The Advantages of Cryotechniques: Application To Bioluminescent Cells

1990 ◽  
Vol 48 (1) ◽  
pp. 486-487
Author(s):  
Gisèle Nicolas ◽  
Jean-Marie Bassot ◽  
Marie-Thérèse Nicolas
Keyword(s):  
Endoplasmic Reticulum ◽  
Striated Muscle ◽  
Light Emission ◽  
Stable State ◽  
Freeze Substitution ◽  
Initial Step ◽  
Point Of Interest ◽  
Cell Components ◽  
Excellent Preservation ◽  
External Water

The use of fast-freeze fixation (FFF) followed by freeze-substitution (FS) brings substantial advantages which are due to the extreme rapidity of this fixation compared to the conventional one. The initial step, FFF, physically immobilizes most molecules and therefore arrests the biological reactions in a matter of milliseconds. The second step, FS, slowly removes the water content still in solid state and, at the same time, chemically fixes the other cell components in absence of external water. This procedure results in an excellent preservation of the ultrastructure, avoids osmotic artifacts,maintains in situ most soluble substances and keeps up a number of cell activities including antigenicities. Another point of interest is that the rapidity of the initial immobilization enables the capture of unstable structures which, otherwise, would slip towards a more stable state. When combined with electrophysiology, this technique arrests the ultrastructural modifications at a well defined state, allowing a precise timing of the events.We studied the epithelium of the elytra of the scale-worm, Harmothoe lunulata which has excitable, conductible and bioluminescent properties. The intracellular sites of the light emission are paracrystals of endoplasmic reticulum (PER), named photosomes (Fig.1). They are able to flash only when they are coupled with plasma membrane infoldings by dyadic or triadic junctions (Fig.2) basically similar to those of the striated muscle fibers. We have studied them before, during and after stimulation. FFF-FS showed that these complexes are labile structures able to diffentiate and dedifferentiate within milliseconds. Moreover, a transient network of endoplasmic reticulum was captured which we have named intermediate endoplasmic reticulum (IER) surrounding the PER (Fig.1). Numerous gap junctions are found in the membranous infoldings of the junctional complexes (Fig.3). When cryofractured, they cleave unusually (Fig.4-5). It is tempting to suggest that they play an important role in the conduction of the excitation.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

scholarly journals A Cellular Reaction to Antibody in Tissue Culture Studied with Electron Microscopy

1959 ◽  
Vol 5 (3) ◽  
pp. 405-410 ◽  
Author(s):  
Harrison Latta
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Normal Serum ◽  
Butyl Methacrylate ◽  
Heart Cells ◽  
Plasma Clot ◽  
Outer Nuclear Membrane ◽  
Cell Components

The reaction of embryonic chick heart cells grown in tissue culture to specific guinea pig antiserum has been studied with electron microscopy. Heart fragments from chick embryos were cultured with a plasma clot. After being tested with antiserum or normal serum, they were fixed with buffered osmium tetroxide and embedded in butyl methacrylate before removal from the glass culture chamber. Thin cells found by phase microscopy to have reacted were sectioned in a plane parallel to the glass surface on which they had grown. The results confirm and extend observations made previously while the reactions were occurring. The plasma membrane, like that of the red cell, becomes disrupted or less resistant to trauma following the action of antiserum. The membranes of mitochondria and endoplasmic reticulum vesiculate and swell. Before nuclear shrinkage becomes prominent, the outer nuclear membrane separates over a large portion of the nuclear envelope and forms one or more large swollen blebs. Thus, the outer nuclear membrane shows a reactivity similar to endoplasmic reticulum. It is suggested that the various physical and chemical changes observed to follow the action of antibody and complement on fibroblasts may be explained by osmotic pressure differences between various cell components. Some basic similarities to the action of hemolytic agents on red cells are noted.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1971 ◽  
Vol 49 (3) ◽  
pp. 899-905 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Carol Pannek ◽  
Daniel S. Friend
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Total Activity ◽  
Thiamine Pyrophosphate ◽  
Transferase Activity ◽  
Galactosyl Transferase ◽  
Thiamine Pyrophosphatase ◽  
Cell Components

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

Cytochemistry of the Golgi Apparatus

1973 ◽  
Vol 31 ◽  
pp. 690-691
Author(s):  
D. James Morré ◽  
E. L. Vigil ◽  
T. W. Keenan
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Vesicle ◽  
Developmental Pathways ◽  
Cell Component ◽  
Membrane Flow ◽  
Membrane Differentiation ◽  
Morphological Studies ◽  
Biochemical Studies ◽  
Cell Components

Concepts of membrane flow and membrane differentiation are combined to explain the formation of eukaryotic endomembranes along a sequence of cell components in subcellular developmental pathways. Membrane differentiation is the gradual conversion of membranes from one type to another and is documented by comparisons of enzymatic activities, lipid composition and progressive modification of the proteins and lipids of membranes along the endoplasmic reticulum (ER)-Golgi apparatus (GA)-secretory vesicle-plasma membrane (PM) export route. The biochemical studies show the transitional nature of GA membranes first revealed by morphological studies. Membrane dimensions and staining characteristics change progressively from ER-like to PM-like across the stacked cisternae from the forming to the maturing face of the apparatus. Membrane flow is the physical transfer of membrane from one cell component to another.

Ultrastrucure of Peruvian and Aleutian Mummified Tissues

1980 ◽  
Vol 38 ◽  
pp. 528-529
Author(s):  
E.R. Rivera ◽  
E.A. Coughlin
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Striated Muscle ◽  
Cytoplasmic Organelles ◽  
Low Viscosity

Electron microscopy has been employed in the study of Egyptian and Aleutian human mumified tissues (1,2). The Egyptian mummification process apparently fixed some tissues well enough to allow observation of some cytoplasmic organelles (1). In observed Aleutian tissues, nucleoli and possibly endoplasmic reticulum have been found in cartilage but not in other tissues (2). The use of TEM methods may provide some indication of the preservation procedure during mummification. Arm muscle and pleura from Peruvian and Aleutian mummies were cut into 1 mm cubes and floated in 5% glutaraldehyde in 0.075 M PIPES buffer for 2 weeks at 4°C. This procedure allowed rehydration of tissue and fixation to proceed simultaneously. These tissues were then post-fixed in 2% OsQ4 and embedded in Spurr’s low viscosity resin (3) and sectioned for TEM. Functional human striated muscle and pleura were similarly prepared as controls.

A comparison of membrane fracture faces of fixed and unfixed glycerinated tissue

1976 ◽  
Vol 21 (3) ◽  
pp. 437-448
Author(s):  
A.S. Breathnach ◽  
M. Gross ◽  
B. Martin ◽  
C. Stolinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Particle Aggregation ◽  
Buccal Epithelium ◽  
Mitochondrial Membranes ◽  
Nuclear Membranes ◽  
General Plasma

Fixed (glutaraldehyde, 3%) and unfixed specimens of rat buccal epithelium, striated muscle, and liver, were cryoprotected with glycerol, freeze-fractured, and replicated without sublimation. A comparison of fracture faces of general plasma membranes, nuclear membranes, mitochondrial membranes, and membranes of rough endoplasmic reticulum revealed no significant differences as between fixed and unfixed material. Apart from some membranes of liver endoplasmic reticulum, there was no evidence of aggregation or redistribution of intramembranous particles in the unfixed material. The results demonstrate that chemical prefixation of tissues for freeze-fracture is not always necessary, or even desirable, and that glycerol may not be as deeply or directly implicated in particle aggregation as previously thought. Fixation with glutaraldehyde alters the cleaving behaviour of plasma membrane at desmosomes and tight junctions, but not at gap junctions.

scholarly journals Polarized compartmentalization of organelles in growth cones from developing optic tectum.

1985 ◽  
Vol 101 (4) ◽  
pp. 1473-1480 ◽  
Author(s):  
T P Cheng ◽  
T S Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Cone ◽  
Smooth Endoplasmic Reticulum ◽  
Freeze Substitution ◽  
Growth Cones ◽  
Distal Segment ◽  
Computer Assisted ◽  
Chemical Fixation ◽  
Coated Pits ◽  
Membrane Bound

We have used computer-assisted reconstructions of continuous serial sections to study the cytoplasmic organization of growth cones in vivo. Optic tecta from 6.25-6.5-d-old chicken embryos were quick-frozen and then freeze-substituted in acetone-osmium tetroxide or, for comparison, prepared by conventional fixation. Images of eight freeze-substituted and two conventionally fixed growth cones were reconstructed from aligned serial micrographs. After freeze-substitution, numerous lumenless membrane-bound sacs arrayed in multilamellar stacks appear to replace the abundant smooth endoplasmic reticulum found after chemical fixation. Microtubule fascicles progressively diverge from their typical fascicular organization in the initial segment of the growth cone and are absent in the varicosity and the more distal segment. Mitochondria, in contrast, are concentrated in the proximal segment of the varicosity; multilamellar stacks and endosome-like vacuoles are in the distal segment; and coated pits and vesicles are concentrated near the terminal filopodium, which is the most distal and organelle-poor domain of the growth cone. These observations suggest that dilation and fusion of the lumenless, membrane-bound sacs that occurs during chemical fixation give rise to the network of smooth endoplasmic reticulum. The three-dimensional reconstructions show that the cytoplasmic components of growth cones, including the membrane-bound sacs and multilamellar stacks revealed by freeze substitution, are polarized along the axis of these growth cones, which suggests that they have a role in recycling of membrane during elongation of the growth cone.

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