The cytoplasmic matrix of the adrenal chromaffin cells of rats under normal and stressed conditions.
ABSTRACT 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.
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ABSTRACT: TEM of any in situ cells in embedment-free sections--regardless of specimen-fixation methods--clearly shows strand-lattices occupying the cytoplasmic matrix. The cytoplasmic matrix is assumed to be a site of soluble proteins; however, it appears indistinct as conventional TEM cannot target it. Strand-lattices similar to the cytoplasmic ones are duplicated in bovine serum albumin as well as solated gelatin fixed at warm temperatures and at appropriate concentrations, while lattices from gelatin gelated by cooling before fixation are much more compact than those from solated gelatin at a given concentration. Based on the finding of the in vitro proteins, a new interpretation of cell ultrastructures in embedment-free section TEM is proposed: first, differences in the compactness of cytoplasmic lattices represent those in the protein concentration in the cytoplasmic matrix; second, when loose and compact lattices are contiguous within a cell, the cytoplasmic matrix domain occupied by the compact lattice is in a gel state while the remaining domain of the same cell is in a sol state. The explanation for the states of the gel and sol based on the lattice-compactness is applicable to changes in the lattice-compactness of the cytoplasmic matrix of neurohypophyseal axons under intense secretion.Advances in colloid and interface science 10/2010; 160(1-2):49-55. · 5.68 Impact Factor
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ABSTRACT: 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.Microscopy Research and Technique 07/2008; 71(6):418-42. · 1.59 Impact Factor
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ABSTRACT: With higher contrast and transparency due to the absence of epon and stereo-viewing effect due to thicker sections than conventional electron microscopy as methodological advantages, the renal glomerular slits were re-examined in embedment-free section electron microscopy. In addition to clear demonstration of strands bridging the slits in forms of ladders with highly irregular intervals and various extension-directions and length, this study disclosed clearly for the first time in the "section" TEM thin sheets which partially spanned the slit together with the strand-ladders. No strands were found to align in forms of typical zippers in normal kidney. Furthermore, en-face ultrastructure of the basal lamina in situ was clearly demonstrated in superimposed sites of the endothelial fenestrae with the slits.Microscopy Research and Technique 02/2011; 74(2):142-7. · 1.59 Impact Factor