scholarly journals The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance.

1984 ◽  
Vol 99 (5) ◽  
pp. 1655-1668 ◽  
Author(s):  
P C Bridgman ◽  
T S Reese
Keyword(s):  
Cultured Cells ◽  
Ground Substance ◽  
Ice Crystal ◽  
Improved Method ◽  
Intact Cells ◽  
Cultured Fibroblasts ◽  
Cytoplasmic Proteins ◽  
Granular Matrix ◽  
Cytoplasmic Ground Substance ◽  
Xenopus Laevis Embryos

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.

scholarly journals Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality.

1979 ◽  
Vol 82 (1) ◽  
pp. 114-139 ◽  
Author(s):  
J J Wolosewick ◽  
K R Porter
Keyword(s):  
Critical Point ◽  
Osmium Tetroxide ◽  
Prolonged Exposure ◽  
Cultured Cells ◽  
Lattice Structure ◽  
Model Systems ◽  
Ground Substance ◽  
Specimen Preparation ◽  
Critical Point Drying ◽  
Cytoplasmic Ground Substance

The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.

Studies on The Cytoplasmic Ground Substance Using High Voltage Electron Microscopy of Whole Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 818-821
Author(s):  
K.R. Porter ◽  
K.J. Luby
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Monolayer Culture ◽  
Cultured Cells ◽  
Ground Substance ◽  
High Quality ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Ground Substance ◽  
Very High

Cells of several types, when grown and maintained in monolayer culture, will spread on the substrate to be not greater than 1 pm thick in their thinner margins. When fixed with glutaraldehyde and OsO4 and then dried by the critical-point method,these cells can be viewed in the HVEM and stereo images of very high quality can be obtained. Grown directly on formvar-coated gold grids, such cells are quickly and easily prepared for microscopy.

Organisation in the Structure of the Cytoplasmic Ground Substance

1978 ◽  
Vol 36 (3) ◽  
pp. 627-639
Author(s):  
K.R. Porter
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Random Distribution ◽  
Ground Substance ◽  
The Matrix ◽  
Cell Processes ◽  
Cytoplasmic Ground Substance

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

Intralysosomal Accumulation of Colloidal Gold-Low Density Lipoprotein Conjugates in Chloroquine-Treated Fibroblasts

1985 ◽  
Vol 43 ◽  
pp. 546-547 ◽  
Author(s):  
Dean A. Handley ◽  
Cynthia M. Arbeeny ◽  
Larry D. Witte
Keyword(s):  
Colloidal Gold ◽  
Cultured Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coated Vesicle ◽  
Low Density ◽  
Cholesterol Esters ◽  
Cultured Fibroblasts ◽  
Surface Receptors ◽  
Ldl Particles

Low density lipoproteins (LDL) are the major cholesterol carrying particles in the blood. Using cultured cells, it has been shown that LDL particles interact with specific surface receptors and are internalized via a coated pit-coated vesicle pathway for lysosomal catabolism. This (Pathway has been visualized using LDL labeled to ferritin or colloidal gold. It is now recognized that certain lysomotropic agents, such as chloroquine, inhibit lysosomal enzymes that degrade protein and cholesterol esters. By interrupting cholesterol ester hydrolysis, chloroquine treatment results in lysosomal accumulation of cholesterol esters from internalized LDL. Using LDL conjugated to colloidal gold, we have examined the ultrastructural effects of chloroquine on lipoprotein uptake by normal cultured fibroblasts.

Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta

1987 ◽  
Vol 241 (2) ◽  
pp. 463-467 ◽  
Author(s):  
J F Krall ◽  
N Jamgotchian
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cultured Cells ◽  
Rat Aorta ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Morphological Properties ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Uptake Activity

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

scholarly journals Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines

1986 ◽  
Vol 235 (3) ◽  
pp. 707-713 ◽  
Author(s):  
L H Rome ◽  
D F Hill
Keyword(s):  
Cultured Cells ◽  
The Other ◽  
Biosynthetic Pathways ◽  
Significant Extent ◽  
Lysosomal Degradation ◽  
Intact Cells ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Glycoprotein Degradation ◽  
Macromolecule Degradation

Lysosomal degradation of the carbohydrate portion of glycoproteins and glycosaminoglycans produces monosaccharides and sulphate, which must efflux from the lysosomes before re-entering biosynthetic pathways. We examined the degradation of glycoproteins and glycosaminoglycans by lysosomes isolated from cultured human diploid fibroblasts. Cells were grown for 24 h in medium containing [3H]glucosamine and [35S]sulphate. When lysosomes are isolated from these cells, they contain label primarily in macromolecules (glycoproteins and glycosaminoglycans). Glycoprotein degradation by isolated lysosomes was followed by measuring the release of tritiated sugars from macromolecules and efflux of these sugars from the organelles. Glycosaminoglycan degradation was monitored by the release of both tritiated sugars and [35S]sulphate. During macromolecule degradation, the total amounts of free [35S]sulphate, N-acetyl[3H]glucosamine and N-acetyl[3H]galactosamine found outside the lysosome parallels the amounts of these products released by degradation. The total degradation of glycoproteins and glycosaminoglycans by intact cultured cells was also examined. The lysosomal contribution to degradation was assessed by measuring inhibition by the lysosomotropic amine NH4Cl. After 48 h incubation, inhibition by NH4Cl exceeded 55% of glycoprotein and 72% of glycosaminoglycan degradation. Recycling of [3H]hexosamines and [35S]sulphate by intact cells was estimated by measuring the appearance of ‘newly synthesized’ radioactively labelled macromolecules in the medium. Sulphate does not appear to be appreciably recycled. N-Acetylglucosamine and N-acetylgalactosamine, on the other hand, are reutilized to a significant extent.

Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes

1992 ◽  
Vol 101 (2) ◽  
pp. 315-322 ◽  
Author(s):  
M. Terasaki ◽  
T.S. Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Cultured Cells ◽  
Protein A ◽  
Epithelial Cell Line ◽  
Cultured Fibroblasts ◽  
Whole Cells ◽  
Membrane Compartments ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding ◽  
Peripheral Regions

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiO6(3) in CV-1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.

Mechanism of Fumonisin Toxicity and Carcinogenesis

1994 ◽  
Vol 57 (7) ◽  
pp. 638-644 ◽  
Author(s):  
RONALD T. RILEY ◽  
KENNETH A. VOSS ◽  
HWAN -SOO YOO ◽  
WENTZEL C.A. GELDERBLOM ◽  
ALFRED H. MERRILL
Keyword(s):  
Rat Liver ◽  
Cell Injury ◽  
Cultured Cells ◽  
Degradation Products ◽  
Fumonisin B1 ◽  
Hepatocyte Proliferation ◽  
Sphingolipid Metabolism ◽  
Sphingoid Base ◽  
Cultured Fibroblasts ◽  
Animal Diseases

What are the molecular events that fumonisin-induced porcine pulmonary edema syndrome and equine leucoencephalomalacia have in common? Do these animal diseases relate mechanistically to fumonisin toxicity in laboratory rats? There is considerable data indicating that disruption of sphingolipid metabolism plays an important early role in all of these diseases. In vitro studies have revealed that fumonisins and structurally related Alternaria alternata f. sp. lycopersici-toxin (AAL-toxin) are potent inhibitors of the enzyme sphinganine (sphingosine) N-acyl transferase (ceramide synthase). Soon after cultured cells or animals are exposed to fumonisins there is a dramatic increase in the free sphingoid base, sphinganine, in tissues, serum and/or urine. Also, free sphingosine concentration increases, complex sphingolipid concentration decreases, and sphingoid base degradation products and other lipid products also increase. It is hypothesized that disruption of sphingolipid metabolism is an early molecular event in the onset and progression of cell injury and the diseases associated with consumption of fumonisins. However, the exact mechanisms responsible for the diseases will not be easily revealed since the role of sphingolipids in cellular regulation is very complex and not yet fully understood. While fumonisin B1 is non-genotoxic it is a complete carcinogen in rat liver. Recent studies indicate that fumonisins inhibit hepatocyte proliferation in rat liver. It has been hypothesized that hepatotoxicity and effects on hepatocyte proliferation are critical determinants for fumonisin B1 cancer initiation and promotion. Alternatively, recent studies have found that fumonisin B1 has mitogenic activity in cultured fibroblasts. It is conceivable that the mitogenic, cytostatic and cytotoxic potential of fumonisin may all contribute to the animal diseases including liver cancer in rats.

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

1997 ◽  
Vol 273 (4) ◽  
pp. C1250-C1258 ◽  
Author(s):  
Ashok K. Grover ◽  
Sue E. Samson
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Muscle Cells ◽  
Intact Cells ◽  
Serca Pump

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated45Ca2+uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 μM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 ± 13 μM, but up to 500 μM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 μM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 ± 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.

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