scholarly journals Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.

1978 ◽  
Vol 79 (3) ◽  
pp. 601-616 ◽  
Author(s):  
A Saito ◽  
C T Wang ◽  
S Fleischer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Tannic Acid ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Thin Sections ◽  
Membrane Asymmetry ◽  
The Asymmetry ◽  
First Time ◽  
Negative Staining Electron Microscopy

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.

Direct visualization of a vast cortical calcium compartment in Paramecium by secondary ion mass spectrometry (SIMS) microscopy: possible involvement in exocytosis

1995 ◽  
Vol 108 (5) ◽  
pp. 1895-1909 ◽  
Author(s):  
N. Stelly ◽  
S. Halpern ◽  
G. Nicolas ◽  
P. Fragu ◽  
A. Adoutte
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Membrane Vesicles ◽  
Cell Periphery ◽  
A Value ◽  
External Reference ◽  
Ion Mass Spectrometry ◽  
First Time ◽  
Secondary Ion

The plasma membrane of ciliates is underlaid by a vast continuous array of membrane vesicles known as cortical alveoli. Previous work had shown that a purified fraction of these vesicles actively pumps calcium, suggesting that alveoli may constitute a calcium-storage compartment. Here we provide direct confirmation of this hypothesis using in situ visualization of total cell calcium on sections of cryofixed and cryosubstituted cells analyzed by SIMS (secondary ion mass spectrometry) microscopy a method never previously applied to protists. A narrow, continuous, Ca-emitting zone located all along the cell periphery was observed on sections including the cortex. In contrast, Na and K were evenly distributed throughout the cell. Various controls confirmed that emission was from the alveoli, in particular, the emitting zone was still seen in mutants totally lacking trichocysts, the large exocytotic organelles docked at the cell surface, indicating that they make no major direct contribution to the emission. Calcium concentration within alveoli was quantified for the first time in SIMS microscopy using an external reference and was found to be in the range of 3 to 5 mM, a value similar to that for sarcoplasmic reticulum. After massive induction of trichocyst discharge, this concentration was found to decrease by about 50%, suggesting that the alveoli are the main source of the calcium involved in exocytosis.

scholarly journals Ultrastructural and molecular characteristics of crayfish photoreceptor membranes.

1976 ◽  
Vol 69 (3) ◽  
pp. 721-732 ◽  
Author(s):  
H R Fernandez ◽  
E E Nickel
Keyword(s):  
Freeze Fracture ◽  
Photoreceptor Cells ◽  
Molecular Characteristics ◽  
Thin Sections ◽  
Freeze Fracturing ◽  
Mean Diameter ◽  
Hexagonal Arrangement ◽  
Photoreceptor Membranes

The ultrastructure of photoreceptor cells of the crayfish (P. clarkii) has been examined by means of thin sections and freeze-fracturing. The study reveals that in the photoreceptor membranes there are particles associated primarily with the A faces of freeze-fracture preparations which have a mean diameter of 80-84 A and a density of 6,600 per per micrometer2. Treatment of the retina with digitonin (a substance capable of extracting visual photopigments) in Ringer's causes marked disruption of the hexagonal arrangement of the microvilli, breakdown of the microvilli into smaller segments, and gradual removal of the particles. The estimated photopigment concentration in the microvillus is 4,000 per micrometer. It is suggested that the observed particles represent the photopigment in situ.

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 Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.

1984 ◽  
Vol 99 (2) ◽  
pp. 390-402 ◽  
Author(s):  
D Zucker-Franklin ◽  
S Petursson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Precursor Cell ◽  
Membrane Domains ◽  
Thin Sections ◽  
Energy Dependent ◽  
First Time ◽  
Later Phase ◽  
Peripheral Cytoplasm

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.

scholarly journals Effect of Ca2+ on the dimeric structure of scallop sarcoplasmic reticulum.

1989 ◽  
Vol 108 (2) ◽  
pp. 511-520 ◽  
Author(s):  
L Castellani ◽  
P M Hardwicke ◽  
C Franzini-Armstrong
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Surface Structure ◽  
Calcium Binding ◽  
Freeze Fracture ◽  
Hill Coefficient ◽  
Half Maximum ◽  
Crystalline Surface ◽  
Maximum Activation ◽  
The Individual

Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze-fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+-ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isolation procedure of SR vesicles does not alter the dimeric organization of the Ca2+-ATPase, although the overall appearance of the tubes seems to be affected: the edges of the vesicles are scalloped and the individual Ca2+-ATPase molecules are not clearly defined. The effect of Ca2+ on isolated scallop SR vesicles was investigated by correlating the enzymatic activity and calcium-binding properties of the Ca2+-ATPase with the surface structure of the vesicles, as revealed by electron microscopy. The dimeric organization of the membrane is preserved at Ca2+ concentrations where the Ca2+ binds to the high affinity sites (half-maximum saturation at pCa approximately 7.0 with a Hill coefficient of 2.1) and the Ca2+-ATPase is activated (half-maximum activation at pCa approximately 6.8 with a Hill coefficient of 1.84). Higher Ca2+ concentrations disrupt the crystalline surface array of the SR tubes, both in the presence and absence of ATP. We discuss here whether the Ca2+-ATPase dimer identified as a structural unit of the SR membrane represents the Ca2+ pump in the membrane.

Electron Microscopic Evidence for the Transmembrane Displa cement of Calcium ATPase

1984 ◽  
Vol 39 (1-2) ◽  
pp. 177-179 ◽  
Author(s):  
Donald J. Scales ◽  
Stefan R. Highsmith
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Freeze Fracture ◽  
Calcium Atpase ◽  
Two Dimensional ◽  
Electron Microscopic ◽  
Microscopic Evidence ◽  
Sarcoplasmic Reticulum Membrane ◽  
First Time

Abstract Incubation of the Ca2+-ATPase in vanadate solutions leads to the formation of two-dimensional arrays in the sarcoplasmic reticulum membrane. Electron micrographic freeze fracture replicas show depressions on the inner leaflet for the first time. This indicates that the ATPase has moved perpendicular to the plane of the membrane. Our results also suggest that aggregation of the Ca2+-ATPase into the two-dimensional arrays occurs before they move into the membrane. These phenomena were observed as soon as 15 minutes after vanadate was added. The effects of vanadate appear to be completely reversible. When SR was incubated in the vanadate solutions and was then diluted into a buffer containing Ca2+ and ATP, the ATPase activity was normal for up to several hours of incubation and only somewhat reduced after 3 days.

The ultrastructure of Entamoeba sp. (Laredo isolate). Observations on thin sections and freeze-fracture preparations

1979 ◽  
Vol 57 (9) ◽  
pp. 1723-1735 ◽  
Author(s):  
Hampik S. Injeyan ◽  
Erwin Huebner
Keyword(s):  
Particle Density ◽  
Freeze Fracture ◽  
Structural Complexity ◽  
Structural Features ◽  
Nuclear Pore ◽  
Pore Density ◽  
Thin Sections ◽  
Membrane Surfaces ◽  
Intranuclear Microtubules ◽  
First Time

The ultrastructure of Entamoeba sp. (Laredo isolate), seen in thin section and in freeze-fracture preparation, indicates that numerous structural features are common to both this E. histolytica-like amoeba and "regular" strains. In addition, heavy meromyosin (HMM)-binding, actin-like microfilaments and intranuclear microtubules and microfilaments are demonstrated for the first time in Entamoeba. Cytoplasmic microtubules were not detected using two different fixation procedures. Replicas of freeze-fractured membrane surfaces revealed particle size and distribution to be heterogeneous on both fracture faces P and E and suggested a similar structural complexity to that previously described for the HK9 strain of E. histolytica. Particle density was determined in plasma and phagosome membranes, and an enrichment of 4.5-fold and 1.6-fold was estimated for the P and E faces, respectively, of phagosome membranes.Nuclear-pore distribution was heterogenous and pore density was highly variable in log-phase cells probably reflecting different cell-cycle stages. Possible differences in nuclear-pore density and distribution relative to published accounts on "regular" strains of E. histolytica are considered.

scholarly journals Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

1989 ◽  
Vol 37 (11) ◽  
pp. 1635-1643 ◽  
Author(s):  
A H Schrijvers ◽  
P M Frederik ◽  
M C Stuart ◽  
K N Burger ◽  
V V Heijnen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Freeze Fracture ◽  
Tissue Preparation ◽  
Unilamellar Vesicles ◽  
Thin Sections ◽  
Multilamellar Vesicles ◽  
Morphological Studies ◽  
Rat Hearts ◽  
Small Unilamellar Vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

scholarly journals THE SPLITTING OF HEPATOCYTE GAP JUNCTIONS AND ZONULAE OCCLUDENTES WITH HYPERTONIC DISACCHARIDES

1974 ◽  
Vol 61 (3) ◽  
pp. 575-590 ◽  
Author(s):  
Daniel A. Goodenough ◽  
Norton B. Gilula
Keyword(s):  
Portal Vein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Chloride Ion ◽  
Thin Sections ◽  
Zonulae Occludentes ◽  
The Times

Mouse livers were perfused in situ through the portal vein with the disaccharides sucrose, lactose, maltose, and cellobiose in hypertonic concentrations (0.5 M). This treatment resulted in plasmolysis of the hepatocytes and splitting of the gap junctions and zonulae occludentes. The junctions split symmetrically, leaving a half-junction on each of the two separated cells. The process of junction splitting is followed using the freeze-fracture technique, since the junctional membranes are indistinguishable from the nonjunctional membranes in thin sections once the splitting occurs. The split junctions are also studied using the freeze-etch technique, allowing a view of the gap junction extracellular surface normally sequestered within the 2-nm "gap." The monosaccharides sorbitol and mannitol did not split the junctions during the times studied (2 min), but substitution of the chloride ion with propionate in the perfusion mixture did result in junction splitting. An envelope of morphologically distinct particles surrounding freeze-fractured gap junctions is also described.

Evidence that tubulin forms an integral membrane skeleton in molluscan gill cilia

1987 ◽  
Vol 88 (4) ◽  
pp. 527-535
Author(s):  
R.E. Stephens ◽  
S. Oleszko-Szuts ◽  
M.J. Good
Keyword(s):  
Critical Micelle Concentration ◽  
Freeze Fracture ◽  
Sedimentation Coefficient ◽  
Membrane Vesicles ◽  
Membrane Skeleton ◽  
Particulate Material ◽  
Cross Reactivity ◽  
Thin Sections ◽  
Octyl Glucoside ◽  
Membrane Tubulin

Controlled extraction of intact gill tissue, isolated cilia or reconstituted membrane vesicles with Nonidet P-40 at greater than 4 times the critical micelle concentration, or with octyl glucoside at the critical micelle concentration, delipidates the membrane, leaving a membrane remnant or skeleton of membrane tubulin and associated proteins. This skeleton consists of a disordered reticular protein network in reconstituted membrane vesicles and a similar but more compact sleeve in cilia of extracted tissue. The membrane skeleton is closely apposed to the axoneme and is attached to the outer doublets by fine radial bridges having a 20–24 nm longitudinal periodicity, supporting earlier observations made utilizing a lipophilic cross-linking agent. Higher concentrations of detergent solubilize the membrane tubulin-protein complex, producing 5–10 nm particulate material of low sedimentation coefficient. Dilution of an octyl glucoside solution to below the critical micelle concentration results in disappearance of the particles and reformation of the membrane, indicating that the particles are protein-detergent micelles and not denatured protein. Freeze-fracture electron microscopy reveals no comparable-sized natural particles in the ciliary membrane proper. The reticular material of the membrane skeleton contains tubulin, demonstrated on Lowicryl K4M thin sections by a rabbit polyclonal antibody to sea-urchin egg cytoplasmic tubulin, using gold-labelled secondary antibody. Minimal cross-reactivity is detected prior to Triton-delipidation, suggesting that most membrane tubulin antigenic sites are buried within the bilayer and that the tubulin is not simply adsorbed to the lipid bilayer.

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