Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder

1976 ◽  
Vol 22 (2) ◽  
pp. 355-370 ◽  
Author(s):  
S. Knutton ◽  
J.D. Robertson
Keyword(s):  
Plasma Membrane ◽  
Urinary Bladder ◽  
Cytoplasmic Membrane ◽  
Membrane Surface ◽  
Hexagonal Lattice ◽  
Freeze Fracture ◽  
Hexagonal Structure ◽  
Unit Cell ◽  
Hexagonal Array ◽  
Thin Sections

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.

scholarly journals Analysis of the structure of intramembrane particles of the mammalian urinary bladder.

1980 ◽  
Vol 86 (2) ◽  
pp. 514-528 ◽  
Author(s):  
J D Robertson ◽  
J Vergara
Keyword(s):  
Urinary Bladder ◽  
Glass Surface ◽  
Hexagonal Lattice ◽  
Freeze Fracture ◽  
Transitional Epithelium ◽  
Thin Sections ◽  
Intramembrane Particles ◽  
Thin Sectioning ◽  
Space Constant ◽  
Dominant Structure

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100 degrees C and replicated at -190 degrees C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface. A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have beeen found in the EF faces. The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure. By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims. Each domain has a central spot of metal encircled by a shadow. The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant. The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model. The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements. The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures.

scholarly journals Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane

1978 ◽  
Vol 77 (2) ◽  
pp. 323-328 ◽  
Author(s):  
WW Franke ◽  
C Grund ◽  
E Schmid ◽  
E Mandelkow
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Membrane Surface ◽  
Hexagonal Lattice ◽  
Interparticle Distance ◽  
Macromolecular Complexes ◽  
Inner Surface ◽  
Cross Bridges ◽  
Plasma Membrane Surface

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.

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 A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus.

1979 ◽  
Vol 83 (2) ◽  
pp. 371-382 ◽  
Author(s):  
J C Linder ◽  
L A Staehelin
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Fluid Secretion ◽  
Contractile Vacuole ◽  
Dense Adhesion ◽  
Thin Sections ◽  
Ultrarapid Freezing ◽  
Leptomonas Collosoma ◽  
Adhesion Plaque

We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.

Ultrastructural changes in chloroplasts resulting from fluctuations in NaCl concentration: freeze-fracture of thylakoid membranes in Dunaliella salina

1975 ◽  
Vol 18 (2) ◽  
pp. 287-299 ◽  
Author(s):  
A.O. Pfeifhofer ◽  
J.C. Belton
Keyword(s):  
Salt Concentration ◽  
Dunaliella Salina ◽  
Unit Area ◽  
Morphological Changes ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Thylakoid Membranes ◽  
Ultrastructural Changes ◽  
Thin Sections ◽  
Number Of Particles

The structure of chloroplasts isolated from Dunaliella salina has been studied with respect to changing concentrations of sodium chloride in the culture medium. Freeze-fracture replicas and thin sections of intact chloroplasts do not exhibit any noticeable changes in structure at concentrations ranging between 3.5 and 25% NaCl. Chloroplasts isolated from algal cells that have been acclimatized to the higher salt concentration show a change in the thylakoid membranes. The thylakoid membranes appear compressed over a major portion of the membrane surface, with only the end of the thylakoid membranes unappressed. The number of particles per unit area on the B face is also altered by the salt concentration. The chloroplasts acclimatized to 25% NaCl have about 3 times the number of particles per unit area on a B face of end-membranes as on a comparable face of thylakoid membranes acclimatized to low (3.5% NaCl) salt concentration. These morphological changes can be reversed if the chloroplasts acclimatized to high or low salt concentrations are returned to a medium of different salt concentration prior to freeze-fracturing.

scholarly journals ISOLATION OF THE PLASMA MEMBRANE OF THE LUMINAL SURFACE OF RAT BLADDER EPITHELIUM, AND THE OCCURRENCE OF A HEXAGONAL LATTICE OF SUBUNITS BOTH IN NEGATIVELY STAINED WHOLE MOUNTS AND IN SECTIONED MEMBRANES

1970 ◽  
Vol 45 (3) ◽  
pp. 542-553 ◽  
Author(s):  
R. M. Hicks ◽  
B. Ketterer
Keyword(s):  
Plasma Membrane ◽  
Membrane Structure ◽  
Mercuric Acetate ◽  
Hexagonal Lattice ◽  
Negative Contrast ◽  
Luminal Surface ◽  
Hexagonal Array ◽  
Bladder Epithelium ◽  
Luminal Membrane ◽  
Whole Mounts

A method of isolating the thick luminal membrane from homogenates of bladder epithelium is described, which entails pretreatment of the epithelium with fluorescein mercuric acetate and centrifugation of the homogenate on sucrose density gradients. A hexagonal array of hexamers is illustrated by negative contrast staining in whole mounts of the isolated thick membrane. Subunits are also shown in tangential sections of this thick membrane, in fixed, embedded bladder epithelium. The significance of the subunits is discussed in the context of membrane structure and permeability.

scholarly journals Membrane differentiations at sites specialized for cell fusion.

1977 ◽  
Vol 72 (1) ◽  
pp. 144-160 ◽  
Author(s):  
R L Weiss ◽  
D A Goodenough ◽  
U W Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Active Role ◽  
Central Dome ◽  
Thin Sections ◽  
Mating Structure ◽  
Putative Site ◽  
Freeze Fracture Electron Microscopy

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

Isolation of the Crystalline Arrays from the Yeast Plasma Membrane

1982 ◽  
Vol 40 ◽  
pp. 100-101
Author(s):  
Gina Sosinsky ◽  
Robert M. Glaeser
Keyword(s):  
Plasma Membrane ◽  
Test Specimen ◽  
Current Model ◽  
Hexagonal Lattice ◽  
Freeze Fracture ◽  
Structural Studies ◽  
Electron Microscopic ◽  
Threefold Axis ◽  
Yeast Plasma Membrane ◽  
Microscopic Techniques

Crystalline arrays of particles found in freeze-fracture replicas of the yeast plasma membrane have been used extensively as a test specimen for investigating artifacts that can occur during the freeze fracture process. No information has yet been obtained about the particles themselves from electron microscopic techniques other than freeze fracture. The current model of the paracrystals consists of three intramembranous particles arranged in a hexagonal lattice: 1) a large 100 K particle on the PF, which has a “volcano” appearance in replicas that are uncontaminated by water vapor, 2) a smaller EF particle at the threefold axis of the hexagonal lattice, and 3) an even smaller EF particle which fits into the crater of the PF volcano particle. We have now isolated the crystalline arrays of the yeast plasma membrane for further structural studies by freeze fracture, surface replication, and negative staining, in order to test this current model.

Structure of the insect flight muscle Z disk

1983 ◽  
Vol 41 ◽  
pp. 456-457
Author(s):  
Deatherage J.F. ◽  
Luke B. ◽  
Sainsbury G.M. ◽  
Bullard B.
Keyword(s):  
Flight Muscle ◽  
Insect Flight ◽  
Hexagonal Lattice ◽  
Unit Cell ◽  
Insect Flight Muscle ◽  
Thin Filaments ◽  
Thin Sections ◽  
Carpenter Bee ◽  
Contour Maps ◽  
Fourier Filtering

The Z disk of striated muscle is a regular planar network of connections linking two sets of oppositely directed thin filaments from adjacent sarcomeres. Ashurst has inspected images of oblique thin sections through insect Z disks, and proposed that parallel overlapping filaments in the Z disk are arranged in hexagonal bundles of six, which alternating filaments enter from opposite sarcomeres. We have now analyzed images of thin-sectioned and negatively-stained Z disks using Fourier filtering and crystallographic image processing methods. Our findings confirm the basic model of Ashurst for the arrangement of filaments inside the Z disk. By evaluating contour maps of crystallographically averaged images at higher resolution, we have obtained additional information about the positions of the filaments and the connections linking them.We have examined Z disks of flight muscles of three orders of insect, Diptera (blowfly), Hemiptera (Lethocerus) and Hymenoptera (bumble and carpenter bee). Thin sections from Lethocerus whole muscle fibers and negatively-stained isolated Z disks from bumble bee were examined in most detail. The connectivities of thin filaments of one sarcomere through the Z disk to the adjacent sarcomere were traced on Fourier-filtered images of oblique thin-sections. These cut the myofibril at an angle off perpendicular, intersecting it on one side of the Z disk, passing through it, and exiting on the other side. The filaments in the Z disk lattice, and the connections between them, were examined on crystallographically averaged images of transverse sections through the Z disk, and of isolated, negatively-stained Z disks.The insect flight muscle Z disk is arranged on a hexagonal lattice with unit cell dimensions of about 465Å. Images of both thin- sectioned and negatively-stained Z disks diffract optically to the fifth or sixth order (about 75Å resolution). The projection symmetry of the Z disk is p3m. Contour maps of crystallographically averaged images of thin- sectioned and isolated Z disks show prominent features arranged in roughly hexagonal bundles of six. These features correspond to extensions of the thin filaments into the Z disk, three from each of the opposing sarcomeres. By analysis of oblique sections we have confirmed that the opposed thin filament lattices of the two sarcomeres meet in three-fold to six-fold register (that is, a third of a unit cell out of register along both crystallographic axes). The relative strengths of features in these images are consistent with long overlap of filaments from opposite sarcomeres inside the Z disk.

Role of freeze-fracture technique in tumour pathology

1992 ◽  
Vol 50 (1) ◽  
pp. 616-617
Author(s):  
T. M. Mukherjee ◽  
J. G. Swift
Keyword(s):  
Plasma Membrane ◽  
Cell Membranes ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Intercellular Junctions ◽  
Structural Components ◽  
Thin Sections ◽  
Human Tumours ◽  
Tumour Pathology

The freeze-fracture technique is unique in its ability to expose extensive face views of the interior of the cell membranes. This feature is particularly useful for studies of events occurring at the plasma membrane, such as exocytosis of secretory granules and of structural components within the membrane such as the intercellular junctions. We have used freeze-fracture preparations in conjunction with conventional thin sections to study the intercellular junctions in a variety of human tumours.

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