal transport of slow-transported proteins in retinal ganglion cells and spinal motor neurons of young rats. Nine proteins that contribute to axonal structures were examined: the neurofilament triplet (NFT), alpha and beta tubulin, actin, fodrin, calmodulin, and clathrin. Axonally transported proteins were pulse-labeled by intraocular or intracord injections of 35S-methionine. After allowing sufficient time for labeled slow-component proteins to enter the spinal or optic nerves, consecutive 2-3 mm nerve segments were subjected to SDS-PAGE. Fluorographs were used as templates for locating the gel regions containing the above polypeptides, and the radioactivity in these regions was measured by liquid-scintillation spectrometry. In retinal ganglion cells, the peak of tubulin labeling advanced at 0.36 mm/d in association with the NFT and fodrin. The cotransport of tubulin and the NFT identified this complex as the slower subcomponent of slow transport, termed slow component a (SCa) and representing the movement of the microtubule-neurofilament network. The peaks of actin and calmodulin labeling were cotransported at 2.3 mm/d in near-register with peaks of fodrin and clathrin labeling. These 4 proteins, moving ahead of the NFT, identified this complex as SCb--the faster subcomponent of slow transport, which represents the movement of the cytoplasmic matrix and microtrabecular lattice. Both subcomponents had the same composition and rate as that reported for the optic axons of guinea pigs and rabbits, establishing a basic mammalian pattern. In spinal motor axons, the SCa tubulin peak advanced at 1.3 mm/d, and the SCb actin and calmodulin peaks were cotransported at 3.1 mm/d. Unlike optic axons, SCa in motor axons was more heavily labeled than SCb, and included labeled peaks of actin, clathrin, and calmodulin moving in register with the SCa tubulin peak. Actin was the most heavily labeled of these SCb proteins moving with SCa, and it left a higher plateau of radioactivity behind the advancing SCa peak. The SDS-PAGE labeling pattern for SCb did not differ from that seen in optic axons, except that some tubulin was found to form a peak that advanced in register with the actin and calmodulin peaks.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence of a microtrabecular cytoskeletal lattice in glandular cells of hydrozoan planulae.

Hydrozoan planulae of Pennaria tiarella and Podocoryne carnea were processed for transmission electron microscopy using diethylene glycol distearate (DGD). The DGD functions as a removable embedding medium to produce embedment-free sections of intact planulae. Images of glandular cells obtained using embedment-free sections were compared with those from conventional Spurr-embedded sections. In unembedded sections a large number of thin anastomosing fibers were observed throughout the cytoplasm of the glandular cell. The fibers appeared to coalesce in certain areas to form thick bundles of fibers that partitioned the glandular cytoplasm into spherical compartments. The meshwork of fibers is three-dimensional and resembles a microtrabecular lattice. Mitochondria are suspended within and attached to the network of fibers, thus suggesting a cytoskeletal role of the fibers. This study documents the presence of a cytoplasmic fiber system within cells of intact invertebrate larvae.

Fracture—Permeation: A technique to assess cytoplasm compactness after glutaraldehyde‐fixation

AbstractWe have developed a method—“fracture‐permeation”—to assess the compactness of the cytoplasm in glutaraldehyde‐fixed cells. Cells or tissues are fixed in glutaraldehyde, impregnated with glycerol, and frozen in liquid nitrogen. The cells are freeze‐fractured, thawed, deglycerinated, and immersed in concentrated solutions of globular proteins. In initial experiments, we used native ferritin (NF) to permeate model matrices made of bovine serum albumin (BSA). We show that permeation depends on the concentraion of proteins within the cross‐linked matrix: NF permeates matrices made from 10 or 15% (w/v) BSA solutions but do not permeate matrices made from solutions with 20% (w/v) protein or more. With freeze‐fractured cells, ferritin molecules were unable to permeate the cross‐linked cytoplasm of fungal zoospores and cysts, used here as examples of resting cells. In human lymphocytes from peripheral blood, permeation of ferritin was limited or absent, but it became massive in cells activated by phytohemagglutinin. Massive permeation of ferritin was also observed within the cytoplasmic matrix of other active cells (fungal sporangia, germinating cysts). In the examined resting cells, glutaraldehyde crosslinks the proteins into a dense matrix that effectively excludes ferritin (diameter 12 nm). These findings cannot be explained by models that envisage all cytoplasmic proteins congregated into a single‐phase microtrabecular lattice with the nature and dimensions proposed by Porter and co‐workers. We conclude that compactness of the cytoplasm matrix depends on the physiological state of the cell: It varies through differentiation and is related to the degree of cellular activity.

Stable intermicrotubule linkers in the isolated cytostomal apparatus of Tetramitus

Detergent lysis of Tetramitus rostratus flagellates liberates intact a "cytostomal apparatus" composed of four flagella, their basal bodies, and several striated flagellar rootlets, as well as an unusually stable array of long cytoskeletal microtubules. Cytostomal apparatuses, purified on 5–15% Ficoll gradients and negatively stained with uranyl acetate, reveal abundant arrays of intermicrotubule linkers (IMTLs). These linkers, which are irregularly spaced vertically at approximately 50-nm intervals, appear to limit intermicrotubule distances to about 120 nm. The IMTLs of Tetramitus are unusually long, unusually abundant, and unusually stable: it is speculated that they represent an exaggerated form of (and hence a means to identify) a universal component of the microtrabecular lattice of eukaryote cells.

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.

The Physiology and Pharmacology of Crustacean Chromatophores

The color change system of crustaceans is being investigated along a broad front. The techniques being used include physiological, pharmacological, biochemical, immunocytochemical, and ultrastructural ones. The problems investigators are seeking answers to include the cellular bases for pigment granule translocation, the number and specificity of the chromatophorotropic hormones responsible for the color changes, and the identity of the neuroregulators (neurotransmitters and neuromodulators) that control the release of these hormones. With respect to the cellular bases of pigment granule translocation, microtubules and a microtrabecular lattice are prime candidates as organelles that might be responsible for the pigment granule movements. Pigment dispersing and pigment concentrating neuropeptides have been identified. Some exhibit no specificity with respect to the chromatophore type they activate. Others show high specificity, affecting only one specific type of chromatophore, such as the melanophore. Several putative neuroregulator candidates have been identified as possibly having a role in controlling chromatophorotropic hormone release. These include 5-hydroxytryptamine, dopamine, norepinephrine, octopamine, and gamma-aminobutyric acid. Some, like the first three, stimulate hormone release, whereas the latter two have inhibitory actions.

The translational mobility of substances within the cytoplasmic matrix.

The translational mobility of fluorescent-labeled molecules injected into the cytoplasm of living cells can be measured by the fluorescence recovery after photobleaching (FRAP) technique. In the fibroblast cytoplasm, the diffusion coefficients, D, of test macromolecules ranging in molecular weight from 12,000 to 440,000 are about 10(-8) cm2/sec and exhibit almost no dependence on molecular weight. FRAP experiments also showed that macromolecular diffusion within Sepharose beads having an effective pore size smaller than the "microtrabecular lattice" is only slightly retarded compared to buffer values--in contrast to the marked retardation measured in the cytoplasm. This leads to the conclusion that diffusion in the cytomatrix is dominated not by steric effects but rather by binding of the diffusing species to elements of the cytomatrix. These diffusion rates were difficult to modulate; cytochalasin, colchicine (except at 5 degrees C), and taxol treatments had little effect. The diffusion rates were not dependent on cellular energy metabolism. However, hypotonic treatment increased the D for bovine serum albumin by nearly 2-fold, whereas hypertonic treatment halved D. Withdrawing the free water from the cell by using 44% polyethylene glycol treatment stopped the translational mobility of the test molecules. A survey of the recent literature is presented, which shows that major differences in the cytomatrix of different cell types exist with respect to the translational diffusion of injected probes. Finally, the spectrum of cytoplasmic translational mobilities ranging from small molecules to organelles is discussed.

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.