scholarly journals Morphology of isolated triads.

1983 ◽  
Vol 96 (4) ◽  
pp. 1017-1029 ◽  
Author(s):  
R D Mitchell ◽  
A Saito ◽  
P Palade ◽  
S Fleischer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Thin Section ◽  
Freeze Fracture ◽  
Structural Features ◽  
Negative Staining ◽  
Transverse Tubule ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Terminal Cisternae

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.

scholarly journals Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.

1984 ◽  
Vol 99 (3) ◽  
pp. 875-885 ◽  
Author(s):  
A Saito ◽  
S Seiler ◽  
A Chu ◽  
S Fleischer
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Thin Section ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Morphological Characteristics ◽  
Negative Staining ◽  
Section Electron ◽  
Terminal Cisternae

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.

Freeze-fracture electron microscopy: relationship of membrane structural features to transport physiology

1977 ◽  
Vol 232 (2) ◽  
pp. F77-F83 ◽  
Author(s):  
J. B. Wade ◽  
W. A. Kachadorian ◽  
V. A. DiScala
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Structural Features ◽  
Toad Urinary Bladder ◽  
Luminal Membrane ◽  
Cautious Interpretation ◽  
Freeze Fracture Electron Microscopy ◽  
Induced Changes ◽  
Relationship Of ◽  
Fracture Electron

Recent observations utilizing freeze-fracturing electron microscopy are discussed which indicate that the membrane structural features visualized by this technique may in some instances be related to specialized membrane transport properties. The occurrence of organized aggregates of intramembrane particles observed in vasopressin or cAMP-treated toad urinary bladder has been found to be closely correlated with induced changes in the permeability of the luminal membrane. Although a cautious interpretation is considered appropriate, these observations raise the possibility that some aspects of hormone action may be restricted to limited regions of membrane. Difficulties in interpretation and some serious limitations of the freeze-fracture technique are discussed.

New Evidence for Membrane Specialization in the Diatom Thalassiosira Antarctica Comber

1980 ◽  
Vol 38 ◽  
pp. 624-625
Author(s):  
Gregory J. Doucette
Keyword(s):  
Electron Microscopy ◽  
Thin Section ◽  
Freeze Fracture ◽  
Marine Diatom ◽  
Supplementary Data ◽  
Ultra Structure ◽  
Membrane Specialization ◽  
Freeze Fracture Electron Microscopy ◽  
New Evidence ◽  
Fracture Electron

The present investigation was undertaken to examine the nonsiliceous ultra-structure of diatoms (BacilIariophyta) by means of freeze fracture electron microscopy. Freeze fracture procedures are complicated by the difficulties encountered in fracturing silica-based components and the removal of these materials subsequent to replica casting. Supplementary data was obtained through conventional thin section methodologies. This report is a prelim¬inary account of observations made on selected nonsiliceous, cellular con¬stituents of Thalassiosira antarctica Comber, a planktonic, marine diatom.

scholarly journals Coalescence of microsomal vesicles from rat liver: a phenomenon occurring in parallel with enhancement of the glycosylation activity during incubation of stripped rough microsomes with GTP.

1980 ◽  
Vol 86 (1) ◽  
pp. 29-37 ◽  
Author(s):  
J Paiement ◽  
H Beaufay ◽  
D Godelaine
Keyword(s):  
Electron Microscopy ◽  
Thin Section ◽  
Rat Liver ◽  
Freeze Fracture ◽  
Endogenous Protein ◽  
Previously Treated ◽  
Nucleotide Sugars ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Rough microsomes from rat liver have been subjected to various treatments and incubated afterwards with UDP-N-acetyl-[14C]glucosamine and GDP-mannose in the presence of GTP (0.5 mM), or of other nucleotides. In agreement with earlier results from this laboratory, the preparations previously treated to strip off the ribosomes and incubated in the presence of GTP assembled dolichol-linked oligosaccharides and transferred these oligosaccharides to endogenous protein acceptors much more actively than untreated preparations, or stripped preparations incubated in the absence of GTP. Thin-section and freeze-fracture electron microscopy have revealed that pyrophosphate-treated preparations incubated with GTP are aggregated and contain numerous vesicles as large as 1-4 micrometer, or more. Such large vesicles were not present before incubation and thus were considered to have been formed through coalescence of regular-sized ones. Like glycosylation, the coalescence phenomenon depends upon the removal of ribosomes, because it occurred whether ribosomes had been stripped, at least partly, with pyrophosphate, KCl, or puromycin, but not when rough microsomes had been washed with 0.25 M sucrose or with KCl and MgCl2. Like glycosylation, it also depends on the addition of GTP and was not induced by ATP, UTP, CTP, and nonhydrolysable analogues of GTP. Rough microsomes coalesced, however, when pyrophosphate-treated preparations were incubated with GTP in the absence of nucleotide sugars, or in the presence f tunicamycin, indicating that the coalescence phenomenon does not result from the glycosylation of some membrane constituents.

scholarly journals Ultrastructure of the calcium release channel of sarcoplasmic reticulum.

1988 ◽  
Vol 107 (1) ◽  
pp. 211-219 ◽  
Author(s):  
A Saito ◽  
M Inui ◽  
M Radermacher ◽  
J Frank ◽  
S Fleischer
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Calcium Release ◽  
Double Staining ◽  
Side View ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Transverse Tubule ◽  
Terminal Cisternae

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.

Paramecium trichocysts isolated with their membranes are stable in the presence of millimolar Ca2+

1989 ◽  
Vol 93 (3) ◽  
pp. 557-564
Author(s):  
OSCAR LIMA ◽  
TADEUSZ GULIK-KRZYWICKI ◽  
LINDA SPERLING
Keyword(s):  
Structural Transition ◽  
External Medium ◽  
Freeze Fracture ◽  
Wild Type ◽  
Water Matrix ◽  
Matrix Expansion ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

We have developed a simple and rapid procedure for the isolation of a pure fraction of Paramecium trichocysts (mature secretory vesicles) with their membranes. Since in wild-type Paramecium cells essentially all trichocysts are docked at pre-formed cortical sites, trichocysts were isolated from cells in which functional trichocysts remain free in the cytoplasm owing to a mutation, tam6, that affects the docking site. Examination of the preparations by freeze-fracture electron microscopy confirms the presence of the membranes. The distribution of particles in the membranes of the isolated trichocysts and in the membranes of wild-type trichocysts in situ are nearly identical and this argues against any rearrangement of the membranes during the isolation procedure. Although the trichocyst matrix undergoes a dramatic structural transition in the presence of Ca2+ and water (matrix expansion), the isolated vesicles with intact membranes are perfectly stable in the presence of millimolar free Ca2+. This result supports a chronology in which the first step in exocytosis is membrane fusion, the swelling of vesicle contents occurring only afterwards, once the contents come into contact with the water and Ca2+ of the external medium. The role of swelling would then be to help disperse, propel or otherwise empty the contents of the vesicle outside the cell.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

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